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https://openalex.org/W2162073279	https://europepmc.org/articles/pmc3684091?pdf=render	English	null	Ankle Fracture Surgery on a Pregnant Patient Complicated by Intraoperative Emergency Caesarian Section	Case reports in orthopedics	2,013	cc-by	2,303	Case Report Ankle Fracture Surgery on a Pregnant Patient Complicated by Intraoperative Emergency Caesarian Section Ran Schwarzkopf,1 Steven C. Gross,2 Allen Coopersmith,3 and Ramesh Gidumal2 1 Department of Orthopaedic Surgery, UC Irvine Medical Center, 101 The City Drive South Pavillion III, Orange, CA 92868, USA 2 Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 301 E. 17th Street, New York, NY 10003, USA 3 Department of Anesthesia, NYU Hospital for Joint Diseases, 301 E. 17th Street, New York, NY 10003, USA Correspondence should be addressed to Ran Schwarzkopf; schwarzk@gmail.com Received 28 April 2013; Accepted 21 May 2013 Academic Editors: K. Erler, I. Han, P. Perrini, and T. Tsurumoto Copyright © 2013 Ran Schwarzkopf 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. We report the case of a woman in the third trimester of pregnancy who sustained an ankle fracture dislocation that could not be adequately closed reduced. After discussions with the patient, her obstetrician, and the anesthesiologists, she was indicated for surgical fixation. A heart tone monitor was used to assess fetal health during the procedure. During surgical incision, the fetus went into distress, and an emergency caesarian section was performed. After delivery of the infant and abdominal closer, surgery was completed. Due to a cohesive team effort, both the patient and her infant had excellent outcomes. There are many important considerations in the surgical management of the pregnant patient with traumatic orthopaedic injuries. Of especial importance to the orthopaedic surgeon is the impact of patient positioning on uteroplacental blood flow. This report discusses factors that should be taken into account by any orthopaedist who plans to operate on a pregnant patient. Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 962794, 4 pages http://dx.doi.org/10.1155/2013/962794 Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 962794, 4 pages http://dx.doi.org/10.1155/2013/962794 Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 962794, 4 pages http://dx.doi.org/10.1155/2013/962794 3. Discussion it A fetal heart rate monitor was used for continuous fetal assessment while in the operating room. Spinal anesthesia was performed with the patient in the lateral decubitus posi- tion, as this was deemed more appropriate than general anes- thesia with relation to the pregnancy. Following the induction of anesthesia, the patient was placed in the supine position with a bump under her right side to displace the uterus. Sterile draping was begun. During this time, the fetal monitor began to show prolonged heart rate decelerations to 50–60 beats per minute, which did not correct with changing the patient’s position. The obstetrical team, who had been previously notified about the case and was present in the hospital, was immediately called. Upon their arrival, the obstetricians decided to perform an emergent cesarean section. Though the fetal heart rate had stabilized by the time they completed their evaluation, they were committed to an emergent delivery in order to avoid further distress to the fetus.h Many pregnant patients in the United States undergo nonob- stetric surgery each year, with current estimates placing the number at greater than 80,000 [4]. Although the vast majority of these procedures are performed without significant com- plication, it is important to take note of the inherent risks involved. While in many situations it is preferable to delay surgery on a pregnant patient until the postpartum period, this is not always possible. The most recent ACOG Commit- tee Opinion on nonobstetric surgery in pregnancy stresses the importance of obtaining preoperative obstetric consulta- tion. It also states that there are inadequate data to make spe- cific guidelines for these procedures, including the utility of using continuous intraoperative fetal heart monitoring [5].h g p g The current evidence for using continuous fetal heart monitoring in order to avoid adverse outcomes is anecdotal [4]. In general it is recommended that pregnant patients who have sustained traumatic injury and exhibit signs of fetal distress, including fetal heart rate of less than 100 beats per minute and/or prolonged decelerations, undergo emergent cesarean section if the fetus is older than 26 weeks [6]. How- ever, this decision can only be made after consultation by an obstetrician. In our case, it seems that use of continuous fetal monitoring detected a significant abnormality and led to a timely intervention, potentially avoiding a poor outcome. 1. Introduction In the United States, traumatic injury remains a significant complication of many pregnancies. Trauma affects as many as 8% of pregnancies and represents the leading nonobstetric cause of maternal death [1–3]. While major injury can poten- tially result in life-threatening sequelae, minor trauma can nonetheless also create diagnostic and therapeutic challenges for the patient and surgeon. We present the case of a thirty- nine-year-old female who was thirty-six weeks pregnant when she sustained a left ankle twisting injury resulting in an ankle fracture dislocation. Her surgical procedure was complicated by fetal heart rate abnormalities resulting in emergent cesarean section. We believe this scenario, while described in the obstetrical literature, is unique in the ortho- paedic literature and illustrative of a few important points related to perioperative management of the pregnant patient. Written consent for publication of this case was obtained from the patient involved. A thirty-nine-year-old woman who was thirty-six weeks pregnant sustained a left ankle injury during a mechanical slip and fall, resulting in significant pain and deformity. Her pregnancy had been uncomplicated up until that point. She had no significant past medical or surgical history and did not take any medications at the time of injury. On initial presentation to the emergency department at an outside institution, the patient was diagnosed with a trimalleolar left ankle fracture dislocation (Figure 1). The fracture pattern was consistent with a Lauge-Hansen supination-external rotation IV injury. The patient’s neurovascular status was intact. She was closed reduced and transferred in a splint from the outside hospital to our institution for further care. Upon arrival at our emergency room, we reviewed the radiographic images, which revealed an incompletely reduced tibiotalar joint and proceeded to remove the splint in order to fully examine the site of injury. At this time, the ankle 2 2 Case Reports in Orthopedics (a) (b) Figure 1: Initial anteroposterior and lateral radiographs showing an ankle fracture dislocation with lateral and posterior displacement of the talus. (a) (b) Figure 2: Postoperative mortise and lateral radiographs taken three months after surgery. (a) (b) (a) (b) (b) (a) (b) (a) Figure 2: Postoperative mortise and lateral radiographs taken three months after surgery. Figure 1: Initial anteroposterior and lateral radiographs showing an ankle fracture dislocation with lateral and posterior displacement of the talus. the procedure well, and there were no further complications. 1. Introduction Postoperatively, the patient was transferred to the postpartum unit, where she progressed well over the ensuing days in terms of rehabilitation and physical therapy. The patient was changed from a plaster splint to a fiberglass cast after several days. She was discharged from the hospital on postoperative day four in good condition. Clinical followups in the twelve months since the operation have shown no complications, with both the patient and her daughter doing well and progressing appropriately. was noted to be highly unstable. Attempts at closed reduction and splinting yielded the same incomplete restoration of the ankle mortise with persistent subluxation of the talus. The patient was counseled that her injury was unstable, incompletely reduced, and that it would require surgical intervention. The risks and benefits of surgery, not only to the patient but also to the fetus, were discussed with her in conjunction with the obstetricians. The decision was reached to proceed the following day with open reduction and inter- nal fixation of the left ankle. 3. Discussion The patient was again placed in the lateral decubitus position in order to receive an epidural catheter. After this was placed, the patient was returned supine, and a caesarian section was performed. A healthy infant female was success- fully delivered, with APGAR scores of eight at one minute and nine at five minutes. The obstetricians closed the uterus and abdomen without incident.t After the anesthesiologists and obstetricians reassessed the patient and determined that she was stable for fur- ther surgery, we proceeded with open reduction and inter- nal fixation of the ankle (Figure 2). The patient tolerated Surgical positioning is another important consideration in the pregnant patient, particularly in the third trimester. 3 Case Reports in Orthopedics Figure 3: Preoperative positioning of a pregnant patient with a bump under the right hip and a lead apron draped over the abdomen and pelvis. anesthesiologist. There are many case-specific factors to con- sider, including patient positioning and method of anesthesia. While there is no consensus recommendation on the use of continuous intraoperative fetal heart monitoring, in our case the monitor helped identify a potential problem before it led to an adverse outcome. As a result of cohesive patient care by multiple providers in the perioperative period, we were successfully able to deliver a healthy infant as well as perform surgical fixation of an orthopaedic injury in a timely and safe fashion. Traumatic injury complicates many pregnancies. Surgical management of orthopaedic injuries in the pregnant patient is complex and requires a team-oriented approach. Consul- tation by an obstetrician is mandatory preoperatively, and an appropriate plan for surgical anesthesia is essential. The use of a heart tone monitor can aid in identifying fetal distress and potentially prevent poor outcomes. The orthopaedic surgeon must be aware of limitations in anesthesia and surgical posi- tioning when operating on a pregnant patient, particularly during the third trimester. Special attention must be given to the effect of patient positioning on uteroplacental blood flow. Through teamwork and careful planning, it is possible to deliver safe and effective surgical care to the injured pregnant patient. Figure 3: Preoperative positioning of a pregnant patient with a bump under the right hip and a lead apron draped over the abdomen and pelvis. 3. Discussion Placing the patient supine during this time can result in com- pression of the inferior vena cava by the uterus, which in turn can reduce maternal cardiac output by up to 30% and cause alterations in uteroplacental blood flow [7]. This compressive effect can be partially relieved or eliminated by angling the patient’s body to the left during positioning (Figure 3). The lateral decubitus position is ideal, but the necessity of certain surgical approaches may prevent its use. In our case, the patient required a lateral incision over the left ankle as part of the planned repair, so a left lateral decubitus position was not possible. However, even though a bump was used under her right side, the fact that our patient was placed supine for the planned ankle fixation may have contributed somewhat to the onset of the observed fetal heart rate abnormalities. This high- lights the importance of the orthopaedic surgeon being cog- nizant and flexible in terms of positioning a pregnant patient. It is recommended to avoid a purely supine position if pos- sible. Placing a wedge under the right hip and/or tilting the operating table can help in positioning if desired. Disclosure No authors received any outside support, including aid from pharmaceutical or industrial sources, in the creation of this paper. Authors’ Contribution Ran Schwarzkopf and Steven C. Gross contributed equally to the paper. [7] A. J. Shah and B. A. Kilcline, “Trauma in pregnancy,” Emergency Medicine Clinics of North America, vol. 21, no. 3, pp. 615–629, 2003. References There are some important distinctions between the goals of obstetric and nonobstetric anesthesia in the pregnant patient. Obstetric anesthesia is designed for pain relief with- out hindering uterine contractility. In contrast, a central func- tion of nonobstetric surgical anesthesia is prevention of pre- mature labor or spontaneous abortion. Also, obstetric anes- thesia should avoid depression of the fetal central nervous system, whereas these concerns are not theoretically relevant in nonobstetric anesthesia [8]. For our patient, the original decision was made to use spinal anesthesia, which is associ- ated with less fetal drug exposure, less impact on fetal heart rate variability than general anesthesia, and absent risk of unintentional dural puncture with an epidural needle [4]. However, the need for emergent cesarean section required that the patient’s anesthesia be augmented with an epidural. Our patient’s clinical situation emphasizes the importance of an appropriate anesthetic plan for pregnant surgical patients. [1] K. Flik, P. Kloen, J. B. Toro, W. Urmey, J. G. Nijhuis, and D. L. Helfet, “Orthopaedic trauma in the pregnant patient,” Journal of the American Academy of Orthopaedic Surgeons, vol. 14, no. 3, pp. 175–182, 2006. [2] J. W. Van Hook, “Trauma in pregnancy,” Clinical Obstetrics and Gynecology, vol. 45, no. 2, pp. 414–424, 2002. [3] B. M. Lo, E. J. Downs, and J. C. Dooley, “Open-book pelvic frac- ture in late pregnancy,” Pediatric Emergency Care, vol. 25, no. 9, pp. 586–587, 2009. [4] T. G. Cheek and E. Baird, “Anesthesia for nonobstetric surgery: maternal and fetal considerations,” Clinical Obstetrics and Gyne- cology, vol. 52, no. 4, pp. 535–545, 2009. [5] ACOG Committee on Obstetric Practice, “ACOG committee opinion: nonobstetric surgery in pregnancy,” Obstetrics & Gyne- cology, vol. 102, no. 2, article 431, 2003. Management of pregnant patients with operative frac- tures is complex. A team-based approach is warranted, incorporating the orthopaedic surgeon, obstetrician, and [6] J. A. Morris Jr., T. J. Rosenbower, G. J. Jurkovich et al., “Infant survival after cesarean section for trauma,” Annals of Surgery, vol. 223, no. 5, pp. 481–491, 1996. Case Reports in Orthopedics 4 [7] A. J. Shah and B. A. Kilcline, “Trauma in pregnancy,” Emergency Medicine Clinics of North America, vol. 21, no. 3, pp. 615–629, 2003. [8] E. S. Steinberg and A. C. Santos, “Surgical anesthesia during pregnancy,” International Anesthesiology Clinics, vol. 28, no. 1, pp. 58–66, 1990.
W4391194632.txt	https://www.degruyter.com/document/doi/10.1515/9783839469811-016/pdf	de		Handbuch zum Kurzfilm Schweigemahl (2020)	transcript Verlag eBooks	2,024	cc-by	5,719	Handbuch zum Kurzfilm Schweigemahl (2020) An Anti-Racism Pedagogy Leona-Maike Wenning Kurzfilme schaffen es, in einer sehr kurzen Zeit ein zentrales Thema in den Fokus zu setzen. Sie konzentrieren sich auf eine Situation oder Problematik, beschreiben diese und regen die Zuschauenden zur Auseinandersetzung mit eben diesem Thema an. Trotz ihrer Kürze können sie durch die Prägnanz ein Thema vertieft darstellen. Das Genre des Kurzfilms umfasst verschiedene Produktionsstile. Häufig wird großer Fokus auf die Gestaltung der Filmästhetik gelegt, wie etwa die Kameraeinstellungen, Schnitte und Bildkompositionen. Diese Arbeit beschäftigt sich speziell mit deutschen Kurzfilmen, die ein zentrales Thema innerhalb der deutschen Kultur repräsentieren. Es soll ein Filmhandbuch entwickelt werden, das orientiert ist an der Zielgruppe US-amerikanischer Studierender des Studienganges German Studies. Bevor die Auswahl getroffen wurde, den Kurzfilm Schweigemahl (http://www.youtube.com/watch?v=3ornrMXetmQ&t=8 s) in den Fokus der Arbeit zu setzen, standen zahlreiche, ebenso interessante und wertvolle Kurzfilme zur Auswahl.1 Bereits die Vorarbeit in der Filmauswahl war sehr interessant, da sie ein Eindenken in die Zielsituation erforderte, um herauszufinden, welche Kriterien relevant sind für die finale Entscheidung für einen Kurzfilm. Wie lang darf der Film sein (es muss die Möglichkeit bestehen, ihn zweimal gucken zu können)? Ist der Film inhaltlich und sprachlich verständlich für die Zielgruppe? Und am schwierigsten – repräsentiert der Film Deutschland? Was soll überhaupt von Deutschland repräsentiert werden? Was verstehen wir Deutsche unter deutscher Kultur, »typisch Deutsch«? Was verstehen die Studierenden bisher unter deutscher Kultur? Wie soll Deutschland von den Studierenden gesehen werden? 1 Die Kurzfilme, die ebenfalls als geeignet und wertvoll bewertet wurden, können über folgenden Link abgerufen werden, online unter http://www.youtube.com/playlist?list=PLl05GkD4 -hBdogKFNg79YtpgzO2rSt3it. 186 Forum on Pedagogy/Fachdidaktik Prolog Mit der Auswahl des Kurzfilms Schweigemahl wurde entschieden, den Fokus auf Rassismus in Deutschland zu lenken. Alltagsrassismus zeigt sich in Deutschland auf vielerlei Art und Weise. Seien es Stereotypen verstärkende ›Komplimente‹, abwertende Blicke oder die immer wieder gestellte Frage: »Wo kommst du denn eigentlich her?« Einige dieser typischen Strukturen des Alltagsrassismus werden in dem Kurzfilm Schweigemahl von Horst Wegener und Arne Schramm thematisiert. Der Kurzfilm ist in Verbindung mit Wegeners Song werden zwölf/zwölf im Jahr 2020 produziert worden und bietet einen Einblick in den Alltag einer Familie mit schwarzer Mutter und weißem Vater. Mit der Mutter und den beiden Kindern werden Situationen aufgezeigt, in denen intentionaler und nicht intentionaler Alltagsrassismus deutlich werden. Der Kurzfilm soll die Zuschauenden zur Reflexion anregen, über eigene Rassismen und Ungleichbehandlungen nachzudenken. Der Film schafft es, authentisch und zeitgemäß ein Bild von Alltagsrassismus zu zeichnen, in dem sich vermutlich viele Zuschauende wiederfinden werden können – als Opfer, aber definitiv auch als Täter. Es werden unterschwellige, aber auch offensichtliche rassistische Ungleichbehandlungen dargestellt, deren Situationen geprägt sind von Naivität, Unwissen, aber auch bewussten Anfeindungen. Der Film spielt viel mit Kamera- und Lichteinstellungen sowie Ton und Schnitten. Dadurch lädt er zur Analyse auf inhaltlicher und gestalterischer Ebene ein. Die Aussage des Films ist zwar nicht versteckt, wird aber auch nicht direkt ausgesprochen und erfordert so ein Weiterdenken vonseiten der Zuschauenden. Der Kurzfilm ist sprachlich einfach gehalten, auch dadurch, dass alltägliche Familienunterhaltungen im Fokus stehen. Außerdem sprechen die Schauspieler/innen klares Deutsch ohne Dialekt oder Akzent und das Audiomaterial wird visuell gut unterstützt. Durch diese Aspekte eignet sich der Film insbesondere auch für den Fremdsprachenunterricht. Das generelle Thema Rassismus stellt allerdings den Hauptaspekt dar, der den Film relevant macht für den Fremdsprachenunterricht oder für Studierende des Studiengangs German Studies an US-amerikanischen Hochschulen, die in dieser Arbeit als Zielgruppe im Fokus stehen. Der Studiengang German Studies umfasst mehr als nur das Lehren der deutschen Sprache und Literatur. Stärker im Fokus steht auch die deutsche Kultur und die fächerübergreifende, also interdisziplinäre Arbeit und das Aneignen selbstkritischer und selbstreflexiver Gewohnheiten (vgl. Donahue et al. 22). Dazu gehört die Auseinandersetzung mit Diskriminierung und Rassismus. Deutschland wird häufig in Bezug auf seine Erinnerungskultur eine besondere Position zugeschrieben, aber auch hier stellt der Alltagsrassismus ein dringendes Problem dar. Am Beispiel Deutschlands soll deutlich gemacht werden, dass, wenn es um das Thema Diskriminierung und Rassismus geht, immer mehr gegen Diskriminierung gemacht werden kann. Rassismus ist Teil der deutschen Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) Geschichte, aber nicht nur in der Vergangenheit, weil eben diese Vergangenheit Auswirkungen auf die Gegenwart hat. Man sollte sich nicht darauf ausruhen, dass in Deutschland die Erinnerungskultur existiert und gelehrt wird, sondern sie muss auch zum aktiven Nach- und Überdenken führen, zum Immer-wiederHinterfragen der eigenen Handlungen (vgl. Moffitt). Der Kurzfilm Schweigemahl ist eine der Möglichkeiten, weiterhin Aufmerksamkeit auf das Thema zu lenken in Deutschland, aber auch in den USA. Insbesondere der ›Stop Woke Act‹, der regulieren soll, wie Identität und Herkunft (›race‹) an US-amerikanischen Schulen, Universitäten und Arbeitsplätzen behandelt wird, würde so eine Auseinandersetzung mit Rassismus in den USA aufhalten (vgl. Svrluga /Rosza). Aus einer liberalen, ›blauen‹ Sicht heraus lässt sich das Gesetz nur als problematisch einstufen, denn mit Blick auf Deutschland wird deutlich, dass eine ausgeprägte Erinnerungskultur und Aufarbeitung nicht ausreichen, Alltagsrassismus in Deutschland einzugrenzen. Wie kann bei so einer Aussicht verboten werden, generell über diese Thematiken zu reden? Der Kurzfilm Schweigemahl findet seine Relevanz also darin, dass er verdeutlicht, wie aktuell das Thema Alltagsrassismus ist. Er fordert Zuschauende zur Selbstreflexion auf und zur Auseinandersetzung mit eigenen Erfahrungen. Er hebt hervor, wie wichtig die Sensibilisierung insbesondere für unterschwelligen Rassismus ist. Mit diesen Thematiken erfüllt er die Forderungen des Studienganges German Studies, nicht nur den Spracherwerb der Studierenden zu fördern, sondern auch deren Entwicklung zu kritischen, selbsthinterfragenden Individuen. Filmanalyse Wie bereits angesprochen, haben die Kameraeinstellung, Bildkomposition und Schnitttechnik einen großen Einfluss auf die Perzeption des Inhalts bei den Zuschauenden. Die folgende Übersicht dieser Faktoren2 soll als Grundlage für eine spätere Analyse dienen. 2 Aufgrund der vielen wechselnden Einstellungen würde es den Rahmen dieser Arbeit übertreffen, alle einzelnen Schnitte aufzulisten. Deswegen werden hier nur Beispiele aufgeführt, die als besonders hervorstechend gesehen wurden. 187 188 Forum on Pedagogy/Fachdidaktik Einstellungsgliederung Time Marker Einheit nach Inhalt Ton & Musik Kameraführung & Montage Mise-en-scéne 0.26–0.31 In der Sequenz: Titel wird eingeblendet Musik stoppt und setzt wieder ein Nahaufnahme 1.10–1.30 Familie sitzt beim Essen am Tisch, Vater greift nach Zeitung Essensgeräusche z.B. Geschirrklappern, sonst Stille Totale Einstellung in Zentralperspektive Sehr aufgeräumtes Esszimmer mit hochwertigen Möbeln ausgestattet, in dunklen Tönen gehalten 1.48–1.55 Tochter im Fokus, Wechsel zur Erinnerung der Tochter in der Schule Schulklingel ertönt Halbnahe Aufnahme Kamera filmt Tochter von links, erst Mutter im Hintergrund, dann ein anderes Kind verschwommen im Vordergrund, Tochter weiterhin im Fokus 1.55–2.40 Sequenz: Gemalte Bilder, Hände, die malen, Tochter greift nach ›hautfarbenem‹ Stift, fragt dann nach dem braunen Hintergrundgeräusche Musik setzt ein (2.12) Nahaufnahme vom Gesicht des Mädchens, Hände der Kinder und Stifte, Halbtotale Einstellung Im Hintergrund Weltkarte, Materialien auf dem Tisch verteilt, sehr dunkel beleuchtet Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) Time Marker Einheit nach Inhalt Ton & Musik Kameraführung & Montage Mise-en-scéne 3.30–3.37 Sohn im Fokus, Wechsel zur Erinnerung des Sohnes im Supermarkt, Frau blickt wieder nach vorne und zieht ihre Tasche vor sich Frau: »Man muss es ihnen ja nicht immer gleich so leicht machen.« Kameraschwenk von unten nach oben, der Tasche folgend, Nahaufnahme Fokus erst auf Tasche, dann auf Gesicht der Frau, die die Kassiererin anblickt, Sohn im Hintergrund 5.55–7.00 Sequenz: Vater putzt sich die Zähne und nach der Reihe erscheinen der Sohn, der sich ins Gesicht fasst, die Tochter, die ihr Gesicht mit weißem Rasierschaum anmalt, und die Mutter, die ihre Haare glättet, im Spiegel Anschwellende Musik, im Hintergrund werden Aussagen wiederholt (z.B. »Das ist mein Sohnemann«; »Ich bin noch nicht fertig mit Ausmalen« und »diese Exotik …«) Amerikanische Einstellung in Zentralperspektive, dann Nahaufnahme der Gesichter im Spiegel; Sohn, Tochter und Mutter sind nur im Spiegel zu sehen, nicht im Badezimmer vor dem Spiegel Badezimmer sehr dunkel gehalten von Farben und Belichtung, Gesichter durch Spiegellicht hell angeleuchtet 8.25–9.00 Sequenz: Vater komplimentiert Mutter, weiter im Gespräch, aber beobachtet, wie Mutter ein Glas in die Hand gedrückt bekommt; sie lässt das Glas fallen Anschwellende Musik, weiterhin Hintergrundgeräusche, aber hallend; Kommentare hörbar wie: »Ihre Frau ist eine wahre Schönheit. Woher kommt sie eigentlich? « Nahaufnahme Vater und Gesprächspartner, amerikanische Einstellung auf die Mutter; Zoom auf Gesicht des Vaters und der Mutter Im Fokus stehen die Mutter und der Vater, die anderen Person rücken in den Hintergrund und auch deren Stimmen 189 190 Forum on Pedagogy/Fachdidaktik Time Marker Einheit nach Inhalt Ton & Musik Kameraführung & Montage 9.40–9.47 Mann auf der Bühne stellt Glas auf ein Tablett, das von der Mutter als Schauspielerin gehalten wird Musik nimmt weiter zu, Gespräch am Tisch, Uhrenticken setzt ein Detailaufnahme des Glases, Geschwindigkeit etwas verzögert, als Glas aufs Tablett gestellt wird, und Kameraschwenk diagonal nach oben auf das Gesicht der Mutter 9.56–10.32 Sequenz: Mutter lässt Glas und Tablett fallen, wendet sich langsam dem Zuschauerraum zu, Vater steht auf Laute Musik, andere Geräusche sind hallend (z.B. Klirren des Glases), Musik schwellt an, als der Vater aufsteht Amerikanische Einstellung auf die Mutter, Kamera auf ihren Rücken gerichtet, zeitlich leichte Verlangsamung, Detailaufnahme des Gesichtes der Mutter, Kameraschwenk, halbnahe Einstellung aus Vogelperspektive auf den Vater, Kamerabewegung mit dem aufstehenden Vater Mise-en-scéne Spotlight auf die Mutter gerichtet auf der Bühne und auf den Vater im Zuschauerraum, als er aufsteht; andere Zuschauer/-innen starren geradeaus auf die Bühne Kurzanalyse Für den gesamten Kurzfilm wird eine Standkamera verwendet. Der Film beginnt mit einer Szenensequenz, die das Zubereiten eines wahrscheinlich afrikanischen Gerichtes zeigt. Dabei werden zunächst Nahaufnahmen verwendet und halbnahe Einstellungen. Durch diesen Wechsel, der immer im Zwei-Sekunden-Takt erfolgt, sowie die schnelle Musik und die Bildkomposition – drei Menschen in einer recht kleinen, vollgestellten Küche – wird man eingeführt in das etwas hektische, aber ty- Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) pische3 Familienleben. Auch die anschließende Szene, die das gemeinsame Essen darstellt, bei dem über Hausaufgaben gesprochen wird und erledigte Einkäufe, vermittelt den Zuschauenden ein vertrautes Gefühl. Der Vater redet laut und offen über die Dinge, die ihn während des Tages geärgert haben, und insbesondere im Kontrast zu seinen eher unwichtig erscheinenden Problemen stehen dann die Erlebnisse der Kinder, die ihnen im Laufe des Tages widerfahren sind. Allerdings scheint es für diese Probleme im Esszimmer keinen Raum zu geben, da Zuschauende nur über Rückblicke, also Erinnerungen, von ihnen erfahren, die jeweils eingeleitet werden durch Nahaufnahmen der Gesichter. Dass die rassistischen Erlebnisse der Kinder und auch der Mutter unausgesprochen bleiben, wird auch in der Badezimmerszene verdeutlicht. Hier hallen all die Momente wider, die der Ausgangspunkt hätten sein können für eine offene Unterhaltung über den erlebten Alltagsrassismus. Aber wieder finden sie nicht ihren Weg in die ›echte‹ Welt, sondern bleiben verbannt im Spiegelbild. Der Sohn fasst sich nachdenklich ins Gesicht, die Tochter ›bemalt‹ ihr Gesicht mit weißem Rasierschaum und die Mutter glättet ihre natürlichen Locken. Das sind die Szenen, die man nur im Spiegel zu sehen bekommt, und sie scheinen in einer Welt stattzufinden, die für den Vater nicht ersichtlich, nicht zugänglich zu sein scheint. Denn in dieser ›Spiegelwelt‹ fühlen sich die anderen, als müssten sie sich verändern, und hinterfragen ihr natürliches Selbst. Der Vater ist in der echten und der Spiegelwelt derselbe. Im Laufe des Kurzfilms wird zudem deutlich, dass ein Glas das Leitmotiv darstellt. Während die Mutter abwäscht, reicht der Vater ihr ein Glas und sie ist kurz davor, über ihre Diskriminierung zu reden, im Theaterfoyer reicht ein fremder Mann ihr ein Glas, der Annahme folgend, dass sie die Bedienung sei, und im Theaterspiel wird ihr erneut ein Glas gereicht, dieses Mal, während sie die Rolle des Hausmädchens spielt. Hier folgt die Verbindung beziehungsweise die Erinnerung der Zuschauenden an die zuvor vorgelesene Kritik, dass die Mutter als Schauspielerin eine »authentische Darstellung ihrer Rolle« abliefere (Wegener/Schramm). Das Glas steht dann im Fokus, wenn zentrale Momente im Erkenntnisprozess des Vaters hervorgehoben werden sollen. Im zweiten Akt ist die Stimmung und Wahrnehmung des Vaters deutlich verändert und nach dem Vorfall im Theaterfoyer beginnt er die Bühnenereignisse auf sich zu beziehen. Dies wird zum Beispiel deutlich durch die anschwellende, Spannung aufbauende Musik und den Schnitt vom Schauspieler auf der Bühne zum Vater im Publikum bei dem Wort »angespannt«. Auch aufseiten der Mutter ist das Glasmotiv entscheidend. Denn auch sie führt den Vorfall im Foyer 3 Ursprüngliche Formulierung, die nun in der Bearbeitung die Frage aufbringt, für wen dieses Szenario eigentlich ›typisch‹ ist. Dieser Gedanke kann auch als Ausgangspunkt für eine Fragestellung im Kurs mit Studierenden sein, aus der eine Diskussion über ›typische‹ Familienbilder in unterschiedlichen Kulturen entwachsen kann. 191 192 Forum on Pedagogy/Fachdidaktik mit ihrer Rolle auf der Bühne zusammen und scheint dann, zumindest in Gedanken, auszubrechen und sich zu wehren. Das Spotlight, das auf die Mutter auf der Bühne gerichtet wird und beleuchtet, wie diese das Tablett und das Glas fallen lässt, gibt den Zuschauenden einen Ausbruch zu erwarten – alle Lichter und Augen sind auf sie gerichtet und warten darauf, was sie zu sagen hat. Und auch der Vater erhebt sich und stellt sich zu ihr ins Spotlight, alle anderen starren stumpf nach vorne, nur er »sieht« den Ausbruch seiner Frau. Er scheint schlussendlich offen zu sein zu hören, was seine Frau ihm versucht zu sagen. Insbesondere in dieser Szene sowie in der Szene im Badezimmer fällt der intendierte Wechsel zwischen Licht und Schatten auf. Einerseits wird dadurch der Fokus der Zuschauenden gelenkt, andererseits wird dadurch auch gezeigt, dass einige der Probleme wortwörtlich im Dunkeln liegen und sie erst ans Licht gebracht werden müssen. Generell ist noch anzufügen, dass der Kurzfilm so produziert ist, dass immer dann eine Nahaufnahme oder ein leichter Zoom erfolgt, wenn die Reaktion einer Person im Fokus stehen soll. Außerdem spielt die Kamera eine Rolle und der Schnitt mit wiederholenden Motiven, wie der Rückblende in die Erinnerungen der Kinder oder die totale Einstellung auf den Esszimmertisch der Familie und den Tisch der Theaterfamilie, wodurch die beiden Szenen in Verbindung gebracht werden. Außerdem wird viel akzentuiert durch anschwellende oder gestoppte Musik. Arbeit mit dem Kurzfilm Es soll nun im Folgenden beispielhaft gezeigt werden, welche Erarbeitungsweisen sich für den Kurzfilm Schweigemahl eignen. Die Zielgruppe der Aufgaben sind Studierende des Studienganges German Studies an US-amerikanischen Hochschulen. Glossar Deutsch Englisch Lächerlich machen (to) become a common laughing-stock Kammerspiel Chamber play/chamber drama Premiere feiern (to) premier, (to) debut SMS-Terror (to) flood sb. with text messages Sohnemann Coll. for son, can be translated with junior or sonny Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) Prise Pinch Hab dich doch nicht so Don’t be like that In höchsten Tönen schwärmen (to) praise sth. or sb. very highly Lokalpresse Local press Leibspeise Favorite food Schlawiner Dodger or mischief Alltagsrassismus Everyday racism or routine racism of everyday life Quotenschwarzer Token black man or woman Fremdzuschreibung Ascription Vergangenheitsaufarbeitung (to) cope with the past – in Germany especially used to describe dealing with the time after 1945 Erinnerungskultur Commemorative culture Intertexte, Anspielungen und Quellen zur Erläuterung Eines der Merkmale des Kurzfilms Schweigemahl ist, dass er leicht ohne viel benötigtes Hintergrundwissen zu verstehen ist. Durch die Kleidung und Produktion wird deutlich, dass der Film zur heutigen Zeit spielt und die einzelnen Rollen, wie Familienverhältnisse, sind auch eindeutig zuzuordnen. Die Sprache ist gut zu verstehen und das Vokabular ist auf einem einfachen Niveau gehalten. Einige umgangssprachliche Wörter, wie zum Beispiel »Sohnemann«, lassen sich im Vornherein erklären. Allerdings wird es hilfreich sein, an die Thematik Rassismus anzuknüpfen und genügend Material zu Rassismus in Deutschland bereitzustellen. Wahrscheinlich werden die Studierenden ungefähr einschätzen können, wie die Lage in Deutschland ist, aber sie werden nicht im Detail die Hintergründe von Rassismus in Deutschland kennen, deren Auswirkungen auf die heutige Zeit oder die verschiedenen Situationen, in denen schwarze Menschen Alltagsrassismus begegnen. Für allgemeine Hintergrundinformationen zum Thema Rassismus in Deutschland eignen sich die Internetseiten der Bundeszentrale für politische Bildung: • Seite des BpB, die zahlreiche weitere Artikel vorschlägt zum Themenbereich Rassismus: http://www.bpb.de/themen/rassismus-diskriminierung/rassismu s/#skip-nav-target 193 194 Forum on Pedagogy/Fachdidaktik • • • Allgemeiner Artikel zum Thema Rassismus. Erklärt grundlegende Begriffe: htt p://www.bpb.de/themen/migration-integration/dossier-migration/223738/ras sismus Artikel zum Thema Alltagsrassismus in Deutschland: http://www.bpb.de/them en/rechtsextremismus/dossier–rechtsextremismus/194569/offensichtlich-und zugedeckt-alltagsrassismus-in-deutschland Verschriftlichung eines Webtalks an einer Schule in Berlin: http://www.bpb.de /themen/rechtsextremismus/dossier–rechtsextremismus/194137/wo-kommstdu-denn-her-alltagsrassismus-in-deutschland Etwas anspruchsvoller, aber nicht weniger informativ sind auch Videos über Alltagsrassismus in Deutschland, die zum Beispiel über YouTube zu streamen sind. ZDF, ein großer öffentlich-rechtlicher Fernsehsender, hat beispielsweise eine afroamerikanische Familie begleitet, die in Deutschland lebt und von Alltagsrassismus betroffen ist (http://www.youtube.com/watch?v=mP64SY9pYnE). Zum Einstieg könnten die Student/-innen auch englischsprachige Hintergrundinformationen sammeln, die das Thema grob zusammenfassen. Ein interessantes Video ist das dokumentarische Racism in Germany, produziert vom Auslandsrundfunk DW (http://www. youtube.com/watch?v=dMk-XpjK98M). Hier lohnt sich auch ein Blick in die Kommentare, in denen andere Betroffene von ihren Erlebnissen berichten. Als Beispiel für Alltagsrassismus in Deutschland wird in dem Kurzfilm das ›Typecasting‹ thematisiert. Viele der schwarzen Schauspieler/-innen in Deutschland beklagen sich genau darüber, dass sie ausschließlich Rollen zugeteilt bekommen, die rassistische Stereotype unterstützen. Auch die Mutter im Kurzfilm erlebt dies in ihrer Rollenbesetzung als Hausmädchen, zu der die Kritik sogar noch ihre »Authentizität« lobt (vgl. Wegener/Schramm). Schauspieler/-innen beklagen, dass sie nicht die Chance erhalten, Rollen wie Akademiker/-innen und Ärzt/-innen zu spielen, und dass generell Theaterprogramme zu wenig divers sind. Einen Einblick in dieses Thema gibt der Artikel »Rassismus an deutschen Bühnen – ein strukturelles Problem?«, veröffentlicht auf der Seite des Auslandsrundfunks Deutsche Welle: http://www.dw.com/de/rassismus-an-deutschen-b%C3%BChnen–einstrukturelles–problem/a-57056069. Ein anderer interessanter Artikel, der auch auf der Seite der Deutschen Welle veröffentlicht wurde, spricht die Kritik an, dass Deutschland nicht als Beispiel für Antirassismus gelten kann (http://www.dw.com/en/germany-anti-racist-nazi-pas t/a-54210136). Insbesondere während der Black-Lives-Matter-Proteste im Frühjahr 2020 wurde der Blick auf Deutschland gelenkt und seinen Umgang mit Denkmälern im Vergleich zu den USA, wo neben Denkmälern auch zahlreiche Statuen an historische rassistische Figuren erinnern. Der Artikel verweist darauf, dass Deutschland trotz Vergangenheitsaufarbeitung der Nazizeit, die Zeit vor dem Naziregime und die Zeit danach und deren Auswirkungen auf den Umgang mit Rassismus heut- Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) zutage vernachlässigt. Auch das Gespräch von Sven Beckstette, Veronika Fuechtner und Oliver Hardt »Rassismus benennen« sieht den Grund für den anhaltenden Rassismus in Deutschland in der Reaktion auf die Nazivergangenheit (http://www .textezurkunst.de/de/113/naming-racism/). Das ist so zu erklären, dass als Reaktion auf unter anderem den Holocaust zum Beispiel der Begriff ›Rasse‹ in Deutschland nicht mehr verwendet wird, da er zu sehr auf die biologische Unterscheidung von Menschen fokussiert. Außerdem kann die stark ausgeprägte Erinnerungskultur, laut Hardt, dazu führen, dass die Kritik an der rassistischen Vergangenheit den gegenwärtigen Rassismus sozusagen verdrängt (vgl. Beckstette). Oberflächlich gesehen, lehnen Deutsche Rassismus demnach ab, zumindest in der Vergangenheit, haben dies aber nicht internalisiert, was nun negative Auswirkungen auf die Gegenwart hat. Deswegen folgt von den Gesprächsteilnehmer/-innen die Aufforderung, nicht nur mit Betroffenen, sondern mit allen über Rassismus zu reden und diesen nicht nur zu tabuisieren. Am Ende des Gesprächs wird noch die Verbindung zu den USA gezogen. Im Gegensatz zu Deutschland gebe es in den USA schon eine »kritische Masse«, die Bewegungen wie ›MeTwo‹ und ›BLM‹ ermöglichen (ebd.). Diese Masse konnte in Deutschland noch nicht erreicht werden. Insbesondere dieser letzte Vergleich kann den Studierenden aufzeigen, wie man aus verschiedenen historisch und gesellschaftlich bedingten Perspektiven auf Rassismus blicken kann. Er regt zu weiteren Vergleichen an und zeigt auf, was man von anderen Ländern lernen kann – oder auch nicht. Auch auf der Seite des Tagesspiegels wurde ein Artikel veröffentlicht, der sich mit den Auswirkungen des Alltagsrassismus auf Opfer und Täter beschäftigt (www.tag esspiegel.de/wissen/alltagsrassismus-in-deutschland-wenn-der-hass-krank-mac ht/25295914.html). Hier werden aktuelle politische Entwicklungen kontextualisiert, Ereignisse basierend auf rassistischen Einstellungen, aber auch Lösungsansätze vorgestellt. Neben den inhaltlichen Informationen ist dieser Artikel auch hilfreich, um einen Einblick in den medialen Umgang mit rassistisch geleiteten Ereignissen zu geben. Diese Artikel und Quellen bereitzustellen, hilft den Studierenden nicht nur dabei, sich thematisch mit dem Thema Rassismus in Deutschland auseinanderzusetzen und sich zu informieren. Weiter zeigt die Menge an Informationsquellen auch, dass Rassismus in Deutschland als relevant gesehen wird, ein aktuelles Problem darstellt und dass zu Veränderungen aufgerufen wird. Besonders wichtig ist dabei zu sehen, dass zahlreiche Quellen von einer staatlichen Behörde (BpB) veröffentlicht werden, die das Ziel verfolgt, politisches Wissen und Bildung in der Bevölkerung zu fördern. Eine damit vergleichbare zentrale Einrichtung existiert in den USA nicht. 195 196 Forum on Pedagogy/Fachdidaktik Prompt – Einstiegsarbeit Es lohnt sich, auch bereits vor dem ersten Gucken, den Studierenden einen ›Prompt‹ bereitzustellen, der den Fokus der Zuschauenden lenkt und spezifiziert. Dafür bietet es sich an, einen Vorschlag zu wählen, der die Studierenden in die Richtung lenkt, in die die anschließende Diskussion starten möchte beziehungsweise die eine Diskussion anfachen könnte. Gleichzeitig sollte der ›Prompt‹ inhaltlich nichts vorwegnehmen. Für den hier bearbeiteten Kurzfilm würde sich ein ›Prompt‹ anbieten, der den Sohn der Familie in den Fokus setzt: »Beobachten Sie beim Gucken des Kurzfilms Schweigemahl insbesondere den Sohn der Familie. Versuchen Sie sich zu überlegen, was in seinem Kopf vorgeht.« Es ist offensichtlich, dass der Kurzfilm den Fokus auf den Vater der Familie lenkt. Er steht am häufigsten im Fokus der Kamera und der Film verdeutlicht seine Erkenntnis über die diskriminierende Behandlung seiner Frau. Im Verlauf des Films wird aber auch deutlich, dass nicht nur der Mutter die Diskriminierung schon lange bewusst ist, sondern auch den Kindern. Der Sohn wird direkt Opfer einer rassistischen Handlung, indem die Frau im Supermarkt ihm unterstellt, er würde ihre Tasche stehlen wollen. Anhand seines Gesichtsausdruckes und der Szene im Bad, wo er sich im Spiegel betrachtet, wird auch den Zuschauenden deutlich, dass er diese Behandlung – zu Recht – als rassistisch oder zumindest als ungerecht bewertet. Auch in der Szene am Esstisch, in der der Vater die Kritik des Kammerspiels vorliest, ist die Kamera auf sein Gesicht gerichtet, aber auch auf das der Mutter, um die Reaktion zu zeigen, die durch die Kritik hervorgerufen wird. Die Mutter bezieht sich in ihrem Kommentar nur auf den Teil der Rezension, der sie eine »Newcomerin« (Wegener/Schramm) nennt, nicht aber auf den Teil, in dem sie beschrieben wird mit »eine Prise Exotik«, ihre Darbietung »authentisch« genannt wird und gelobt wird, dass sie »nicht nur Farbe« auf die Bühne bringe (ebd.). Dadurch, dass gezeigt wird, dass der Sohn selbst bereits Rassismus erleben musste, kann man davon ausgehen, dass nicht nur die Mutter4 , sondern auch er mehr Problematiken in der Kritik sieht, als dass sie eine »Newcomerin« genannt wird. Man kann nicht sicher sein, ob der Sohn mitbekommt, wie die Mutter im Foyer das Glas in die Hand gedrückt bekommt, in der Annahme eines Zuschauenden, dass sie die Bedienung sei, da er in dem Moment nicht mehr im Bildrahmen zu sehen ist. Allerdings kann man davon ausgehen, dass er zumindest das Glas fallen 4 Bei dem Hinweis, dass sie Farbe auf die Bühne bringt, verweilt die Kamera auf ihrem Gesicht und zeigt, wie sie in ihrem Handeln stoppt und leicht geschockt auf ihren Mann blickt. Diese Reaktion kam nicht bei der Beschreibung »Newcomerin«, weshalb man davon ausgehen kann, dass ihr die weiteren rassistischen Bemerkungen durchaus bewusst sind, sie aber sich dazu entschließt, nicht darauf einzugehen. Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) gehört hat und aufmerksam geworden ist. Mit diesen Anmerkungen im Hinterkopf bleibt nun offen, welchen Gedanken der Sohn bei dem Zuschauen der Theaterszene nachhängt. Eine Antwort des ›Prompts‹ könnte also in die Richtung gehen, dass dem Sohn die Diskriminierung seiner Mutter wahrscheinlich bewusst ist, da er selbst bereits Opfer rassistischer Behandlung wurde. Deswegen werden seine Gedanken am Essenstisch vermutlich davon handeln, dass die Kritik über seine Mutter nicht so zufriedenstellend ist, wie sein Vater es einschätzt. Außerdem könnten seine Gedanken davon handeln, dass er keinen Sinn darin sieht, seiner Familie von seinen Erlebnissen im Supermarkt zu erzählen. Er bekommt anhand der Reaktion seiner Mutter – die den Rassismus nicht direkt anspricht – vermittelt, dass der Essenstisch nicht der geeignete Ort zu sein scheint, die diskriminierenden Handlungen anzusprechen. Arbeitsblätter für den Unterricht Schweigemahl (2020) Kurzfilmanalyse Regie: Arne Schramm und Horst Wegener a) Beobachten Sie beim Gucken des Kurzfilms Schweigemahl insbesondere den Sohn der Familie. Versuchen Sie sich zu überlegen, was in seinem Kopf vorgeht. Machen Sie Ihre Beobachtungen am Film fest, indem Sie zum Beispiel eine Tabelle erstellen mit den Spalten ›Handlung im Film‹ und ›Gedanken/Emotionen des Sohnes‹. b) Gucken Sie sich erneut die Szene am Esstisch und die Szene im Badezimmer an. Beschreiben Sie die Erlebnisse der Familienmitglieder mit Alltagsrassismus und ihre Reaktion darauf. Füllen Sie die Tabelle aus. In welchen Momenten wird deutlich, dass nicht alle Familienmitglieder den Alltagsrassismus erkennen? Wie wird dies durch die Kameraarbeit und Montage unterstützt? (Z.B. Kamerafokus, Nahaufnahmen, Ton …) Vater Mutter Sohn Tochter … … … … 197 198 Forum on Pedagogy/Fachdidaktik c) Schauen Sie sich folgendes Musikvideo an: Horst Wegener – werden 12/12 ft. Roger Rekless & Ami Warning (http://www.youtube.com/watch?v=RtlM5m-4jE U). d) Das Video ist im Zusammenhang mit dem Kurzfilm Schweigemahl entstanden. Inwiefern kann man die Lyrics des Songs mit dem Kurzfilm zusammenführen? Gibt es einen Vers, der für Sie die Problematik am besten zusammenfassen kann? (in Partnerarbeit) Songtext zu werden zwölf/zwölf : https://genius.com/Horst-wegener-roger-rekless-and-ami-warning-werden-zwo lf-zwolf-lyrics 1. Hier finden Sie einen informierenden Artikel von der Bundeszentrale für politische Bildung zum Thema Alltagsrassismus in Deutschland: http://www.bpb. de/themen/rechtsextremismus/dossier-rechtsextremismus/194569/offensichtl ich-und-zugedeckt-alltagsrassismus-in-deutschland/ Dieser gibt einen guten Einblick in die aktuelle Lage in Deutschland. Diskutieren Sie, ob und inwiefern der Zusatz ›Alltags-‹ rassistische Taten und dessen Folgen verharmlost. 2. In dem Interview »Rassismus benennen« von Sven Beckstette, Veronika Fuechtner und Oliver Hardt wird unter anderem der Zusammenhang von Rassismus und der deutschen Vergangenheit beleuchtet. a) Lesen Sie den Artikel und fokussieren Sie sich auf die Zusammenhänge, die genannt werden zwischen dem aktuellen Deutschland und seiner Geschichte. b) Äußern Sie sich zu dem Gedanken, dass der Ursprung von Rassismus in Deutschland oft auf die Zeit unter dem Nationalsozialismus zurückgeführt wird. Stimmen Sie zu? Warum oder warum nicht? Link zum Artikel: http://www.textezurkunst.de/de/113/naming-racism/ Abschließende Gedanken The analysis of the short film not only asks students to reflect on the issue of daily racism – whether in the USA or Germany – but also the lecturer who presents the unit. In the context of preparing this short film, I have read many articles and watched many videos addressing racism in Germany or in general around the world. And even before that, I thought a lot about topics and issues that would not only be interesting but also relevant to an American classroom. One starts to take another perspective on one’s own country and culture. Where are intersections or Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) differences between Germany and the USA; where do the differences stem from; what impact can it have to talk about these intersections and differences? The challenge is also to find an adequate film in terms of length and language level and in ratio of language to visual elements. As I am convinced that there are many additional films that would have been suitable for this context, I am also sure that the film I picked would not be suitable for every student group. However, I would argue that it is always important to prepare the material, provide background information, listen to the students’ opinions, and discuss together the advantages and disadvantages of the film and the way it conveys its message. Every time I watch the short film I discover something new about the film as my focus is on the smallest details. That might be something the characters say or how the camera focuses on a specific object or person. The film also has encouraged me to reflect on my own behavior and look out for issues I might be oblivious to at the moment – starting with not naming a light color »skin-colored,« or even questioning whether the term »Alltagsrassismus« is suitable, or if it perhaps downplays racist actions. I think the change of perspective is one of the most useful tools when it comes to this kind of reflection. Several literary courses I took at university before highlight this thought. In my bachelor thesis for example, I focused on the use of medieval literature in German classes in order to develop a sensitive perception of others. There I argued that the finding of ›otherness‹ in one’s history fosters students’ »interculturality« insofar as the perception of themselves and their culture is positively unsettled. Literary courses in general teach about how a change of perspective can bring these insights, and one of my English pedagogy seminars highlights this as one of the aspects of global education. Global education means that nowadays students are expected to be able to act globally. Teaching English is not only about teaching the language, but concerned to foster the students’ development to become intercultural speakers and better citizens. Kroschweski (2015) for example refers to awareness of the positive value of diversity, cultural appreciation and empathy as a part of global education. One of the things I was taught and that stuck with me was the appreciation for various accents. American English is only the standardized version of one dialect and from an objective perspective there is nothing ›better‹ about it than an English spoken with a Spanish accent. These are also relevant considerations when it comes to studying German as a second or foreign language. In the courses concerning multilingual classrooms, we often discuss how we can include students’ language background and how the inclusion of the first language can for example foster motivation when the students are not yet fluent in German. Therefore, we talk about creating fair situations, how to avoid language-based discrimination or picturing students whose parents were born in other countries than Germany as ›others‹. Every one of these aspects is relevant to expose and fight (institutional) racism. 199 200 Forum on Pedagogy/Fachdidaktik When including the short film Schweigemahl in an American German Studies classroom, one could expect the students to already have some knowledge about racism in Germany. I would also argue that the scenes in which the discrimination of the children are portrayed are quite easily understood without the need of background information. Only the racism the mother has to face might be a bit troubling, at least watching it for the first time. The scene in which she is handed the glass in the foyer of the theater is also understandable without words or context. However, one might not catch the function of the glass as a leitmotif during the first time watching. The discrimination towards the mother also becomes more complex when one takes into account the review that was read aloud or the talk she has with the father/husband in the kitchen. Overall, the purpose of the film is to show daily racism people of color encounter in Germany. The producers chose to portray mostly subliminal or »subtle« discrimination. Meaning, they do not show scenes of physical violence or other open forms of racism (excluding the scene in the supermarket). Some students might response to the film saying the depiction of racism is not ›alarming‹ enough – that there are worse forms of racism and discrimination. This is definitely true; however, I would argue that the portrayed form of discrimination might be even more alarming. For example, even the father, who is married to a Black woman, appears not to be aware of the discrimination his family faces and even contributes to that discrimination. As explained above, many Germans declare themselves against racism and discrimination and praise the German commemorative culture and continue to mistreat non-white Germans. The short film is able to portray this predicament in an accountable way. Many white Germans favor being open-minded, yet are not aware of underlying issues like institutional or daily racism. As the film was produced in 2020, it is very up to date in its depiction. Another scene that could lead to good discussion is the scene in which the mother gets the glass pushed into her hands in the theater foyer at intermission. As she is wearing a maid’s costume in this scene – because of her role in the play – one could argue that the man mistook her for a waitress because of her clothing. However, the camera work – zooming in on the husband, blurry sound – and the following reactions by the protagonists show the audience that this act indicates a larger underlying issue. As the moment of the father’s awareness, it places importance on discriminatory behavior. Even if students were to see it as less egregious than more obviously harmful (physical) acts of racism, one could mention the prior part of the film in which the critics praised the play for its »authentic cast,« which of course type-cast the mother as maid. This reveals the same racist stereotype, only that it first appears in written form and then, during the intermission, is »acted out.« Such a discussion of daily racism in Germany might reveal how American students feel about the critical handling of racism in Germany and the USA in general. Leona-Maike Wenning: Handbuch zum Kurzfilm Schweigemahl (2020) The short film and its analysis will spark a conversation about this issue and will invite the students to compare a problem in two countries that differ so greatly in their history. It will be interesting to see how the students’ own experiences, perceptions and knowledge will influence the discussion. I would argue that there is no other ›goal‹ for the discussion but the development of a holistic approach to global issues within a global perspective. Literatur Auma, Maureen Maisha (2022): Rassismus. Dossier am 30.11.2017, online unter http://www.bpb.de/themen/migration-integration/dossier-migration/2237 38/rassismus. Beckstette, Sven (2022): Rassismus benennen. In: Texte zur Kunst 113 (2019), online unter http://www.textezurkunst.de/de/113/naming-racism. Der Tagesspiegel (2022): Wenn der Hass krank macht. Beitrag am 04.12.2019, online unter http://www.tagesspiegel.de/wissen/alltagsrassismus-in-deutschlan d-wenn-der-hass-krank-macht/25295914.html. Donahue, William Collins et al. (2021): Transatlantic German Studies in the American College Classroom. Student Voices on Race, Complicity, and Ideology via Short German Film. In: andererseits 9/10, S. 21–32. DW Documentary (2022): Racism in Germany. Videobeitrag auf YouTube veröffentlicht 2021, online unter http://www.youtube.com/watch?v=dMk-XpjK98M. DW (2022): Rassismus an deutschen Bühnen. Ein strukturelles Problem. Artikel am 22.04.2022, online unter www.dw.com/de/rassismus-an-deutschen-b%C3%BC hnen-ein-strukturelles-problem/a-57056069. Haruna, Hadija (2022): Wo kommst du denn her? Alltagsrassismus in Deutschland. Artikel am 17.11.2014, online unter http://www.bpb.de/themen/rechtsextremis mus/dossier-rechtsextremismus/194137/wo-kommst-du-denn-her-alltagsrassi smus-in-deutschland. Kroschewski, Annette (2015): From British Diversity to Global Diversity: Perspectives for the EFL Classroom. In: Global Education. Perspectives for English Language Teaching. Hg. von Christiane Lütge. Münster, S. 93–126. Moffitt, Ursula (2022): Germany is not the anti-racist model the US is looking for. 17.07.2020, online unter www.dw.com/en/germany-anti-racist-nazi-past/a-54 210136. Nguyen, Toan Quoc (2022): Offensichtlich und zugedeckt. Alltagsrassismus in Deutschland. 06.11.2014, online unter http://www.bpb.de/themen/rechtsextre mismus/dossier-rechtsextremismus/194569/offensichtlich-und-zugedeckt-allt agsrassismus-in-deutschland. 201 202 Forum on Pedagogy/Fachdidaktik Svrluga /Rosza: In Florida, De Santis’s plans for colleges rattle some academics. 01.06.2022. Washington Post. Wegener, Horst (2022): werden 12/12. Videobeitrag auf YouTube veröffentlicht 2020, online unter www.youtube.com/watch?v=RtlM5m-4jEU. WUPPERwerft (2022): Schweigemahl. Videobeitrag auf YouTube veröffentlicht 2020, online unter http://www.youtube.com/watch?v=3ornrMXetmQ. ZDF Heute Nachrichten (2022): Der alltägliche Rassismus in Deutschland. Videobeitrag auf YouTube, veröffentlicht 2020, online unter http://www.youtube.com/w atch?v=mP64SY9pYnE.
https://openalex.org/W2550996618	https://europepmc.org/articles/pmc5138035?pdf=render	English	null	An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation	eLife	2,016	cc-by	16,160	An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation Max E Harner1,2, Ann-Katrin Unger3, Willie JC Geerts4, Muriel Mari5, Toshiaki Izawa1, Maria Stenger3, Stefan Geimer3, Fulvio Reggiori5, Benedikt Westermann3, Walter Neupert1,6* Max E Harner1,2, Ann-Katrin Unger3, Willie JC Geerts4, Muriel Mari5, Toshiaki Izawa1, Maria Stenger3, Stefan Geimer3, Fulvio Reggiori5, Benedikt Westermann3, Walter Neupert1,6* 1Max Planck Institute of Biochemistry, Martinsried, Germany; 2Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universita¨t Mu¨ nchen, Martinsried, Germany; 3Cell Biology and Electron Microscopy, Universita¨t Bayreuth, Bayreuth, Germany; 4Biomolecular Imaging, Bijvoet Center, Universiteit Utrecht, Utrecht, Netherlands; 5Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands; 6Department of Anatomy and Cell Biology, Biomedical Center, Ludwig-Maximilians Universita¨t Mu¨ nchen, Martinsried, Germany Abstract Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution. *For correspondence: Neupert@ biochem.mpg.de Competing interests: The authors declare that no competing interests exist. DOI: 10.7554/eLife.18853.001 Introduction Mitochondria have a multitude of functions in the eukaryotic cell. They perform respiration-depen- dent energy transduction to generate proton-motive force and ATP, and house a large number of metabolic enzymes. They have their own genetic system but are closely connected with the rest of the cell by a number of pathways, such as metabolite, protein and lipid transport, protein quality control, autophagy and apoptosis. Light microscopy has revealed mitochondria as threadlike bodies that form three-dimensional networks. Mitochondria are dynamic organelles undergoing continual fission and fusion (Bereiter-Hahn and Vo¨th, 1994). The dynamics of mitochondria is essential for their function as well as maintenance, inheritance and integrity of their DNA (mtDNA). Recent research has revealed the involvement of this organelle in a multitude of human pathological condi- tions, including neurodegeneration, cardiomyopathies, metabolic diseases and cancer (Chan, 2012; Costa and Scorrano, 2012; Itoh et al., 2013; Mourier et al., 2014; Nunnari and Suomalainen, 2012; Pickrell and Youle, 2013; Youle and van der Bliek, 2012). Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany RESEARCH ARTICLE Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany Copyright Harner et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. 1 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Research article Research article Research article Biochemistry Cell Biology eLife digest Cells contain compartments called mitochondria, which are often called the powerhouses of the cell because they provide energy that drives vital cellular processes. Mitochondria have two membranes: an outer and an inner membrane. The outer membrane separates the mitochondria from the rest of the cell. The inner membrane is elaborately folded and the folds – called cristae – create a larger space to accommodate all of the protein machinery involved in producing energy. The cristae can be shaped as flat sac-like structures called lamellar cristae or as tubes known as tubular cristae. Mitochondria are dynamic and are constantly fusing with other mitochondria and splitting up. Even though the internal architecture of mitochondria was first revealed around 60 years ago, it is still not clear how the cristae form. Harner et al. now address this question in yeast cells by combining imaging, biochemistry and genetic approaches. The experiments show that lamellar cristae form when two mitochondria fuse with each other. The outer membranes merge and then the inner membranes start to fuse around their edges to generate the sac-like structure of lamellar cristae. A yeast protein called Mgm1 (known as Opa1 in mammals) drives the fusion of the inner membranes, but this process only takes place when enzymes called F1FO-ATP synthases on the inner membrane form pairs with one another. These F1FO-ATP synthase pairs stabilize the cristae membranes as they curve to form the sac-like structure. Later on, the formation of a group of proteins called the MICOS complex halts the fusion process to prevent the lamellar cristae from completely separating from the rest of the inner membrane. Harner et al. also found that tubular cristae form using a different mechanism when the inner membrane of the mitochondria grows inwards. This process also requires pairs of F1FO-ATP synthases and the MICOS complex, but does not involve Mgm1/Opa1. Together, these findings show that lamellar and tubular cristae in yeast form using two different mechanisms. Since the key components of these mechanisms are also found in virtually all other eukaryotes, the findings of Harner et al. Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany are also likely to apply to many other organisms including animals. DOI: 10.7554/eLife.18853.002 Mitochondrial ultrastructure, also referred to as the mitochondrial architecture, is intimately linked to the function and homeostasis of this organelle, and has therefore been investigated in a large number of cell types, tissues and organisms (Fawcett, 1981). There is a considerable diversity in mitochondrial architecture, but common basic structural elements can be recognized (Man- nella, 2008; Perkins et al., 2010; Sun et al., 2007). Mitochondria are delimited from the cytosol by the outer membrane (OM). The inner membrane (IM) is composed of the inner boundary membrane (IBM) and the crista membranes. The cristae are invaginations of the IM into the interior of the mito- chondria (Figure 1). In most cases they form lamellae, but tubules are also observed (Faw- cett, 1981). Crista junctions (CJs) join the cristae with the IBM. They represent small ring or slot like openings that connect the intermembrane space with the intracrista space. The IM encloses the matrix space. Cristae do not cross the matrix space completely, but they are closed by strongly bent crista rims. Septa completely cross the matrix space and separate it into distinct compartments. They are virtually absent in wild type (WT) cells, but are frequent in a variety of mutant cells. Continu- ous protein and lipid import leads to generation of curved septa which can be seen as onion-like structures in cross sections (Figure 1). Over the past 20 years a number of proteins have been identi- fied which play key roles in mitochondrial function, dynamics and homeostasis. Still, our knowledge concerning the molecular mechanisms that determine the formation and maintenance of mitochon- drial architecture is quite limited. A molecular understanding of these complex processes is possible only when biochemical and genetic analyses are combined with 3D electron microscopy (EM) analysis, whose resolution is suffi- cient to precisely evaluate the ultrastructure of mitochondrial membranes and localize proteins with nano-scale precision. With this as a guiding principle, we have addressed a central question of the molecular biology of mitochondria, the mechanisms of the biogenesis and maintenance of cristae. This question is open ever since the structural organization of mitochondria was discovered (Pal- ade, 1953; Sjostrand and Hanzon, 1954). Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 2 of 25 Research article Biochemistry Cell Biology Figure 1. Schematic representation of the ultrastructure of the mitochondria. Mgm1 plays a direct role in cristae formation Mgm1 plays a direct role in cristae formation The dynamin-related GTPase, Mgm1 (yeast)/Opa1 (higher eukaryotes), is essential for fusion of the mitochondrial IM. Its deletion in yeast leads to fragmentation of mitochondria and loss of respira- tion-dependent growth as well as of mtDNA (Cipolat et al., 2004; Jones and Fangman, 1992; Meeusen et al., 2006; Song et al., 2009; Wong et al., 2000). Previous EM analyses showed an altered IM structure in Dmgm1 cells that includes the loss of cristae (Sesaki et al., 2003). Consistent with these observations, quantitative EM of Dmgm1 cells revealed mitochondrial profiles that were mainly empty or contained one or a few septa. Vesicular and crista-like membranes were present only to a minor extent (Figure 2—figure supplement 1). Moreover, the levels of mitochondrial respiratory components were strongly reduced (Figure 2—figure supplement 2). These observations raised the possibility that Mgm1 is required for the formation of cristae. Cris- tae membranes accommodate the respiratory chain complexes which consist of both nuclear and mitochondria-encoded subunits. Thus, it is conceivable that loss of mtDNA first leads to the loss of respiratory chain complexes and then indirectly also to the loss of cristae. Alternatively, Mgm1 might be required for cristae formation, and in the absence of cristae mtDNA is not maintained. To dis- criminate between these two scenarios, we made use of the temperature sensitive mgm1-5 mutant in which a shift to non-permissive temperature leads to the inactivation of the protein and concomi- tant fragmentation and alteration of mitochondrial ultrastructure (Meeusen et al., 2006; Wong et al., 2000). We performed quantitative EM of WT and mgm1-5 cells grown at 25˚C, shifted to 37˚C for 25 min, and back again to 25˚C for 30 min. In WT cells almost only cristae were present and no significant changes were observed upon exposure to 37˚C and return to 25˚C (Figure 2A and B). In mgm1-5 cells grown at 25˚C, cristae made up about 70%; apparently the temperature sen- sitive mutant was leaky. Exposure to 37˚C and thus inactivation of Mgm1 led to a drastic loss of cris- tae (Figure 2A and B). We expected that a time period of 25 min, which is much less than one generation time of yeast, would be too short to result in loss of mtDNA. Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany Upper panel, a wild type mitochondrion containing a crista. Lower left panel, a mitochondrion containing a septum. A septum separates the matrix into two discontinuous subcompartments. Lower right panel, a mitochondrion containing an extended curved septum, appearing as an onion-like profile in EM. The drawings illustrate the reconstruction of the 3D structures from the 2D EM sections whose directions are indicated by the grey planes. Blue line, outer membrane (OM); green line, inner membrane (inner boundary membrane (IBM), crista membrane (CM) and septa membranes); small light rings, assembled MICOS complex forming a crista juncti In the lower panels, the inner boundary membranes of two matrix subcompartments are drawn in red and green to point out that the matrix is divide by a septum. DOI: 10 7554/eLife 18853 003 Figure 1. Schematic representation of the ultrastructure of the mitochondria. Upper panel, a wild type mitochondrion containing a crista. Lower left panel, a mitochondrion containing a septum. A septum separates the matrix into two discontinuous subcompartments. Lower right panel, a mitochondrion containing an extended curved septum, appearing as an onion-like profile in EM. The drawings illustrate the reconstruction of the 3D structures from the 2D EM sections whose directions are indicated by the grey planes. Blue line, outer membrane (OM); green line, inner membrane (inner boundary membrane (IBM), crista membrane (CM) and septa membranes); small light rings, assembled MICOS complex forming a crista junction. In the lower panels, the inner boundary membranes of two matrix subcompartments are drawn in red and green to point out that the matrix is divided by a septum. DOI: 10.7554/eLife.18853.003 Our approach has led us to an entirely novel hypothesis on the formation and homeostasis of cris- tae and CJs. Based on our results we present a model that postulates the existence of two different pathways of crista formation and is fully consistent with published data on mitochondrial structure, function and dynamics in yeast and mammalian cells. In the first pathway, two IBM sheets are con- verted into a single lamellar crista upon fusion of mitochondria. This process not only requires 3 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Research article Research article Biochemistry Cell Biology Mgm1, the dynamin-like fusion protein of the IM, but also the dimeric form of the F1FO-ATP syn- thase (F1FO) and the MICOS complex. Reviewing editor: Nikolaus Pfanner, University of Freiburg, Germany Mgm1 appears to mediate fusion of the IM only when dimeric F1FO is available to stabilize bending of the newly formed crista membrane and to thereby generate crista rims. Assembly of the MICOS complex is proposed to limit the fusion process by forming a CJ. In the second pathway, crista formation is independent of Mgm1, but dependent on dimeric F1FO and MICOS. It entails formation of tubular cristae and not of lamellar cristae. Our results suggest a mechanism by which growth of membranes from the IBM, driven by the influx of newly synthesized proteins and lipids, creates these tubular cristae. Importantly, all proteins found to be involved in crista biogenesis are conserved from yeast to human, emphasizing the general importance of this process. Research article Mgm1 plays a direct role in cristae formation Indeed, staining of mtDNA and test on respiratory competence revealed no loss of functional mtDNA upon exposure to 37˚C for 25 min (Figure 2C and Figure 2—figure supplement 3). However, longer exposure (72 hr) of mgm1-5 cells to non-permissive temperature led to inhibition of cell growth on respiratory medium (Figure 2—figure supplement 3). Strikingly, upon return of the mgm1-5 cells to 25˚C for 30 min cris- tae reappeared and septa were reduced, comparable to the situation before incubation at non-per- missive temperature (Figure 2A and B). Interestingly, mitochondrial respiratory complexes in both WT and mutant, as determined for Complex III and IV, remained intact during the temperature shifts (Figure 2—figure supplement 4). In parallel to the ultrastructure of the IM, the morphology of mitochondria was determined by fluorescence microscopy. While the normal mitochondrial tubular network was not altered by the temperature shift in WT, it was converted to numerous small fragments in almost all mgm1-5 cells that were exposed to 37˚C. Normal mitochondrial morphology was largely restored in mgm1-5 cells during recovery at 25˚C (Figure 2D). Although the structural analysis of the mgm1-5 mutant strongly suggested that crista formation and maintenance depends on Mgm1, reproducibly about 20% of the mitochondrial cristae remained intact after inactivation of Mgm1 in the various experiments (Figure 2B). To analyze the structure of these cristae we used electron tomography. Notably, exposure of mgm1-5 cells to 37˚C led to a Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 4 of 25 Research article Research article Biochemistry Cell Biology Figure 2. Mgm1 controls mitochondrial ultrastructure. (A) Inactivation of Mgm1 leads to rapid loss, and reactivation to the r WT cells and cells expressing the temperature sensitive mgm1-5 mutant were grown in YPD medium at 25˚C to logarithmic cultures were incubated for 25 min at either 25˚C or 37˚C; further aliquots were incubated for 25 min at 37˚C and shifted bac were analyzed by EM. Scale bars, 0.2 mm. (B) Quantitative evaluation. 150–200 mitochondrial profiles were analyzed for each Figure 2 continued on next page Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Figure 2. Mgm1 controls mitochondrial ultrastructure. (A) Inactivation of Mgm1 leads to rapid loss, and reactivation to the rapid regeneration of cristae. WT cells and cells expressing the temperature sensitive mgm1-5 mutant were grown in YPD medium at 25˚C to logarithmic phase. Figure 2 continued We conclude that the formation of lamellar and tubular cristae relies on two dif- ferent pathways. Moreover, Mgm1 plays a direct role in the formation and maintenance of lamellar but not of tubular cristae. To further study the role of Mgm1 in cristae formation we first explored where Mgm1 is exactly located in mitochondria. Quantitative immuno-EM revealed that the majority of Mgm1 was localized to the OM/IBM and only about one third to the matrix/crista space (Figure 3A). Mgm1 is present in two isoforms (Shepard and Yaffe, 1999; Wong et al., 2000). l-Mgm1 contains a transmembrane helix that anchors it to the IM; in contrast, s-Mgm1 lacks the transmembrane helix and was reported to be present at the IBM or OM (Sesaki et al., 2003; Wong et al., 2000) and a short form of Opa1 was found to be co-localized with the OM fission machinery (Anand et al., 2014). Importantly, both l-Mgm1 and s-Mgm1 forms are required for Mgm1 function in mitochondrial fusion (DeVay et al., 2009; Zick et al., 2009). To test where s-Mgm1 is located, we generated sub-mitochondrial vesicles by sonication and separated them by gradient density centrifugation. Remarkably, s-Mgm1 co-frac- tionated with the outer membrane marker Tom70, indicating that it is associated with the outer membrane (Figure 3B). These data suggest that Mgm1 could interact with the OM in the course of IM fusion. Thus, as outlined below, it is possible that after fusion of the OM a septum is formed by IMs that are not yet fused, and that Mgm1 at IM/OM contact sites initiates the conversion of these Video 1. 3D reconstruction with modelling of a mitochondrion in wild type cells. Grey, OM; green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM. DOI: 10.7554/eLife.18853.009 Video 2. 3D reconstruction with modelling of a mitochondrion in mgm1-5 cells incubated for 25 min at 37˚C. Grey, OM; green, IBM and cristae connected to the IBM. DOI: 10.7554/eLife.18853.010 Video 1. 3D reconstruction with modelling of a mitochondrion in wild type cells. Grey, OM; green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM. DOI: 10.7554/eLife.18853.009 Video 2. 3D reconstruction with modelling of a mitochondrion in mgm1-5 cells incubated for 25 min at 37˚C. Grey, OM; green, IBM and cristae connected to the IBM. DOI: 10.7554/eLife.18853.010 Video 1. Figure 2 continued Figure 2 continued mtDNA in the mgm1-5 mutant upon exposure to 37˚C. WT and mgm1-5 cells were grown in YPD medium at 25˚C and incubated at 37˚C for 25 min. The percentage of cells containing mtDNA was determined by DAPI staining. (D), Mitochondrial morphology in WT and in the mgm1-5 cells expressing mitochondrially targeted GFP. Cells were treated as described in (A). The morphology of the mitochondrial network in 100 cells per sample was analyzed by fluorescence microscopy. (E) EM tomographic reconstruction of a mitochondrion of a WT yeast cell. Green, IBM and lamellar cristae connected to the IBM; blue, tubular crista. (F) Tomographic reconstruction of a mitochondrion of a mgm1-5 cell grown at 25˚C and shifted to 37˚C for 25 min. Green, IBM and cristae connected to the IBM. DOI: 10.7554/eLife.18853.004 The following figure supplements are available for figure 2: Figure supplement 1. Mgm1 is required for wild type inner membrane structure. DOI: 10.7554/eLife.18853.005 Figure supplement 2. Mgm1 is required for wild type protein composition. DOI: 10.7554/eLife.18853.006 DOI: 10.7554/eLife.18853.006 Figure supplement 3. Incubation of mgm1-5 cells for 25 min at non-permissive temperature does not lead to loss of functional mtDNA. DOI: 10.7554/eLife.18853.007 Figure supplement 3. Incubation of mgm1-5 cells for 25 min at non-permissive temperature does not lead to loss of functional mtDNA. DOI: 10.7554/eLife.18853.007 ges of mitochondrial ultrastructure upon inactivation of Mgm1 do not affect the assembly state of respiratory cha Figure supplement 4. Changes of mitochondrial ultrastructure upon inactivation of Mgm1 do not affect the assembly state of respiratory chain supercomplexes. O / f Figure supplement 4. Changes of mitochondrial ultrastructure upon inactivation of Mgm1 do not affect the assembly state of respiratory chain supercomplexes. decrease of lamellar and an increase of tubular cristae as compared to WT (Figure 2E and F; Videos 1 and 2). These results show that the disappearance and reappearance of cristae are rapid and depend on functional Mgm1. We conclude that the formation of lamellar and tubular cristae relies on two dif- ferent pathways. Moreover, Mgm1 plays a direct role in the formation and maintenance of lamellar but not of tubular cristae. decrease of lamellar and an increase of tubular cristae as compared to WT (Figure 2E and F; Videos 1 and 2). These results show that the disappearance and reappearance of cristae are rapid and depend on functional Mgm1. Research article Biochemistry Cell Biology Research article Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Mgm1 plays a direct role in cristae formation Aliquots of the cultures were incubated for 25 min at either 25˚C or 37˚C; further aliquots were incubated for 25 min at 37˚C and shifted back to 25˚C for 30 min. Cells were analyzed by EM. Scale bars, 0.2 mm. (B) Quantitative evaluation. 150–200 mitochondrial profiles were analyzed for each sample. (C) Maintenance of Figure 2 continued on next page Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 5 of 25 Research article R Figure 2 continued 3D reconstruction with modelling of a mitochondrion in wild type cells. Grey, OM; green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM. DOI: 10.7554/eLife.18853.009 Video 1. 3D reconstruction with modelling of a mitochondrion in wild type cells. Grey, OM; green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM. DOI: 10.7554/eLife.18853.009 Video 2. 3D reconstruction with modelling of a mitochondrion in mgm1-5 cells incubated for 25 min at 37˚C. Grey, OM; green, IBM and cristae connected to the IBM. 6 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Research article Research article Biochemistry Cell Biology Figure 3. Mgm1 is present at the mitochondrial inner and outer membranes. (A) Distribution of Mgm1 between OM/IBM and cristae. Cells expressing 3xHA tagged Mgm1 were analyzed by immuno-EM. Left panel, four mitochondrial profiles (M) showing gold particles at the IBM. Scale bar, 0.2 mm. Right panel, quantification of gold particles. (B) Distribution of Mgm1 between OM and IM. Mitochondrial vesicles generated by sonication were subjected to sucrose density gradient centrifugation followed by fractionation, SDS-PAGE and immunoblotting. Arrow, s-Mgm1. The membrane anchor of l-Mgm1 is notorious for being clipped off during isolation of mitochondria (c.f. Figures 5C and 7C). Therefore, l-Mgm1 was not detected after mitochondrial subfractionation. Cor2, subunit two of respiratory chain complex III. DOI: 10.7554/eLife.18853.011 Figure 3. Mgm1 is present at the mitochondrial inner and outer membranes. (A) Distribution of Mgm1 between OM/IBM and cristae. Cells expressing 3xHA tagged Mgm1 were analyzed by immuno-EM. Left panel, four mitochondrial profiles (M) showing gold particles at the IBM. Scale bar, 0.2 mm. Right panel, quantification of gold particles. (B) Distribution of Mgm1 between OM and IM. Mitochondrial vesicles generated by sonication were subjected to sucrose density gradient centrifugation followed by fractionation, SDS-PAGE and immunoblotting. Arrow, s-Mgm1. The membrane anchor of l-Mgm1 is notorious for being clipped off during isolation of mitochondria (c.f. Figures 5C and 7C). Therefore, l-Mgm1 was not detected after mitochondrial subfractionation. Cor2, subunit two of respiratory chain complex III. DOI: 10.7554/eLife.18853.011 Dimeric F1FO-ATP synthase is preferentially located at crista membranes, but the monomeric form is present at septa and inner boundary membrane Dimeric F1FO provides positive curvature to crista membranes (Dudkina et al., 2005; Strauss et al., 2008; Thomas et al., 2008). The subunits of the FO sector, Su e/Atp21 and Su g/Atp20, mediate the formation of F1FO dimers (Arnold et al., 1998; Habersetzer et al., 2013). Deletion of Su e or Su g leads to the formation of membrane sheets that are crossing the matrix space completely and to 7 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Research article Research article Biochemistry Cell Biology Figure 4. Localization and dimerization of F1FO are relevant to mitochondrial architecture. (A) Distribution of F1FO b WT cells expressing 3xHA tagged F1b were subjected to immuno-EM and quantified. (B) Distribution of F1FO betwe cells. Dsu e cells expressing 3xHA tagged F1b were analyzed as in (A). (C) Distribution of Su e between IBM and crist 3xHA tagged Su e were analyzed as in (A). Scale bars, 0.2 mm. f Figure 4. Localization and dimerization of F1FO are relevant to mitochondrial architecture. (A) Distribution of F1FO between IBM and cristae WT cells expressing 3xHA tagged F1b were subjected to immuno-EM and quantified. (B) Distribution of F1FO between IBM and cristae/sept cells. Dsu e cells expressing 3xHA tagged F1b were analyzed as in (A). (C) Distribution of Su e between IBM and cristae in WT. WT cells expr 3xHA tagged Su e were analyzed as in (A). Scale bars, 0.2 mm. DOI: 10.7554/eLife.18853.012 Figure 4. Localization and dimerization of F1FO are relevant to mitochondrial architecture. (A) Distribution of F1FO between IBM and cristae in WT cells. WT cells expressing 3xHA tagged F1b were subjected to immuno-EM and quantified. (B) Distribution of F1FO between IBM and cristae/septa in Dsu e cells. Dsu e cells expressing 3xHA tagged F1b were analyzed as in (A). (C) Distribution of Su e between IBM and cristae in WT. WT cells expressing 3xHA tagged Su e were analyzed as in (A). Scale bars, 0.2 mm. DOI: 10.7554/eLife.18853.012 onion-like structures. Examination of a large number of EM sections showed that these membrane sheets are not cristae as previously assumed, but septa. Onions represent cross sections of curved septa (Figure 1). This is also apparent upon tomographic reconstruction of isolated mitochondria from cells lacking dimeric F1FO (Davies et al., 2012). Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 The internal membranes in MICOS deficient mitochondria are structurally and functionally similar to crista membranes The MICOS complex is located in the mitochondrial IM of yeast and higher eukaryotes, where it is required for the formation of CJs as well as the formation of contact sites with the OM. Mitochondria lacking Mic60 contain characteristic stacked membranes in the matrix that have no connections to the IBM (Alkhaja et al., 2012; Darshi et al., 2011; Harner et al., 2011; Hoppins et al., 2011; Jans et al., 2013; John et al., 2005; Rabl et al., 2009; von der Malsburg et al., 2011). Since CJs are essential elements of cristae, we asked whether MICOS has a function in the formation of cristae. A so far unanswered question is whether the cristae in WT and the stacked membranes in the Dmic60 mutant differ in their functions. The protein composition of these mitochondria is very similar to that of WT cells (Harner et al., 2014). Importantly F1FO is present in the dimeric form (Rabl et al., 2009). To analyze whether there is a difference in the distribution of IM proteins in the stacked membranes in the Dmic60 mutant as compared to WT cristae, we examined the distribution of F1b and the cytochrome c oxidase subunit Cox5a by immuno-EM. F1b was found almost exclusively in the IM stacks of Dmic60 mitochondria as in WT (Figure 5A). Likewise, the distribution of Cox5a between the IBM and the cristae in WT cells and between IBM and the internal membranes in the Dmic60 mutant was almost identical (Figure 5B). The fact that the distribution of these two proteins is not changed in mitochondria of Dmic60 cells suggests that CJs are not required for assembly of protein complexes in the mitochondria and integration into crista membranes. Consistently, mito- chondria of the Dmic60 mutant are able to perform oxidative phosphorylation (OXPHOS) and thus maintain mtDNA. Deletion of Su e did not lead to a reduction of MICOS subunits both during respiratory growth and fermentative growth (Figure 5C). The level of the assembled MICOS complex, however, was strongly diminished (Figure 5D). This suggests that MICOS is partly dissociated when cristae are absent. Interestingly, the MICOS subunit Mic10 was found to be strongly enriched at the sites where septa membranes meet the IBM and the bending of septa is strongest (Figure 5E). Dimeric F1FO-ATP synthase is preferentially located at crista membranes, but the monomeric form is present at septa and inner boundary membrane A fundamental difference between cristae and septa is that crista membranes are strongly bent to generate crista rims and thus the sac-like crista structure, whereas septa are not closed but divide the matrix into membrane-limited subcompart- ments. In spite of this drastic alteration of mitochondrial architecture, growth is only moderately retarded under respiratory conditions (Paumard et al., 2002; Rabl et al., 2009). It is presently unknown how and when during crista formation this bending is taking place. We localized fully assembled F1FO by detection of its subunit F1b and found it almost exclusively in the crista mem- branes of WT mitochondria (Figure 4A). In the Su e deletion mutant, however, the fraction of mono- meric F1FO detected in the IBM was increased by a factor of three in comparison to WT (Figure 4B). Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 8 of 25 Research article Research article Research article Biochemistry Cell Biology Figure 5. The stacked membrane sheets in mitochondria lacking Mic60 are functionally similar to crista membranes. (A) Distributi cells. Dmic60 cells expressing 3xHA tagged F1b were analyzed by immuno-EM and quantified. (B) Distribution of Cox5A in Dmic60 expressing 3xHA tagged Cox5A were analyzed as in (A). (C) Levels of MICOS subunits in Dsu e cells. WT cells and cells lacking Su (left) or YPD (right). Aliquots of mitochondrial protein were analyzed by SDS-PAGE and immunoblotting. Arrow head, l-Mgm1; arr Figure 5 continued on next page Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Figure 5. The stacked membrane sheets in mitochondria lacking Mic60 are functionally similar to crista membranes. (A) Distribution of F1b in Dmic60 cells. Dmic60 cells expressing 3xHA tagged F1b were analyzed by immuno-EM and quantified. (B) Distribution of Cox5A in Dmic60 cells. Dmic60 cells expressing 3xHA tagged Cox5A were analyzed as in (A). (C) Levels of MICOS subunits in Dsu e cells. WT cells and cells lacking Su e were grown on YPG (left) or YPD (right). Aliquots of mitochondrial protein were analyzed by SDS-PAGE and immunoblotting. Arrow head, l-Mgm1; arrow, s-Mgm1; asterisk, Figure 5 continued on next page Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 9 of 25 Research article R Biochemistry Cell Biology Research article Figure 5 continued Figure 5 continued cross-reaction of the Mic60 antibody. (D) Assembly state of MICOS subunits in Dsu e cells. Mitochondria were isolated and lysed using digitonin. MICOS complex was analyzed by BN-PAGE and immunoblotting. Dimeric F1FO-ATP synthase is preferentially located at crista membranes, but the monomeric form is present at septa and inner boundary membrane Arrow, assembled MICOS complex. The result of one of four independently performed experiments is shown. (E) Distribution of Mic10 in Dsu e cells. Dsu e cells expressing 3xHA tagged Mic10 were analyzed as in (A). SM, septum membrane; SJ, septum junction. Scale bars, 0.2 mm. DOI: 10.7554/eLife.18853.013 This observation is compatible with the assumption that the distribution of F1FO in cristae and IBM correlates with its oligomeric state. Similar to F1b, Su e was present in crista membranes of WT mito- chondria (Figure 4C). Very low levels of F1b, monomeric F1FO and unassembled Su e may exist in the IBM into which new components are continually being inserted, but dimeric F1FO is present almost exclusively in cristae and does not assemble in the planar IBM. Thus, the assembly of dimeric F1FO is likely to take place during the formation of cristae when the bending of membranes has to occur. The internal membranes in MICOS deficient mitochondria are structurally and functionally similar to crista membranes This observation is in line with the finding that this MICOS subunit has an important role in the formation of the nar- row ring or slot like structure of the crista junctions (Barbot et al., 2015; Bohnert et al., 2015; Milenkovic and Larsson, 2015). Taken together, this suggests that cristae and stacks are functionally similar if not identical. Fur- thermore, in the absence of the dimeric ATP synthase assembled MICOS is not sufficient to allow the generation of cristae. These findings raise the questions of how the internal membrane stacks originate and how proteins reach them in view of the virtually complete absence of connections with the IBM. Figure 6. Structure and protein composition of cristae in cells deficient in Dnm1. (A) Ultrastructure and quantitative evaluation of mitochondria in Ddnm1 cells grown on YPG (scale bars, 0.2 mm). (B) Tomographic reconstruction of a mitochondrion of a Ddnm1 cell. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM. (C) Steady state levels of mitochondrial proteins of Ddnm1 cells. WT and Ddnm1 Figure 6 continued on next page Mitochondria lacking Dnm1 show altered crista structure 3D reconstruction revealed that the vesicular profiles mostly represent inter- connected highly convoluted and branched tubular structures (Figure 6B; Video 3). These tubules seem to have the ability to fuse and to branch. Interestingly, we identified tubular cristae also in WT cells by tomography, albeit much less frequently (Figure 2E; Video 1). Normal levels of components of OXPHOS as well as assembled MICOS and dimeric F1FO were present in these mitochondria (Figure 6C and D). Notably, the level of the assembled MICOS complex in the Ddnm1 mutant was significantly increased. This is consistent with the higher number of cristae and CJs in this mutant. The necessity for the accommodation of OXPHOS and other membrane proteins likely requires an increase of the number of cristae since the surface area of tubular cristae compared to lamellar cris- tae is much lower. In summary, we reasoned that the mitochondrial network is in an almost completely fused state in the absence of Dnm1, and thus the rate of fusion is strongly decreased. The diminished Mgm1 activity might then result in a drastically reduced formation of lamellar cristae and massive production of tubular cristae. Mitochondria lacking Dnm1 show altered crista structure Analysis as in Figure 4D. Arrow head, assembled MICOS complex. DOI: 10.7554/eLife.18853.014 observation appeared to contradict our suggestion that Mgm1 is directly required for crista forma- tion and maintenance. To resolve this apparent discrepancy, we first analyzed the ultrastructure of mitochondria in the Ddnm1 mutant. We observed that mitochondrial architecture differed signifi- cantly from that in WT. These mitochondria are considerably enlarged, densely filled with vesicular profiles and short tubular cristae, while CJs are abundant along the IBM (Figure 6A). In comparison to WT, the number of cristae with lamellar structure was found to be much lower as highlighted by electron tomography. 3D reconstruction revealed that the vesicular profiles mostly represent inter- connected highly convoluted and branched tubular structures (Figure 6B; Video 3). These tubules seem to have the ability to fuse and to branch. Interestingly, we identified tubular cristae also in WT cells by tomography, albeit much less frequently (Figure 2E; Video 1). Normal levels of components of OXPHOS as well as assembled MICOS and dimeric F1FO were present in these mitochondria (Figure 6C and D). Notably, the level of the assembled MICOS complex in the Ddnm1 mutant was significantly increased. This is consistent with the higher number of cristae and CJs in this mutant. The necessity for the accommodation of OXPHOS and other membrane proteins likely requires an increase of the number of cristae since the surface area of tubular cristae compared to lamellar cris- tae is much lower. In summary, we reasoned that the mitochondrial network is in an almost completely fused state in the absence of Dnm1, and thus the rate of fusion is strongly decreased. The diminished Mgm1 activity might then result in a drastically reduced formation of lamellar cristae and massive production of tubular cristae. observation appeared to contradict our suggestion that Mgm1 is directly required for crista forma- tion and maintenance. To resolve this apparent discrepancy, we first analyzed the ultrastructure of mitochondria in the Ddnm1 mutant. We observed that mitochondrial architecture differed signifi- cantly from that in WT. These mitochondria are considerably enlarged, densely filled with vesicular profiles and short tubular cristae, while CJs are abundant along the IBM (Figure 6A). In comparison to WT, the number of cristae with lamellar structure was found to be much lower as highlighted by electron tomography. Mitochondria lacking Dnm1 show altered crista structure Dnm1 (in yeast)/Drp1 (in higher eukaryotes), a dynamin-related GTPase, is an essential component of the fission machinery. Its deletion leads to the formation of a highly branched and interconnected mitochondrial network (Bleazard et al., 1999; Otsuga et al., 1998; Smirnova et al., 1998). Loss of Mgm1 in cells depleted of Dnm1 does not lead to loss of cristae (Sesaki et al., 2003). This surprising Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 10 of 25 10 of 25 Research article Research article Biochemistry Cell Biology Figure 6. Structure and protein composition of cristae in cells deficient in Dnm1. (A) Ultrastructure and quantitative evaluation of mitochondria in Ddnm1 cells grown on YPG (scale bars, 0.2 mm). (B) Tomographic reconstruction of a mitochondrion of a Ddnm1 cell. Green, IBM and cristae con to the IBM; blue, tubular cristae without visible connection to the IBM. (C) Steady state levels of mitochondrial proteins of Ddnm1 cells. WT and D Figure 6 continued on next page Figure 6. Structure and protein composition of cristae in cells deficient in Dnm1. (A) Ultrastructure and quantitative evaluation of mitochondria in Ddnm1 cells grown on YPG (scale bars, 0.2 mm). (B) Tomographic reconstruction of a mitochondrion of a Ddnm1 cell. Green, IBM and cristae connecte o the IBM; blue, tubular cristae without visible connection to the IBM. (C) Steady state levels of mitochondrial proteins of Ddnm1 cells. WT and Ddnm Figure 6 continued on next page Figure 6. Structure and protein composition of cristae in cells deficient in Dnm1. (A) Ultrastructure and quantitative evaluation of mitochondria in Ddnm1 cells grown on YPG (scale bars, 0.2 mm). (B) Tomographic reconstruction of a mitochondrion of a Ddnm1 cell. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM. (C) Steady state levels of mitochondrial proteins of Ddnm1 cells. WT and Ddnm1 Figure 6 continued on next page 11 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Research article R Biochemistry Cell Biology Research article Biochemistry Cell Biology Figure 6 continued cells were grown on YPG, aliquots of mitochondrial protein were analyzed by SDS-PAGE and immunoblotting. (D) Assembly state of MICOS and of F1FO in Ddnm1 cells. Analysis as in Figure 4D. Arrow head, assembled MICOS complex. DOI: 10.7554/eLife.18853.014 cells were grown on YPG, aliquots of mitochondrial protein were analyzed by SDS-PAGE and immunoblotting. (D) Assembly state of MICOS and of F1FO in Ddnm1 cells. Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Mitochondrial fusion/fission dynamics contributes to the formation of lamellar cristae In order to obtain more insight into the role of Mgm1 in the formation of cristae, we analyzed the biochemical composition and ultrastructure of mitochondria in the Ddnm1Dmgm1 double deletion mutant. Growth of the Ddnm1 and Ddnm1Dmgm1 strains on non-fermentable carbon sources was similar (Figure 7A). On fermentable carbon source, however, the double deletion mutant displayed a high rate of loss of mtDNA, pointing to a residual function of Mgm1 in mitochondria of the Ddnm1 mutant (Figure 7B). The steady state levels of a number of mitochondrial proteins were similar in WT, Ddnm1, and Ddnm1Dmgm1 strains (Figure 7C). The interior of Ddnm1Dmgm1 mitochondria, like that of the Ddnm1 mutant, was full of tubular cristae. Strikingly, lamellar cristae were not observed in mitochondria of the Ddnm1Dmgm1 mutant (Figure 7D and E; Video 4). Thus, Mgm1 appears to be essential for the formation of Video 4. 3D reconstruction with modelling of a mitochondrion in the Ddnm1Dmgm1 double deletion mutant. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM; yellow, tubular cristae without connection to the IBM. DOI: 10 7554/eLife 18853 018 Video 3. 3D reconstruction with modelling of a mitochondrion in the Ddnm1 mutant. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM; yellow, tubular cristae without connection to the IBM. DOI: 10.7554/eLife.18853.015 Video 4. 3D reconstruction with modelling of a mitochondrion in the Ddnm1Dmgm1 double deletion mutant. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM; yellow, tubular cristae without connection to the IBM. Video 3. 3D reconstruction with modelling of a mitochondrion in the Ddnm1 mutant. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM; yellow, tubular cristae without connection to the IBM. DOI: 10.7554/eLife.18853.015 DOI: 10.7554/eLife.18853.018 DOI: 10.7554/eLife.18853.018 DOI: 10.7554/eLife.18853.018 12 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Research article Research article Figure 7. Growth, structure and protein composition of cristae in mutants lacking either Mgm1, Mic10, Mic60 or Su e in the background. (A) Growth of double mutant strains deficient in Dnm1 and Mgm1, Mic10, Mic60 or Su e. The strains were cult logarithmic phase followed by growth analysis on the indicated media by drop dilution assay. (B) mtDNA maintenance in ce dimeric F1FO or MICOS in the Ddnm1 background. lamellar cristae, but is not required for the formation of tubular cristae and CJs. To further study the role of mitochondrial fusion in the formation of lamellar cristae, we analyzed mitochondrial architecture in 4dnm14fzo1 cells, which have a block of outer membrane fusion and fission. Indeed, we observed mitochondria with numerous tubular cristae (Figure 7—figure supple- ment 1), supporting the idea that tubular cristae are the predominant type in the absence of fusion activity. However, this phenotype was observed to a much lesser degree than in the 4dnm14mgm1 mutant. Strikingly, 4dnm14fzo1 cells quite frequently contained constricted mitochondria and mito- chondria with septa (Figure 7—figure supplement 1). Since it has been repeatedly suggested that a separate Dnm1-independent fission mechanism exists for the IM (Gorsich and Shaw, 2004; Ishihara et al., 2013; Westermann, 2010), we consider it possible that these structures represent division events of the IM in the absence of OM fusion. This could then provide Mgm1 with suitable inner membrane structures for the formation of lamellar cristae and explain why the cristae pheno- type of 4dnm14fzo1 is less severe than that of 4dnm14mgm1. Together, these observations sug- gest that mitochondrial fusion is important for generating crista structure. We further investigated the mitochondria in the Ddnm1Dmic60 mutant; they displayed a virtually complete absence of cristae and CJs. Vesicular-tubular profiles, often located close to the IBM and associated with sheet-like membrane structures, were present in the interior of the mitochondria. Tomographic reconstructions revealed a rather corrugated and perforated appearance, in contrast to the planar structure of WT lamellar cristae. Therefore, these membranes are most likely formed by association of extended tubules, which partly fuse with each other (Figure 7F; Video 5). Interest- ingly, in contrast to the Ddnm1Dmgm1 mutant, growth of Ddnm1Dmic60 cells was very slow on respi- ratory medium (Figure 7A) and the rate of mtDNA loss was higher in this mutant, indicating that in the absence of fully functional Mgm1, MICOS and thereby CJs and tubular cristae contribute to maintenance of mtDNA (Figure 7B). The steady state levels of the mitochondrial proteins analyzed were similar to those in Ddnm1 cells. MICOS components were an exception, as they were reduced to different degrees. This was, however, also observed with the 4mic10 or 4mic60 single deletion mutants. The level of Cox2, a mitochondrial gene product, was also reduced (Figure 7C). Research article Biochemistry Cell Biology Figure 7 continued percentage of cells containing mtDNA was determined by DAPI staining. Mean values of three independent experiments. Error bars, standard deviation. (C), Steady state levels of mitochondrial proteins in the Ddnm1 double mutant cells. Cells were grown on YPG medium, proteins were extracted and their levels were analyzed by SDS-PAGE and immunoblotting. Arrow head, l-Mgm1; arrow, s-Mgm1. (D) Mitochondrial ultrastructure of Ddnm1Dmgm1 cells analyzed by EM. (E) Tomographic reconstruction of a mitochondrion of a Ddnm1Dmgm1 cell. Green, IBM and cristae connected to the IBM; blue, tubular cristae without visible connection to the IBM; yellow, tubular cristae without connection to the IBM. (F) Tomographic reconstruction of a mitochondrion of a Ddnm1Dmic60 cell. Green, IBM; other colors, perforated tubular-sheet like membrane structures in the matrix without connections to the IBM. To be able to discriminate between the different tubular elements they are shown in different colors. (G) Ultrastructure of mitochondria in Ddnm1Dsu e cells. (H) Quantitative evaluation of the EM analysis of the indicated mutant strains. Scale bars, 0.2 mm. DOI: 10.7554/eLife.18853.016 The following figure supplement is available for figure 7: Figure supplement 1. Mitochondrial outer membrane fusion is important but not essential for the formation of lamellar cristae. DOI: 10.7554/eLife.18853.017 nt 1. Mitochondrial outer membrane fusion is important but not essential for the formation of lamellar cristae. 18853 017 mellar cristae, but is not required for the formation of tubular cristae and CJs. Mitochondrial fusion/fission dynamics contributes to the formation of lamellar cristae Strains were grown on YPG and transferred to YPD medium. At time 0 h Figure 7 continued on next page Research article Biochemistry Cell Biology Figure 7. Growth, structure and protein composition of cristae in mutants lacking either Mgm1, Mic10, Mic60 or Su e in the Dnm1 deletion background. (A) Growth of double mutant strains deficient in Dnm1 and Mgm1, Mic10, Mic60 or Su e. The strains were cultured on YPG medium to logarithmic phase followed by growth analysis on the indicated media by drop dilution assay. (B) mtDNA maintenance in cells lacking either Mgm1, dimeric F1FO or MICOS in the Ddnm1 background. Strains were grown on YPG and transferred to YPD medium. At time 0 hr and after 48 hr the Figure 7 continued on next page Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 13 of 25 Research article Research article R Discussion Major advances have been made in our understanding of the biogenesis of mitochondrial protein complexes and supercomplexes, but the biogenesis of mitochondrial architecture, the next higher level of organization, has been investigated to a much lesser degree. In particular, organization and formation of the cristae are poorly understood. We report here on novel and basic insights into the molecular mechanisms underlying the formation of lamellar and of tubular cristae, and the key fac- tors involved in these pathways. Three factors have decisive functions in the biogenesis of cristae: Mgm1, the dimeric F1FO-ATP synthase, and the MICOS complex. Based on our findings and the literature about these three fac- tors and their interactors, we developed a hypothesis that postulates the existence of two distinct mechanisms for the generation of lamellar and tubular cristae. Regarding the formation of lamellar cristae, a first important issue is how Mgm1 performs fusion of the mitochondrial IM (Figure 8A). It is known that dysfunction of the components that mediate mitochondrial membrane fusion, Fzo1, Ugo1, and Mgm1, leads to loss of mtDNA and loss of cristae, concomitant with septa formation and inhibition of IM fusion (Hermann et al., 1998; Hoppins et al., 2009; Meeusen et al., 2006; Sesaki et al., 2003). It was shown that Fzo1 in the OM interacts with the IM, and Mgm1 in the inner membrane interacts physically with Fzo1 and Ugo1 in the OM (Fritz et al., 2001; Sesaki et al., 2003; Wong et al., 2003). Both l-Mgm1 anchored in the IM and s-Mgm1 bound to the OM are required for fusion (DeVay et al., 2009; Zick et al., 2009). These observations suggest a mechanism by which progression of IM fusion takes place at sites where IM and OM are in close contact (Figure 8A). Fusion of the OM by Fzo1 would first generate a mitochondrion with a planar septum consisting of non-fused IM. Tethering of these two IMs by Mgm1 could take place at the IM surface in contact with the OM. According to our hypothesis, the fusion of the IM is then initiated by Mgm1 and proceeds along the IM-OM contacts, thereby generating a membrane sac protruding into the matrix. Shifting the temperature sensitive mgm1-5 mutant to non-permissive temperature leads to rapid fragmentation of mitochondria (Meeusen et al., 2006; Wong et al., 2000). lamellar cristae, but is not required for the formation of tubular cristae and CJs. We sug- gest that in Ddnm1Dmic60 mutant, in contrast to the Dmic60 single mutant in which lamellar cristae can be made, the proteins and lipids imported into the mitochondria invaginate the IBM. This results in irregular networks which, as indicated by the biochemical and the growth phenotype, lack the characteristic properties of cristae. Deletion of both Dnm1 and Su e resulted in cells that grew extremely slowly on non-fermentable carbon source (Figure 7A). Mitochondria contained many highly branched septa and multi-layered onion-like profiles (Figure 7G). In addition, mtDNA was lost at a high rate, indicating that dimeric F1FO is important for maintenance of mtDNA when Mgm1 has no obvious role (Figure 7B). Strik- ingly, mitochondria in the Ddnm1Dmgm1Dmic60 and the Ddnm1Dsu eDmic60 triple mutants showed even more severe ultrastructural alterations than the Ddnm1Dmgm1, Ddnm1Dmic60, or Ddnm1Dsu e double mutants. Fewer internal sheet-like membranes were present and approximately 50% of mito- chondrial profiles were completely empty (Figure 7H). Loss of mtDNA in the Ddnm1Dmgm1Dmic60 triple mutant was particularly high (Figure 7B). Attempts to generate a respiratory competent Ddnm1Dmgm1Dsu e triple or Ddnm1Dmgm1Dmic60Dsu e quadruple mutant by either strain crossing Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 14 of 25 Research article Research article Biochemistry Cell Biology or direct deletion were not successful, suggesting that the simultaneous loss of Mgm1, F1FO dimers and MICOS is deleterious for crista formation and/or maintenance of mtDNA. We conclude that the pathway of tubular crista formation does not need the fusion activity of Mgm1, but does require both MICOS and dimeric F1FO. In contrast, the pathway leading to the for- mation of lamellar cristae, in addition to MICOS and dimeric F1FO, appears to depend on Mgm1. Furthermore, the drastically altered membrane structures present in cells lacking Dnm1 and at the same time MICOS core components or Su e cannot substitute for lamellar and tubular cristae to maintain mtDNA and respiratory growth. Mgm1 mediates Video 5. 3D reconstruction with modelling of a mitochondrion in the Ddnm1Dmic60 double deletion mutant. Green, IBM and cristae connected to the IBM; blue and red, fused tubular structures and perforated tubular-sheet like membrane structures in the matrix without connections to the IBM; yellow, ER-type tubules. DOI: 10.7554/eLife.18853.019 Our hypothesis proposes that fusion of IM is halted by the assembly of MICOS complexes, leading to the generation of a CJ and thereby represents the final step in the formation of lamellar cristae (Figure 8A). In agreement with this speculation, we found that the MICOS component Mic10 is preferentially located at sites where septa merge with the IMS, the septum junctions. Our hypoth- esis does not exclude a stabilizing function of MICOS for cristae. Since membrane proteins and pro- tein complexes can shuttle between cristae and the IBM (Vogel et al., 2006), it seems likely that a major reason for crista stability is the continued presence and activity of Mgm1. In addition, our hypothesis provides a rational explanation for the generation of the stacked closed membrane sheets lacking connection with the IBM that are formed in the absence of functional MICOS com- plex. In the absence of the MICOS core components, Mic60 and Mic10, fusion proceeds as in WT cells, but cannot be halted (Figure 8A). Interestingly, a very similar mechanism was suggested for the fusion of vacuoles, the yeast homolog of lysosomes (Wickner, 2010). Upon fusion of vacuoles an internal vesicle is generated (Wang et al., 2002). It might well be that these vesicles are generated like the internal stacks in Mic10 or Mic60 deletion mutants. According to our hypothesis, during fusion septa membranes and the nascent crista membrane form a continuum. This allows free distri- bution of proteins between the IBM and the nascent crista membrane. This would also explain the similarity of the composition of intramitochondrial membrane sheets, made in the absence of MICOS, to crista membranes as well as their full functionality in mediating OXPHOS. After completion of fusion and formation of CJs, cristae may grow by uptake of newly synthesized proteins, along with assembly of subunits of the F1FO-ATP synthase, which extend the crista rims. Thus, our hypothesis also offers an explanation of how the characteristic shape of lamellar cristae is generated and how cristae can expand. Our results also suggest the existence of a second pathway of crista biogenesis (Figure 8B). Discussion Quantitative EM analysis of this process reveals that, in addition to fragmentation, a rapid and extensive loss of cris- tae takes place, while septa are formed. These findings strongly suggest that the activity of Mgm1 not only is required for fusion but also for maintenance of cristae. Importantly, cristae are rapidly regenerated upon reactivation of Mgm1. Equally important, mtDNA is retained during inactivation of Mgm1 and remains functional. Thus, it is not the loss of mtDNA that causes loss of cristae and for- mation of septa, emphasizing a role of Mgm1 in crista maintenance. At the same time these findings raise the question how this rapid reformation of cristae can be explained. The fusion rate of mitochondria in wild type cells has been measured by live cell micros- copy (Jakobs et al., 2003). Under steady-state conditions at least one fusion and one fission event was observed per minute and cell. This rate is apparently determined by regulatory processes that ensure a balance of fusion and fission. In contrast, fusion of the IM in mgm1-5 mutant cells by reacti- vation of Mgm1 is likely to reflect an entirely different situation. Here, the conversion of a septum to a crista requires only the time of IM fusion by Mgm1 which upon its reactivation is immediately ready to function. The overall process of mitochondrial fusion requires a multitude of steps, including fusion of the OM. Fusion of the IM likely represents one of the fastest steps. Notably in this context, fusion intermediates have not been identified convincingly in intact cells. These conclusions are in good agreement with observations that acute ablation of Opa1 in mouse embryonic fibroblasts leads to disorganized cristae and reduced respiratory function, but maintenance of mtDNA (Cogliati et al., 2013). The conversion of two membranes into a single one by Mgm1 entails the necessity to bend t membrane. Very interestingly, recent experiments with reconstituted vesicles have revealed th Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 15 of 25 Research article Research article Biochemistry Cell Biology Biochemistry Cell Biology Mgm1 mediates membrane bending (Rujiviphat et al., 2015). Bending of crista mem- branes takes place only when the F1FO-ATP syn- thase can dimerize (Davies et al., 2012; Rabl et al., 2009). Therefore, it is reasonable to assume that bending of crista membranes takes place concurrently with the fusion process, and dimeric F1FO-ATP synthase is required to stabilize the bending of the membrane. Discussion Importantly, according to our model, in the absence of Su e, a dimerization component of the F1FO-ATP syn- thase, fusion of IM by Mgm1 cannot take place. Rather, OM fusion still occurs in the absence of IM fusion and septa are generated. The continual import of proteins and lipids then leads to expan- sion of the septa membranes. This expansion may cause the curved structures of septa that in EM sections appear as onion-like profiles (Figure 1). Mgm1 Mgm1 In this pathway, import of newly synthesized proteins and lipids into the IBM and passage through CJs leads to the formation of tubular cristae. Assembly of CJs can take place since a pool of MICOS sub- units is present in the IBM (Harner et al., 2011). Interestingly, oligomeric forms of the MICOS sub- unit Mic10 can induce membrane curvature in vitro and were suggested to form CJs in vivo (Barbot et al., 2015; Bohnert et al., 2015; Milenkovic and Larsson, 2015). We propose that the shape of CJs formed by the MICOS complex determines the tubular shape of nascent cristae. Dimer- ization of F1FO would stabilize bending of the tubular cristae, very much like it stabilizes the rims of lamellar cristae. Interestingly, regular helical zipper-like structures of presumably dimeric F1FO were observed at the surface of the tubular cristae of the protist Paramecium multimicronucleatum by scanning EM (Allen et al., 1989). Remarkably, in the absence of both Dnm1 and functional MICOS, tubules are formed. We suggest that they are generated by the continual influx of proteins and lipids and shaped by dimeric F1FO. Since CJs are not formed, these tubules are rather unstable, having a high tendency to fuse and to generate bizarre corrugated networks, and are not functional in Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 16 of 25 Research article Research article Biochemistry Cell Biology Figure 8. Model of the formation of cristae by the fusion dependent and independent pathways. (A) Formation of lamellar crist dependent pathway. Steps in the conversion of the septa membranes of two fusing mitochondria into a crista membrane. (a) Tw fusion; (b) after fusion of OM and before fusion of IM; (c) tethering of the two septa membranes; (d–e) progressing fusion interm of fusion after formation of a crista junction; (g) formation of a closed crista-like membrane vesicle without connection to the IBM Figure 8 continued on next page Figure 8. Model of the formation of cristae by the fusion dependent and independent pathways. (A) Formation of lamellar cristae by the fusion dependent pathway. Steps in the conversion of the septa membranes of two fusing mitochondria into a crista membrane. Mgm1 (a) Two mitochondria before fusion; (b) after fusion of OM and before fusion of IM; (c) tethering of the two septa membranes; (d–e) progressing fusion intermediates; (f) final stage of fusion after formation of a crista junction; (g) formation of a closed crista-like membrane vesicle without connection to the IBM in mutants deficient in Figure 8 continued on next page Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 17 of 25 17 of 25 Research article R Research article Biochemistry Cell Biology Figure 8 continued Mic60 or Mic10. (B) Formation of tubular cristae by the fusion independent pathway. (C) Formation of bizarre membrane assemblies in mitochondria of MICOS deficient cells by the fusion independent pathway. Blue lines, OM; green lines, IBM, septa membrane and crista membrane; light green, matrix space. Mic60 or Mic10. (B) Formation of tubular cristae by the fusion independent pathway. (C) Formation of bizarre membrane assemblies in mitochondria of MICOS deficient cells by the fusion independent pathway. Blue lines, OM; green lines, IBM, septa membrane and crista membrane; light green, matrix space. DOI 10 7554/ Lif 18853 020 DOI: 10.7554/eLife.18853.020 DOI: 10.7554/eLife.18853.020 DOI: 10.7554/eLife.18853.020 OXPHOS and maintenance of mtDNA (Figure 8C). This points to a correlation between presence of cristae and mtDNA. In fact, mtDNA, condensed in nucleoids, was reported to be bound to crista membranes, possibly to crista rims (Kopek et al., 2012; Kukat and Larsson, 2013). OXPHOS and maintenance of mtDNA (Figure 8C). This points to a correlation between presence of cristae and mtDNA. In fact, mtDNA, condensed in nucleoids, was reported to be bound to crista membranes, possibly to crista rims (Kopek et al., 2012; Kukat and Larsson, 2013). p y ( p ) This second pathway of crista biogenesis suggested in our hypothesis becomes apparent when fission is compromised. In this situation, the mitochondrial network is virtually completely fused and Mgm1-dependent fusion activity should be drastically reduced. The lack of requirement of Mgm1 in this pathway provides a rational explanation for the most surprising observation that Mgm1 can be deleted in the Dnm1 deletion background without loss of cristae. Residual formation of lamellar cris- tae in the absence of Dnm1 can be explained by residual fusion and fission activity of mitochondria (Gorsich and Shaw, 2004; Ishihara et al., 2013; Westermann, 2010). The virtually complete lack of lamellar cristae in cells deficient in both Dnm1 and Mgm1 further supports this assumption. Mgm1 Most interestingly, a study on the role of Opa1 in mitochondrial cristae remodeling showed that mitochon- dria isolated from mouse embryonic fibroblasts lacking functional Opa1 lacked lamellar cristae; instead, these mitochondria contained short tubular-type cristae (Frezza et al., 2006). These obser- vations are not only in very good agreement with our conclusions on a direct role of Mgm1 in crista formation, but also point to the conservation of its role in crista biogenesis in mammalian cells. g Can both pathways of crista biogenesis coexist? This is clearly the case in yeast, as we observed lamellar and, to a lower extent, tubular cristae in WT cells. Inspection of a large number of published EM images of mitochondria from different cell types reveals a bias to lamellar vs. tubular cristae. For example, lamellar cristae dominate in pancreatic cells and hepatocytes, whereas in steroidogenic cells tubular cristae are the principle architectural feature. A mixed situation prevails in certain types of muscle cells where both lamellar and tubular profiles can be recognized (Blinzinger et al., 1965; Fawcett, 1981; Hanaki et al., 1985). Tomographic reconstruction is the optimal approach to deter- mine precisely the distribution of these two types of cristae. Such a study has been performed with cerebellar neuronal cells and revealed lamellar and tubular cristae that are regularly connected by sites of fusion (Perkins et al., 1997). Furthermore, the differentiation of isolated zona glomerulosa cells of rat adrenal cortex triggered by treatment with adrenocorticotropic hormone leads to a dras- tic change of the corticoid hormones synthesized and to a concomitant transformation of the crista structure from mainly “lamellar-tubular” to “tubular-convolute” (Andreis et al., 1990). Interestingly, steroidogenesis in human trophoblasts was reported to be dependent on the activity of Opa1. Dur- ing developmental induction of the steroidogenic pathway, Opa1 levels decreased and steroid for- mation increased. Manipulations of cells to increase Opa1 levels led to decreased efficiency of steroidogenesis. At the same time, crista structure was observed to be remodeled (Wasilewski et al., 2012). Altogether, many basic questions need to be answered, including how mitochondrial architecture is modulated in response to metabolic and developmental requirements, how the state of cellular differentiation affects mitochondrial ultrastructure and why specific crista structures are necessary for specific cellular functions. In conclusion, our investigation of the determinants of mitochondrial architecture has led us to an entirely novel concept of pathways of mitochondrial biogenesis. Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Yeast strains and growth conditions S. cerevisiae strains YPH499 or W303 were used as wild type (WT). Genetic manipulations were per- formed according to standard procedures (Longtine et al., 1998). The genotypes are listed in Table 1. Cells were grown as indicated on YPD (1% yeast extract, 2% peptone, 2% glucose), YPGal (1% yeast extract, 2% peptone, 2% galactose) or YPG (1% yeast extract, 2% peptone, 3% glycerol) (Sherman, 1991). mgm1-5 cells were kept at 25˚C to avoid loss of mtDNA. Loss of Mgm1 function was induced by resuspension of cells in pre-warmed liquid medium and subsequent incubation at 37˚C for 25 min. For restoration of Mgm1 function, cells were resuspended in liquid medium (25˚C) and incubated for additional 30 min. Mgm1 Strain Genotype W303 WT MATa or MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100 W303 mgm1-5 MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100, mgm1-5 (G408 to D408) (Wong et al., 2000) YPH499 WT MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, F1b-3xHA::HISI3 Dsu e F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3, F1b-3xHA::TRP1 Su e-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; S e-3xHA::HIS3 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mgm1::KAN Mgm1-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, MGM1-3xHA::TRP1 Dmic60 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, F1b-3xHA::TRP1 Cox5A-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; COX5A-3xHA::KAN Dmic60 Cox5A- 3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, COX5A-3xHA::KAN Ddnm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN Dmic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic10::KAN Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic60::HIS3 Ddnm1 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mgm1::hphNT1 Ddnm1 Dfzo1 MATa, ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::HIS3, fzo1::KAN, rho+ Ddnm1 Dmic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mic10::hphNT1 Ddnm1 Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mic60::HIS3 Ddnm1 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, su e::HIS3 Ddnm1 Dmgm1 Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; dnm1::KAN, mic60::HIS3; mgm1::hphNT1 Ddnm1 Dsu e Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; dnm1::KAN, mic60::HIS3; su e::HIS3 / f Research article Biochemistry Cell Biolo Biochemistry Cell Biology Strain Genotype W303 WT MATa or MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100 W303 mgm1-5 MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100, mgm1-5 (G408 to D408) (Wong et al., 2000) YPH499 WT MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, F1b-3xHA::HISI3 Dsu e F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3, F1b-3xHA::TRP1 Su e-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; Su e-3xHA::HIS3 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mgm1::KAN Mgm1-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, MGM1-3xHA::TRP1 Dmic60 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, F1b-3xHA::TRP1 Cox5A-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; COX5A-3xHA::KAN Dmic60 Cox5A- 3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, COX5A-3xHA::KAN Ddnm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN Dmic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic10::KAN Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic60::HIS3 Ddnm1 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mgm1::hphNT1 Ddnm1 Dfzo1 MATa, ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::HIS3, fzo1::KAN, rho+ Ddnm1 Dmic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mic10::hphNT1 Ddnm1 Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mic60::HIS3 Ddnm1 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, su e::HIS3 Ddnm1 Dmgm1 Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; dnm1::KAN, mic60::HIS3; mgm1::hphNT1 Ddnm1 Dsu e Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; dnm1::KAN, mic60::HIS3; su e::HIS3 Dmic60 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, F1b-3xHA::TRP1 Cox5A-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; COX5A-3xHA::KAN Dmic60 Cox5A- 3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3 Dmic60 Cox5A- 3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, COX5A-3xHA::KAN Ddnm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN Ddnm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0 mic10::KAN Dmic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic10::KAN Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic60::HIS3 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0 mic60::HIS3 Ddnm1 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, m Ddnm1 Dfzo1 MATa, ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::HIS3, fzo1::KAN, rho+ Ddnm1 Dfzo1 MATa, ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::HIS3, fz mic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mic10::hphN mic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mic60::HIS3 Ddnm1 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, su e::HIS3 u e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, su e::HIS3 Ddnm1 Dsu e Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; dnm1::KAN, mic60::HIS3; su e::HIS3 DOI: 10.7554/eLife.18853.021 Mgm1 Strain Genotype W303 WT MATa or MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100 W303 mgm1-5 MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100, mgm1-5 (G408 to D408) (Wong et al., 2000) YPH499 WT MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, F1b-3xHA::HISI3 Dsu e F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3, F1b-3xHA::TRP1 Su e-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; Su e-3xHA::HIS3 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mgm1::KAN Mgm1-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, MGM1-3xHA::TRP1 Dmic60 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, F1b-3xHA::TRP1 Cox5A-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; COX5A-3xHA::KAN Dmic60 Cox5A- 3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, COX5A-3xHA::KAN Ddnm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN Dmic10 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic10::KAN Dmic60 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; mic60::HIS3 Ddnm1 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, dnm1::KAN, mgm1::hphNT1 Research article Biochemistry Cell Biology Table 1. Genotypes of the strains used in the study. Left column, names of the stains. Right column, genotypes of the strains. Mgm1 Since the identified key players are highly conserved from yeast to human, our study has immediate relevance for the relationship of structure and function of mammalian mitochondria of different tissues under normal and dysfunc- tional conditions. Finally, our results emphasize that understanding inheritance and homeostasis of mitochondria requires an integrated view on their three-dimensional architecture and the molecular topological functions of the various key players involved. 18 of 25 Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 Table 1. Genotypes of the strains used in the study. Left column, names of the stains. Right column, genotypes of the strains. Strain Genotype W303 WT MATa or MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100 W303 mgm1-5 MATa, ade2-1, leu2-3, his3-11,15, trp1-1, ura3-1, can1-100, mgm1-5 (G408 to D408) (Wong et al., 2000) YPH499 WT MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0 Dsu e MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, F1b-3xHA::HISI3 Dsu e F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, su e::HISI3, F1b-3xHA::TRP1 Su e-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; Su e-3xHA::HIS3 Dmgm1 MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mgm1::KAN Mgm1-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, MGM1-3xHA::TRP1 Dmic60 F1b-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1, mic60::His3, F1b-3xHA::TRP1 Cox5A-3xHA MATa; ura3-52, lys2-801amber, ade2-101ocre, trp1-D63, his3-D200, leu2-D1} or BY4724 {MATa; his3D1; leu2D0; lys2D0; ura3D0; COX5A 3xHA::KAN Research article Biochemistry Cell Biology Table 1. Genotypes of the strains used in the study. Left column, names of the stains. Right column, genotypes of the strains. Isolation of mitochondria Mitochondria were washed once with SM buffer (20 mM MOPS, pH 7.4, 0.6 M sorbitol) and finally resuspended in SM buffer. Subfractionation of mitochondria Generation and separation of membrane vesicles were performed as described previously (Harner et al., 2011). 10 mg of freshly isolated mitochondria were resuspended in SM buffer, then 20 ml swelling buffer (20 mM MOPS, pH 7,4, 0.5 mM EDTA, 1 mM PMSF, 1x Roche complete prote- ase inhibitor) were added dropwise, followed by incubation under continuous stirring for 30 min on ice. 5 ml 2.5 M sucrose were added and the suspension was incubated for 15 min on ice. The sus- pension was subjected to sonication for 3 times 30 s with 30 s breaks on ice at 60% duty cycle and output control 0 (Branson Sonifier 250; Branson, Danbury, USA) using a microtip. Residual intact mitochondria were removed by centrifugation for 20 min at 20,000 xg and 4˚C. The submitochon- drial vesicles were concentrated on a 2.5 M sucrose cushion by high speed centrifugation (120,000 xg for 100 min at 4˚C). The vesicle pellet was resuspended, loaded under a continuous sucrose gra- dient (0.8-1.25 M sucrose in 20 mM MOPS, pH 7.4, 0.5 mM EDTA) and separated by centrifugation for 24 h at 200,000 xg and 4˚C. The gradient was fractionated in 500 ml fractions, the proteins were TCA precipitated and analyzed by SDS-PAGE and immunoblotting. Generation of yeast cell extract For analysis of cell extracts the strains were grown to logarithmic phase. Cells (OD600 12.5) were har- vested by centrifugation, washed with water and resuspended 1ml water containing 100 mg PMSF. Cells were lysed by incubation for 10 min in 0.25 M NaOH and 1 % b-mercapto ethanol (final con- centration). Proteins were subjected to TCA precipitation and acetone washed. The pellets were resuspended in 100 ml Laemmli buffer. Maintenance of mtDNA and DAPI staining g Yeast strains were grown on YPG plates to select for mtDNA, transferred to liquid YPD medium to allow for loss of mtDNA and continuously kept in the logarithmic growth phase. At time point 0 and after 48 hr the percentage of cells containing mtDNA was determined by DAPI staining. DAPI stain- ing was performed as described (Harner et al., 2014). Drop dilution assay Cells were grown in YPG liquid medium and kept in logarithmic phase, washed once with water, diluted in water to an OD600 of 0.3. Afterwards serial dilutions were performed (1:10; 1:100; 1:1000; 1:10,000). 3 ml of each dilution were spotted on agar plates containing the indicated media and were incubated at 30˚C. Isolation of mitochondria Mitochondria were isolated as described (Lee et al., 1998). Cells were cultured to a final OD600 of 0.8–1.2 and harvested by centrifugation for 5 min at 2000 xg at RT. Cells were washed with 40 ml water and the wet weight was measured. They were resupended in 30 ml of alkaline solution (100 mM Tris (pH not adjusted), 10 mM DTT) and incubated for 30 min at 30˚C under gentle agitation. Cells were harvested, washed once with spheroplast buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 19 of 25 Research article Research article Biochemistry Cell Biology 1.2 M sorbitol), resuspended in spheroplast buffer (10 ml/g wet weight) containing 6.6 mg Zymo- lyase per g wet weight and incubated for 30 min at 30˚C. Spheroplasts were harvested by centrifu- gation at 2000 xg at 4 ˚C for 5 min and washed twice with 15 ml ice-cold lysis buffer (20 mM MOPS- KOH pH 7.2, 1 mM EDTA, 0.6 M sorbitol, 0.2 % (w/v) BSA, 1 mM PMSF). Spheroplasts were resus- pended in 15 ml ice-cold lysis buffer by pipetting using a Pipetman P5000 (Gilson, Middleton, USA) with 1cm cutoff P5000 tips. Unbroken spheroplasts were harvested by centrifugation at 2000 xg at 4 ˚C for 5 min and again resuspended in ice-cold lysis buffer. The supernatants were collected, pooled and mitochondria were harvested by centrifugation at 12,000 xg and 4˚C for 5 min. Mitochondria were washed once with SM buffer (20 mM MOPS, pH 7.4, 0.6 M sorbitol) and finally resuspended in SM buffer. 1.2 M sorbitol), resuspended in spheroplast buffer (10 ml/g wet weight) containing 6.6 mg Zymo- lyase per g wet weight and incubated for 30 min at 30˚C. Spheroplasts were harvested by centrifu- gation at 2000 xg at 4 ˚C for 5 min and washed twice with 15 ml ice-cold lysis buffer (20 mM MOPS- KOH pH 7.2, 1 mM EDTA, 0.6 M sorbitol, 0.2 % (w/v) BSA, 1 mM PMSF). Spheroplasts were resus- pended in 15 ml ice-cold lysis buffer by pipetting using a Pipetman P5000 (Gilson, Middleton, USA) with 1cm cutoff P5000 tips. Unbroken spheroplasts were harvested by centrifugation at 2000 xg at 4 ˚C for 5 min and again resuspended in ice-cold lysis buffer. The supernatants were collected, pooled and mitochondria were harvested by centrifugation at 12,000 xg and 4˚C for 5 min. Blue native gel electrophoresis (BN-PAGE) 75 mg of mitochondria were pelleted by centrifugation and resuspended in BN-lysis buffer (Wittig et al., 2006). For analysis of F1FO-ATP synthase or MICOS complex 1 % (w/v) or 3 % (w/v) digitonin were used. Cleared lysates were supplemented with Native PAGE 5% G-250 Sample Addi- tive and subjected to BN-PAGE (Native PAGE 3–12% Bis-Tris; Life Technologies, Carlsbad, CA, USA). After blotting on PVDF membranes (Roth, Karlsruhe, Germany) immuno-decoration using the indicated antibodies was performed. Electron microscopy Electron microscopy of chemically fixed cells was performed as described (Bauer et al., 2001). Ultra- thin sections were collected on Pioloform-coated copper slot grids (Plano, Wetzlar, Germany) and stained with uranyl acetate and lead citrate (Reynolds, 1963). For electron microscopy after cryo- sectioning (Tokuyasu method), cells were grown to exponential phase in YPD or YPG medium, chem- ically fixed, embedded in 12% gelatin and cryo-sectioned as described previously (Griffith et al., 2008). Ultrathin cryo-sections were collected with a 1:1 mixture of 2% methylcellulose (in ddH2O) and 2.3 M, sucrose 120 mM PIPES, 50 mM HEPES, pH 6.9, 4 mM MgCl2, 20 mM EGTA and layered on Formvar/carbon coated copper 100 mesh grids. Structural phenotypes were analyzed by evalua- tion of 100 mitochondrial profiles unless otherwise indicated. Immunological reactions were Harner et al. eLife 2016;5:e18853. DOI: 10.7554/eLife.18853 20 of 25 Research article Research article Research article Biochemistry Cell Biology Biochemistry Cell Biology performed with monoclonal anti-HA (RRID:AB_514505) and a protein A-gold 10 nm conjugate. Pro- tein localization gold particles present in 100 cells were counted. Samples for the electron tomogra- phy analyses were processed as previously described (Mari et al., 2014). performed with monoclonal anti-HA (RRID:AB_514505) and a protein A-gold 10 nm conjugate. Pro- tein localization gold particles present in 100 cells were counted. Samples for the electron tomogra- phy analyses were processed as previously described (Mari et al., 2014). Additional information Funding Funder Grant reference number Author Max-Planck-Gesellschaft Max E Harner Ann-Katrin Unger Toshiaki Izawa Walter Neupert Carl Friedrich von Siemens Stiftung Walter Neupert Jung-Stiftung fu¨ r Wissenschaft und Forschung Max E Harner Ludwig-Maximilians-Universita¨t Mu¨ nchen Max E Harner Netherlands organization for Scientific Research DN82-303 Fulvio Reggiori Deutsche Forschungsge- meinschaft DN82-303 Fulvio Reggiori Schweizerischer Nationalfonds zur Fo¨ rderung der Wis- senschaftlichen Forschung CRSII3_154421 Fulvio Reggiori ZonMw ZonMW VICI Fulvio Reggiori Netherlands organization for Scientific Research 822.02.014 Fulvio Reggiori The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions MEH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article; A-KU, WJCG, MM, TI, MS, Acquisition of data, Analysis and interpretation of Acknowledgements We thank Dr. Jodi Nunnari for providing us with the mgm1-5 mutant, Matthew Harmey for discus- sion and Monika Krause, MPI for Biochemistry, for the artwork. WN is grateful to the Max Planck Society for a Senior Fellowship and the Carl Friedrich von Siemens Stiftung for financial support. MEH and WN acknowledge the generous support and advice by Dr. Michael Kiebler, BMC, Univer- sity of Munich. MEH thanks the Jung-Stiftung fu¨ r Wissenschaft und Forschung and LMUexcellent for financial support. FR is supported by ALW Open Program (822.02.014), DFG-NWO cooperation (DN82-303), SNF Sinergia (CRSII3_154421) and ZonMW VICI (016.130.606) grants. 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https://openalex.org/W2771520803	https://europepmc.org/articles/pmc5815701?pdf=render	English	null	Endovascular stent graft repair of aortogastric fistula caused by peptic ulcer after esophagectomy	Medicine	2,017	cc-by	3,423	Abstract Abstract Rationale:Aortogastric ﬁstula (AGF) is a rare but devastating clinical complication after esophagectomy. In a recent report, nearly all AGF patients died of massive hemorrhage or aspiration of massive hematemesis. Therefore, timely appropriate treatment of AGF remains a challenge.Herein, we report a case of AGF that resulted from peptic ulceration after esophagectomy and was successfully treated with endovascular stent graft placement. Patient concerns: A 59-year-old man had undergone video-assisted thoracoscopic esophagectomy for squamous cell carcinoma and esophageal reconstruction using a gastric tube 14 months previously. He suddenly experienced massive hematemesis and unstable circulatory dynamics, Infusion was performed to treat critical hemorrhagic shock but was ineffective. We informed the patient and his family members of the situation, and once written informed consent to treatment was provided, we rushed him to the operating room. Diagnoses: Contrast medium permeated into the gastric cavity through a ﬁstula between the abdominal aorta and gastric tube at the 11th thoracic level, Based on this, we made a diagnosis of AGF resulting from a peptic ulcer, and this diagnosis was further conﬁrmed by high pressure angiography combined with computed tomography (CT) imaging. Interventions: An endovascular stent graft was placed under the guidance of digital subtraction angiography and followed by antibiotic therapy to prevent infection and proton pump inhibitor therapy to inhibit gastric acid secretion. Outcomes: The patient recovered uneventfully after the procedure. Four months after surgery, the patient died of organ failure caused by retroperitoneal lymph node metastasis and multiple intrahepatic metastases, with no postoperative bleeding linked to the endovascular stent graft repair. Lessons: Our case supports the notion that endovascular stent graft repair is a feasible alternative in treatment of AGF with several advantages in addition to surgical intervention, although more such cases should be collected and analyzed in the future to corroborate our observations. Abbreviations: AGF = aortogastric ﬁstula, DSA = digital subtraction angiography, Hb = hemoglobin, Hct = hematocrit, ICU = intensive care unit, NSAIDs = nonsteroidal anti-inﬂammatory drugs, PPI = proton pump inhibitor. eywords: aortogastric ﬁstula, endovascular stent graft repair, esophagectomy, gastric tube ulceratio Medicine ® OPEN Clinical Case Report OPEN 1. Introduction and trachea, ulceration-induced penetration of the gastric tube into any of these structures can be consequently serious and fatal.[1,2] Surgical intervention has been typically used to treat AGF after esophagectomy, but mortality rates have been high. However, conservative treatment was always accompanied by a lethal outcome. Graft replacement appears to be a promising approach.[2] Here, we report an unusual case of AGF that was effectively treated with placement of an endovascular stent graft. We obtained written informed consent from the patient to report this case. Aortogastric ﬁstula (AGF), an abnormal communication between the aorta and the gastrointestinal tract, is a rare but fatal disorder after esophagectomy. Because the reconstructed gastric tube is adjacent to the major vessels and organs such as the heart, aorta, Endovascular stent graft repair of aortogastric ﬁstula caused by peptic ulcer after esophagectomy A case report g Wei, MD, Lei Song, MD ∗, Xue-Song Zhang, MD, Kui-Yang Wang, MD, Jie Wu, MD Xiao-Qing Wei, MD, Lei Song, MD ∗, Xue-Song Zhang, MD, Kui-Yang Wang, M Xiao-Qing Wei, MD, Lei Song, MD ∗, Xue-Song Zhang, MD, Kui-Yang Wang, MD, Jie Wu, MD Editor: Bülent Kantarçeken. The authors have no conﬂicts of interest to disclose. Interventional Therapy Department, The Second Hospital of Dalian Medical University, Dalian, China. ∗Correspondence: Lei Song, Interventional Therapy Department, The Second Hospital of Dalian Medical University, Shahekou District, Dalian, China (e-mail: Songlei_1975@126.com). 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. Medicine (2017) 96:50(e8959) Received: 21 June 2017 / Received in ﬁnal form: 2 November 2017 / Accepted: 8 November 2017 http://dx.doi.org/10.1097/MD.0000000000008959 2. Case presentation A 59-year-old man with a history of type 2 diabetes and smoking (an average of 20 cigarettes per day) but no history of high blood pressure and heart disease underwent video-assisted thoraco- scopic esophagectomy for squamous cell carcinoma (stage PT1N1Mx) of the distal esophagus with an esophageal reconstruction using a gastric tube through the posterior mediastinal route 14 months previously. The surgical resection margins were negative, and right lung nodule metastasis was not 1 Medicine Wei et al. Medicine (2017) 96:50 Figure 1. A peptic ulcer is observed by gastrointestinal endoscopy on the posterior wall of the distal part of the gastric tube. Figure 1. A peptic ulcer is observed by gastrointestinal endoscopy on the posterior wall of the distal part of the gastric tube. Figure 2. Emergency abdominal aortography (A) and CT (B) revealing remarkable extravasation of contrast medium that ﬂowed out of the gastric tube. CT = computed tomography. Figure 2. Emergency abdominal aortography (A) and CT (B) revealing remarkable extravasation of contrast medium that ﬂowed out of the gastric tube. CT = computed tomography. tography (A) and CT (B) revealing remarkable extravasation of contrast medium that ﬂowed out of the gastric tube. CT = Figure 2. Emergency abdominal aortography (A) and CT (B) revealing remarkable extravasation of contrast medium that ﬂowed out of the gastric tube. CT = computed tomography Figure 2. Emergency abdominal aortography (A) and CT (B) revealing remarkable extravasation of contrast mediu computed tomography. observed. The adjuvant radiotherapy (chemoradiotherapy for the original esophageal tumor area and lymphatic drainage area) and chemotherapy were completed. At 7 months postoperation, the patient occasionally experienced episodes of epigastric discom- fort. A gastrointestinal endoscopic biopsy revealed a gastric ulcer on the posterior wall of the distal part of the gastric tube, but his vital signs were stable and routine blood, liver, and kidney function test results were within the normal ranges. This patient did not receive continuous treatment with a proton pump inhibitor. Five months previously, he experienced epigastric discomfort again and had blood in the stool. Gastrointestinal endoscopic examination revealed a round deep ulcer, 1.5mm in diameter, in the gastric body, with visible blood vessels in the middle (Fig. 1A and B). However, there was no evidence of recurrence over the 1 year following the operation. 3. Discussion Because of the patient’s unstable circulatory dynamics, we classiﬁed him as high risk for open surgery and decided to use a stent graft repair as the ﬁrst feasible option for urgent treatment. Diagnostic angiography and endovascular stent graft repair were performed under local anesthesia. After access was obtained via the femoral artery, a 4-Fr catheter was placed into the abdominal aorta, and we found that contrast medium permeated into the gastric cavity through a ﬁstula between the abdominal aorta and gastric tube at the 11th thoracic level (Fig. 2). Based on this, we made a diagnosis of AGF resulting from a peptic ulcer, and this diagnosis was further conﬁrmed by high pressure angiography combined with computed tomography (CT) imaging. We considered the AGF to be the primary cause of upper gastrointestinal bleeding. Thereafter, angiography was per- formed in the aortic arch by puncturing the left femoral artery and entering the marked pigtail tube, and the AGF was located. A stent (COOK ZTEG-2PT-32–160) was then passed through the right femoral artery into the aorta, positioned on the ﬁstula location and released. After stenting, the graft was in good condition, no contrast medium was permeating into the stomach, and hematemesis ceased (Fig. 3). The patient’s circulatory dynamics also stabilized immediately following the procedure, and he was then transferred to the intensive care unit (ICU) where he was kept for 1 day before being returned to the general ward (Fig. 4). The full procedure was performed under the guidance of digital subtraction angiography (DSA). Subsequent treatments included pumping norepinephrine, blood transfusion, infusion booster, sufentanil analgesia, and ceftriaxone. Also, omeprazole AGF is an uncommon but recognized life-threatening complica- tion of esophagectomy. Anastomotic leakage and peptic ulcers are the 2 major causes for AGF after esophagectomy .[2] There is an increasing number of reports of ulcerative lesions in reconstructed gastric tubes, which previously had been compar- atively rare.[1] Ide et al[3] reported that the incidence of such lesions was 13%. The differences in the incidence rates reported by different studies can be attributed to the fact that an ulcer of the reconstructed gastric tube is often asymptomatic and thus goes unnoticed.[4,5] If an ulceration is caused by penetration of the gastric tube into structures such as pericardium, aorta, and trachea, AGF can be serious and fatal. 2. Case presentation Two days after the endoscopic examination, he suddenly experienced massive hematemesis and unstable circulatory dynamics, with a blood pressure of 90/45 mm Hg and a pulse of 150beats/min. Blood tests revealed severe anemia, with hemoglobin (Hb) of 50 g/L and hematocrit of 15.3%. Infusion was performed to treat critical hemorrhagic shock but was ineffective. We informed the patient and his family members of the situation, and once written informed consent to treatment was provided, we rushed him to the operating room. Figure 3. Aortography after stent graft placement around the ﬁstula revealing hemostasis. Figure 3. Aortography after stent graft placement around the ﬁstula revealing hemostasis. 2 Wei et al. Medicine (2017) 96:50 www.md-journal.com Figure 4. Changes in Hb levels before and after stent implantation in the patient. Hb = hemoglobin. and octreotide were continuously administered to inhibit gastric acid secretion. The stent implantation, Hb concentration, and vital signs of this patient were relatively stable, and the patient did not have hematemesis, melena, or any other active bleeding. Considering that this patient had a tumor and was in a hypercoagulable state, in order to avoid thrombosis, he did not continue antibleeding treatment. At 2 months postoperation, in order to monitor the patency of the stent and active bleeding and related complications, we recommend that patients should have endoscopic review, but this patient and his family declined. Contrast-enhanced CT revealed no extravasation and no other complications such as hematemesis, infection, and pseudoaneur- ysm (Fig. 5). Four months after surgery, the patient died of organ failure caused by retroperitoneal lymph node metastasis and multiple intrahepatic metastases, with no postoperative bleeding linked to the endovascular stent graft repair. Figure 4. Changes in Hb levels before and after stent implantation in the patient. Hb = hemoglobin. Figure 4. Changes in Hb levels before and after stent implantation in the patient. Hb = hemoglobin. References Recent studies reported successful treatments of AGF with endovascular stent graft repair.[2,19] The advantages of using endovascular techniques include achieving rapid control of bleeding with minimal injury, avoiding intervention in a hostile abdomen, and eliminating the complications such as operative trauma and stress associated with open surgical repair[19,21] The need for only local anesthesia is also one of the advantages. It was quite remarkable that our patient had a quick recovery and short hospital stay (∼2 weeks) compared with other successfully treated AGF cases in the literature.[2,5] Regarding pseudoaneurysm recurrence, placement of an endovascular stent graft is also a useful backup modality if sterilization has been achieved.[22] Considering the lethal course of this case, it should be stressed that celiac artery coverage needs to be considered very carefully, especially in patients with a known gastric ulcer as it may lead to a secondary AGF. One limitation of using endovascular stent graft repair is the high skill requirement for the practitioner. Another limitation of using this technique is that endovascular stent graft repair has been correlated with a high risk of infection, which is a factor contributing to poor outcome after endovascular repair.[22] This also raises another concern of long-term safety and efﬁcacy of this technique by placing a new prosthetic material in an already or potentially infected ﬁeld. Thus, aggressive antibiotic therapy should be utilized following this procedure.[23–25]. In the present case, although there were no signs of infection, a prophylactic antibiotic therapy (i.e., ceftriaxone) was still prescribed because of the potential infection. [1] Mochizuki Y, Akiyama S, Koike M, et al. A peptic ulcer in a reconstructed perforating the thoracic aorta after esophageal replace- ment. Jpn J Thorac Cardiovasc Surg 2003;51:448–51. [2] Okamura A, Kawakubo H, Takeuchi H, et al. Successful treatment of aortogastric ﬁstula after esophagectomy. Esophagus 2015;12:387–91. [3] Ide H, Eguchi R, Nakamura T, et al. Late management of patients after esophagectomy and reconstruction for esophageal cancer (Eng abstr). Nihon Shokaki Geka Gakkai Zasshi 1995;28:2028–61. [4] Katsoulis IE, Veloudis G, Exarchos D, et al. Perforation of a gastric tube peptic ulcer into the thoracic aorta. Dis Esophagus 2001;14:76–8. [5] Takebayashi T, Okushiba S, Ohno K, et al. Peptic ulcer-induced acute aortogastric ﬁstula occurring 7 years after a pharyngogastrostomy following a resection for carcinoma of the esophagus: report of a case. Surg Today 2004;34:777–9. [6] Takemura M, Higashino M, Osugi H, et al. Medicine diagnosis and treated the patient with an endovascular stent graft repair. We carried out the full procedure under the guidance of DSA, which allowed us to see the stent location clearly. Although the patient presented in a critical condition, the choice of the endovascular stent graft repair turned out to be very effective, as evidenced by the facts that no contrast leakage inside the stomach was observed and that the patient’s hematemesis ceased as soon as the procedure was completed. During the disease progression, it is common for a gastric tube to continuously secrete acid even though the vagus nerve has been divided.[26] Therefore, 24-hour pH surveillance of the gastric tube after an esophagectomy and a urease test should be carried out, and treatment to inhibit gastric acid production should be administered. In addition, endoscopic observation for the early diagnosis and appropriate management of AGF is also crucial for a favorable outcome. Previous studies have reported an interval between esophagogastrectomy and hematemesis in patients with peptic ulcers ranging from 14 months to 7 years.[5] Hence, the best precautionary approach should be close follow-up of such patients during this period after esophagectomy. We considered the treatment of AGF in this patient with endovascular stent graft repair successful because the patient did not die from any events related to the endovascular stent graft repair but from cancer complications during the follow-up period. acute onset of mid-thoracic pain, heartburn, and sudden small volume or massive hematemesis. AGF should be highly suspected in patients having one of the aforementioned etiologies with at least one of those symptoms. Early accurate diagnosis is vital for survival. The most effective approach to diagnose an AGF is still CT scanning, which can reveal a number of characteristics such as the existence of novel nonhomogenous masses between the aorta and the stomach, air in the mediastinum and/or inside the aneurysm sac, as well as contrast extravasation from the aorta into the stomach.[10] The deﬁnitive diagnosis can be established when ulcerations accompanied by coagulations are detected on endoscopy, although endoscopy- induced hypertension may increase the risk of further hemor- rhage.[11] In this particular case, the patient had mid-thoracic pain and sudden massive hematemesis, and an AGF was diagnosed by angiography in time. In a recent report, nearly all AGF patients died of massive hemorrhage or aspiration of massive hematemesis.[7,12–17] Therefore, timely appropriate treatment of AGF remains a challenge. Medicine The traditional management goals for AGF are the control of hemorrhage and sepsis and maintenance of lower limb perfusion. An aggressive surgical strategy consists of suturing the perforated aorta or aortic reconstruction via open surgery, which appears to be the sole management with appreciable long-term outcomes. Nevertheless, these surgical procedures have dis- advantages including operation-linked stress and general anes- thesia, which can exacerbate preexisting gastritis and stomach ulceration.[13–19,20] Moreover, if the gastric tube is strongly adhered to the aorta around hiatus, an additional fenestration for the celiac artery would make the procedure even more complex and would prolong the operation time, thereby increasing the perioperative risk.[16] Also, it was reported that pseudoaneur- ysms can develop after the operation.[5] In conclusion, we report here the use of endovascular stent graft repair to treat an unusual case of ulcer-linked AGF after esophagectomy. Our case supports the notion that endovascular stent graft repair is a feasible alternative in treatment of AGF with several advantages in addition to surgical intervention, although more such cases should be collected and analyzed in the future to corroborate our observations. 3. Discussion Peptic ulcer formation has many etiologies including destruc- tion of the mucosal barrier from ischemia in the gastric tube, hypersecretion of gastric acid, pulsation of the descending aorta that delays gastric emptying, Helicobacter pylori infection, and usage of nonsteroidal anti-inﬂammatory drugs (NSAIDs).[6,7] The histological impact of postoperative radiotherapy has also been regarded as a signiﬁcant contributor, as this treatment interferes with the histological responses necessary for healing.[8] The above-mentioned factors, except H pylori infection, were present in our case. Moreover, our patient had a history of gastric ulcer. In addition, this patient had postoperative bleeding for 1 year and upper gastrointestinal angiography showed anastomotic patency. Generally, no speciﬁc clinical manifestations are associated with the formation of an AGF after intrathoracic esophagogastric anastomosis,[9] except some vague symptoms and signs including Figure 5. Contrast-enhanced CT showing no evidence of extravasation. CT = computed tomography. Figure 5. Contrast-enhanced CT showing no evidence of extravasation. CT = computed tomography. 3 Wei et al. Medicine (2017) 96:50 Medicine References Five cases of peptic ulcer of gastric tube after radical esophagectomy for esophageal carcinoma and analysis of Helicobacter pylori infection at gastric tube (in Japanese with English abstract). Nihon Kyoubugeka Gakkaizasshi (J Jpn Assn Thorac Surg) 1997;45:1992–7. [7] Begin LR, Sheiner NM. Anastomotic ulcer-induced aortoenteric ﬁstula after esophagogastroplasty. Ann Thorac Surg 1992;54:564–5. 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https://openalex.org/W2789624191	https://europepmc.org/articles/pmc5879134?pdf=render	English	null	Melatonin Regulates Apoptosis and Autophagy Via ROS-MST1 Pathway in Subarachnoid Hemorrhage	Frontiers in molecular neuroscience	2,018	cc-by	9,808	Melatonin Regulates Apoptosis and Autophagy Via ROS-MST1 Pathway in Subarachnoid Hemorrhage 1Department of Neurosurgery, Second Afﬁliated Hospital of Zhejiang University School of Medicine, Hangzhou, China, 2Brain Research Institute, Zhejiang University, Hangzhou, China, 3Collaborative Innovation Center for Brain Science, Zhejiang University, Hangzhou, China Compelling evidence has indicated that imbalance between apoptosis and autophagy may be involved in subarachnoid hemorrhage (SAH). We aimed to investigate the effects and mechanisms of melatonin in the homeostasis of apoptosis and autophagy. One-hundred and forty-eight male Sprague-Dawley rats were intraperitoneally injected with melatonin or vehicle 2 h after SAH induction. Western blotting and an immunoﬂuorescent assay were performed to detect the expression of apoptosis- and autophagy-related proteins. The neuroprotective effect of melatonin attenuating SAH-induced neurological deﬁcit and brain edema may be associated with the suppression of SAH-induced neuronal apoptosis and autophagy. Furthermore, melatonin inhibited the cleavage of mammalian sterile 20-like kinase 1 (MST1) protein by reducing reactive oxygen species (ROS) content. These effects of melatonin on regulating the homeostasis between apoptosis and autophagy could be reversed by an MST1 agonist, chelerythrine, via enhancement of MST1 cleavage. In conclusion, exogenous melatonin alleviates SAH-induced early brain injury (EBI) by suppressing excessive neuronal apoptosis and autophagy. The underlying mechanism may, at least in part, involve the ROS-MST1 pathway. Edited by: Karsten Hiller, Technische Universität Braunschweig, Germany Reviewed by: Zhouguang Wang, Wenzhou Medical University, China Federico Herrera, Instituto de Tecnologia Quàmica e Biológica (ITQB-NOVA), Portugal Reviewed by: Zhouguang Wang, Wenzhou Medical University, China Federico Herrera, Instituto de Tecnologia Quàmica e Biológica (ITQB-NOVA), Portugal Correspondence: Jianmin Zhang zjm135@zju.edu.cn Jun Yu dr_yujun@zju.edu.cn Correspondence: Jianmin Zhang zjm135@zju.edu.cn Jun Yu dr_yujun@zju.edu.cn †These authors have contributed equally to this work. INTRODUCTION †These authors have contributed equally to this work. Despite the tremendous progress made in intensive neuroprotective therapy and invasive surgical strategies, aneurysmal subarachnoid hemorrhage (SAH) remains a devastating disorder with a high risk of mortality and severe disability (Laiwalla et al., 2016; van Gijn et al., 2007). Early brain injury (EBI) occurs in the first 72 h following SAH, contributing to a poor prognosis (Cahill et al., 2006; Guo et al., 2017). Compelling evidence has indicated that an intrinsic apoptotic or autophagic cell death pathway is involved in EBI (Jing et al., 2012; Chen et al., 2014). Although an appealing hypothesis has been proposed that apoptosis and autophagy may be regulated by a common cellular factor (Dhingra and Kirshenbaum, 2013), no study has explored this missing link between apoptosis and autophagy in EBI after SAH. Received: 26 November 2017 Accepted: 09 March 2018 Published: 26 March 2018 Keywords: melatonin, MST1, apoptosis, autophagy, subarachnoid hemorrhage ORIGINAL RESEARCH published: 26 March 2018 doi: 10.3389/fnmol.2018.00093 SAH Model The filament perforation model of SAH were performed according to previous studies (Marbacher, 2016). Briefly, after the rats were anesthetized with 40 mg/kg pentobarbital sodium via intraperitoneal injection, the left carotid artery and its branches were explored by microsurgical isolation. The distal left external carotid artery was transected and reflected in line with the ipsilateral internal carotid artery. A sharpened 4–0 nylon monofilament suture was placed lightly into the internal carotid artery from the ipsilateral external carotid artery and advanced through the internal carotid artery until the end of the suture reached the bifurcation of the anterior and middle cerebral arteries. The suture was advanced 1 mm to further to perforate the bifurcation of the anterior and middle cerebral arteries. After approximately 10 s, the suture was withdrawn. Sham rats received similar surgical procedures, but without perforation. All the experimental rats were maintained at 37◦C using a heating pad throughout the surgical procedure. Mammalian sterile 20-like kinase 1 (MST1) is a crucial serine-threonine kinase that constitutes a critical component of the Hippo signaling pathway (Chan et al., 2011). Compelling evidence has indicated that MST1 may be a switch that dually regulate apoptosis and autophagy (Dhingra and Kirshenbaum, 2013). In a crucial work, investigators showed that MST1 preferentially phosphorylates Beclin1 at Thr108 during ischemic stress in mouse hearts (Maejima et al., 2013). The phosphorylation of Beclin 1 impairs the formation of Beclin 1-Atg14L-Vps34, resulting in a marked reduction in autophagy (Maejima et al., 2013). In addition, the phosphorylated form of Beclin 1 can combine with Bcl-2 to destroy Bcl-2-Bax inhibitory complexes, resulting in an increase the amount of free cellular Bax available to induce mitochondria-dependent apoptosis (Maejima et al., 2013). In addition to the abovementioned mechanism, MST1 can be cleaved to produce a 36-kDa N-terminal constitutively active fragment (cl-MST1) during cell oxidative stress (Ura et al., 2001). Subsequently, the cl-MST1 is transferred into the nucleus and phosphorylates several histones, inducing neuronal cell apoptosis (Cheung et al., 2003). Interestingly, the enhancement of autophagy and reduction of apoptosis in response to melatonin can be reversed by blocking the MST1 pathway (Hu et al., 2017). Moreover, melatonin, as a potent free-radical scavenger, may regulate the activity of MST1 by scavenging reactive oxygen species (ROS; Lehtinen et al., 2006). However, the role of MST1 in melatonin-regulated apoptosis and autophagy in an SAH model has not been elucidated. Experimental Design Two separate experiments were conducted in this study. Experiment I was designed to assess the effect of melatonin on EBI after SAH induction and determine the optimal dose of melatonin for subsequent mechanistic experiments. The rats were randomly divided into four groups: the Sham group, the SAH+Vehicle (1% ethanol in 1 mL of saline) group, the SAH+Melatonin low-dose (5 mg/kg) group, and the Drug Administration Melatonin (N-acetyl-5-methoxytryptamine) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in vehicle (1% ethanol in 1 mL saline) to create solutions for the doses of 5 mg/kg and 10 mg/kg. The melatonin and the vehicle (1% ethanol in 1 mL saline) were administered intraperitoneally at 2 h after SAH induction. Chelerythrine, an MST1 agonist, was dissolved in dimethyl sulfoxide (1 mmol/L) and administered immediately via intraventricular administration after SAH. Dimethyl sulfoxide was administered as vehicle control. The present study was designed to verify the hypothesis that melatonin can regulate the homeostasis of apoptosis and autophagy by inhibiting the ROS-MST1 pathway in EBI after SAH induction. SAH Model The severity of the SAH was evaluated by a previously reported SAH grading system (Marbacher, 2016). The SAH grades represented the amount of bleeding in the subarachnoid space around the basilar artery rings and brainstem (Wu et al., 2016). Possible total scores range from 3 to 18, in which a higher score means more serious SAH. In the present study, we included only those rats with SAH grade scores ≥8 at 24 h after SAH induction. Citation: Shi L, Liang F, Zheng J, Zhou K, Chen S, Yu J and Zhang J (2018) Melatonin Regulates Apoptosis and Autophagy Via ROS-MST1 Pathway in Subarachnoid Hemorrhage. Front. Mol. Neurosci. 11:93. doi: 10.3389/fnmol.2018.00093 Melatonin is an evolutionarily conserved neuro-hormone that is predominantly synthesized in and secreted from the pineal gland (Hu et al., 2017). Experimental evidence has demonstrated that both melatonin and its metabolites are potent free-radical scavengers and broad- spectrum antioxidants (Zhang et al., 2012), which maintain cellular homeostasis and survival March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 1 Shi et al. Melatonin Regulates Apoptosis and Autophagy by modulating inflammation, apoptosis, or autophagy following different types of brain injury (Fernández et al., 2015). Owing to its highly lipophilic nature, melatonin may be a promising agent that can easily pass through the blood–brain barrier (BBB) and play a neuroprotective role in various pathophysiological situations (Carloni et al., 2016). Previous investigators have reported that injection of melatonin after SAH induction reduced brain edema, decreased mortality and improved neurological function (Chen et al., 2014; Dong et al., 2016). At the molecular level, melatonin can inhibit neuronal apoptosis via up-regulation of the pro-survival protein Bcl-2 and down-regulation of its pro-apoptotic cognate Bax (Fernández et al., 2015). Furthermore, melatonin reduced the translocation of Bax to the mitochondria and inhibited mitochondria-dependent apoptosis by enhancing autophagy (Chen et al., 2014). However, no study has explored the detailed mechanisms underlying the regulation of apoptosis by melatonin-mediated autophagy. 148 male Sprague-Dawley rats weighing 300–320 g. All rats were housed on 12-h/12-h light/dark cycles under temperature- and humidity-controlled conditions. Animal body temperature was maintained at 37◦C. This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. The animal protocol was approved by the Institutional Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine. Brain Edema Measurement Brain Edema Measurement Intravital brain edema was examined by magnetic resonance imaging (MRI) as previously described (Guo et al., 2017; Dang et al., 2017). At 24 h after SAH induction, imaging was carried out in a 3.0-Tesla GE Discovery MR750 scanner (General Electric Company, Boston, MA, USA). T2 fast spin-echo sequences were taken using a field of view of 60 × 60 mm, a matrix of 256 × 256 mm and nine coronal slices (2 mm thick). All MRI data were analyzed by a blinded observer using NIH ImageJ software. The degree of edema in the excised brain was measured as brain water content according to previous studies (Wu et al., 2016). After the rats were sacrificed at 24 h following SAH induction, their brains were removed and separated into the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was weighed (wet weight) and promptly dried for 72 h at Intravital brain edema was examined by magnetic resonance imaging (MRI) as previously described (Guo et al., 2017; Dang et al., 2017). At 24 h after SAH induction, imaging was carried out in a 3.0-Tesla GE Discovery MR750 scanner (General Electric Company, Boston, MA, USA). T2 fast spin-echo sequences were taken using a field of view of 60 × 60 mm, a matrix of 256 × 256 mm and nine coronal slices (2 mm thick). All MRI data were analyzed by a blinded observer using NIH ImageJ software. The degree of edema in the excised brain was measured as brain water content according to previous studies (Wu et al., 2016). After the rats were sacrificed at 24 h following SAH induction, their brains were removed and separated into the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was weighed (wet weight) and promptly dried for 72 h at FIGURE 1 \| Both low and high doses of exogenous melatonin alleviated brain edema and improved neurological function 24 h after subarachnoid hemorrhage (SAH) induction. (A) The rats were randomly divided into four groups: the Sham group, the SAH+Vehicle (1% ethanol in 1 mL of saline) group, the SAH+Melatonin low-dose (5 mg/kg) group, and the SAH+Melatonin high-dose (10 mg/kg) group (n = 18 for each group). (B) SAH grade (n = 18 for each group): similar SAH grades were observed among SAH rats. Neurobehavioral Function Assessment Neurobehavioral Function Assessment Neurobehavioral function was blindly assessed according to a modified Garcia scoring system as in previous studies (Marbacher, 2016). Briefly, this evaluation contains six test items: spontaneous activity (0–3), climbing (1–3), forelimb stretching (0–3), spontaneous movements of all limbs (0–3), body proprioception (1–3), and response to vibrissae touch (1–3). Possible scores range from 3 to 18, according to the severity of neurological deficits. A lower score means worse neurological deficits induced by SAH. Neurobehavioral Function Assessment Neurobehavioral function was blindly assessed according to a modified Garcia scoring system as in previous studies (Marbacher, 2016). Briefly, this evaluation contains six test items: spontaneous activity (0–3), climbing (1–3), forelimb stretching (0–3), spontaneous movements of all limbs (0–3), body proprioception (1–3), and response to vibrissae touch (1–3). Possible scores range from 3 to 18, according to the severity of neurological deficits. A lower score means worse neurological deficits induced by SAH. SAH+Melatonin high-dose (10 mg/kg) group. Neurobehavioral functions, brain water content, brain ROS content, and neuronal apoptosis were examined at 24 h after SAH induction. Eighteen rats per group were applied to evaluate neurobehavioral functions, and then separately sacrificed to assess brain water content (n = 6 per group), brain ROS content (n = 6 per group), and neuronal apoptosis and autophagy (n = 6 per group). Experiment II was designed to explore the molecular mechanism by which melatonin regulates the homeostasis of apoptosis and autophagy in EBI after SAH induction. The rats were randomly divided into four groups: the Sham+Vehicle (1% ethanol in 1 mL of saline, intraperitoneal administration; dimethyl sulfoxide, intraventricular injection) group, the SAH+Vehicle (1% ethanol in 1 mL of saline, intraperitoneal administration; dimethyl sulfoxide, intraventricular injection) group, the SAH+Melatonin (10 mg/kg, intraperitoneal administration) + Vehicle (dimethyl sulfoxide, intraventricular injection) group, and the SAH+Melatonin (10 mg/kg, intraperitoneal administration) + Chelerythrine (1 mmol/L, 10 µL, intraventricular injection) group. All rats were sacrificed at 24 h after SAH induction. Western blotting was performed to examine the expression of several target proteins including MST1, cl-MST1, Bcl-2, Bax, Beclin 1, LC3B-I, LC3B-II and cleaved-Caspase 3 (n = 6 per group), and immunofluorescence was used to detect apoptotic and autophagic neurons (n = 6 per group). Animals All experimental animals were purchased from SLAC Laboratory Animal Company Limited (Shanghai, China). We used a total of March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 2 Shi et al. Melatonin Regulates Apoptosis and Autophagy Brain Edema Measurement Both low and high doses of melatonin, compared with vehicle, appeared to alleviate brain edema (red pane) in rats that had undergone SAH. In addition, SAH rats, regardless of whether they received vehicle or melatonin, had larger lateral ventricles than sham rats (n = 6 for each group). FIGURE 2 \| Both low and high doses of exogenous melatonin alleviated brain edema as measured by magnetic resonance imaging (MRI). Both low and high doses of melatonin, compared with vehicle, appeared to alleviate brain edema (red pane) in rats that had undergone SAH. In addition, SAH rats, regardless of whether they received vehicle or melatonin, had larger lateral ventricles than sham rats (n = 6 for each group). FIGURE 2 \| Both low and high doses of exogenous melatonin alleviated brain edema as measured by magnetic resonance imaging (MRI). Both low and high doses of melatonin, compared with vehicle, appeared to alleviate brain edema (red pane) in rats that had undergone SAH. In addition, SAH rats, regardless of whether they received vehicle or melatonin, had larger lateral ventricles than sham rats (n = 6 for each group). Technology, CST#3682), active Caspase 3 (Abcam, ab49822), Bcl-2 (Cell Signaling Technology, CST#2876), Bax (Santa Cruz Biotechnology, SC-493), Beclin 1 (Proteintech, 11306-1-AP), and LC3B-I/II (Cell Signaling Technology, CST#4108). 105◦C (dry weight). The brain water content was defined as follows: brain water content = (wet weight −dry weight)/wet weight × 100%. Immunoﬂuorescence Staining After SAH and sham rats were sacrificed at 24 h following endovascular perforation, brain tissue samples from around the basal cortical area on the injured side were obtained for assessment. Immediately, the total ROS level was measured with an ROS/RNS assay kit (Cell Biolabs Inc., San Diego, CA, USA), following the manufacturer’s instruction. After 24 h following SAH induction, rats were perfused with PBS followed by 4% paraformaldehyde. The whole brain was immersed in 4% paraformaldehyde for 24 h, and then transferred to 30% sucrose solution for dehydration. Subsequently, the brain samples were frozen and cut into coronal slices (slice thickness: 8 µm) with a cryostat microtome (Leica CM3050S-3-1-1, Bannockburn, IL, USA). The sections were incubated overnight at 4◦C with primary antibodies, including antibodies against NeuN (Abcam, ab177487 or ab104224) and LC3 (Cell Signaling Technology, CST#4108) followed by the appropriate fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). TUNEL staining was performed according to the protocol from the manufacturer (Roche Inc., Switzerland) as described in previous studies (Ying et al., 2016). The sections were visualized with a fluorescence microscope (LSM-710; Brain Edema Measurement (C) Garcia test (n = 18 for each group): garcia score decreased at 24 h after SAH induction. Both low and high doses of exogenous melatonin signiﬁcantly increased Garcia scores compared with those of the SAH+Vehicle group. (D) Brain water content (n = 6 for each group): Low-dose melatonin treatment had no effect on brain water content. High-dose melatonin treatment signiﬁcantly decreased brain water content in the left hemisphere, the right hemisphere, and the cerebellum. Error bars represented the mean ± standard deviation (SD). ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. FIGURE 1 \| Both low and high doses of exogenous melatonin alleviated brain edema and improved neurological function 24 h after subarachnoid hemorrhage (SAH) induction. (A) The rats were randomly divided into four groups: the Sham group, the SAH+Vehicle (1% ethanol in 1 mL of saline) group, the SAH+Melatonin low-dose (5 mg/kg) group, and the SAH+Melatonin high-dose (10 mg/kg) group (n = 18 for each group). (B) SAH grade (n = 18 for each group): similar SAH grades were observed among SAH rats. (C) Garcia test (n = 18 for each group): garcia score decreased at 24 h after SAH induction. Both low and high doses of exogenous melatonin signiﬁcantly increased Garcia scores compared with those of the SAH+Vehicle group. (D) Brain water content (n = 6 for each group): Low-dose melatonin treatment had no effect on brain water content. High-dose melatonin treatment signiﬁcantly decreased brain water content in the left hemisphere, the right hemisphere, and the cerebellum. Error bars represented the mean ± standard deviation (SD). ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 3 Shi et al. Melatonin Regulates Apoptosis and Autophagy FIGURE 2 \| Both low and high doses of exogenous melatonin alleviated brain edema as measured by magnetic resonance imaging (MRI). Both low and high doses of melatonin, compared with vehicle, appeared to alleviate brain edema (red pane) in rats that had undergone SAH. In addition, SAH rats, regardless of whether they received vehicle or melatonin, had larger lateral ventricles than sham rats (n = 6 for each group). FIGURE 2 \| Both low and high doses of exogenous melatonin alleviated brain edema as measured by magnetic resonance imaging (MRI). Both Low and High Doses of Melatonin Treatment Inhibited Brain ROS Accumulation, Neuronal Apoptosis and Neuronal Autophagy During the experimental period, no significant difference was observed in body weight, mean arterial blood pressure, or temperature among the experimental groups (data not shown). The total mortality within 24 h after surgery was 14.19% (21 of 148 rats). Mortality showed no significant difference among SAH groups: 23.33% (7 of 30 rats) in the SAH+Vehicle group, 22.92% (11 of 48 rats) in the SAH+Melatonin group, and 25.00% (3 of 12 rats) in the SAH+Melatonin+Chelerythrine group. In addition, a total of seven SAH rats were excluded for insufficient bleeding (SAH grade <8). Brain ROS content was significantly higher in SAH rats receiving vehicle than in sham rats (1.544 ± 0.1636 and 1.000 ± 0.1254, respectively; P < 0.01; Figure 3). SAH rats receiving either low- or high-dose melatonin showed a significant inhibition of brain ROS accumulation (1.272 ± 0.1659 for low-dose melatonin, 1.155 ± 0.1983 for high-dose melatonin, vs. 1.544 ± 0.1636 for SAH rats receiving vehicle; P < 0.05; Figure 3). At 24 h after SAH induction, TUNEL staining was used to detect neuronal apoptosis, and LC3 staining was used to examine neuronal autophagy. SAH rats receiving vehicle showed higher percentage of TUNEL-positive (32.54 ± 4.877% vs. 4.203 ± 0.9677%, P < 0.01; Figure 4) and LC3-positive (26.36 ± 4.357% vs. 3.771 ± 1.016%, P < 0.01; Figure 5) neurons than sham rats did. SAH rats receiving either low- or Western Blotting After SAH and sham rats were sacrificed at 24 h following endovascular perforation, brain tissue samples from around the basal cortical area on the injured side were obtained for assessment. Western blotting was performed as previously described (Pariente et al., 2016). Proteins were extracted from the brain tissue samples with RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies used were antibodies against MST1 (Cell Signaling Technology, Danvers, MA, USA, CST#3682), cl-MST1 (Cell Signaling March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 4 Melatonin Regulates Apoptosis and Autophagy Shi et al. Zeiss, Oberkochen, Germany). The percentage of TUNEL- positive or LC3-positive neurons was calculated in a blinded manner. Figure 1D). High-dose melatonin alleviated brain edema in the left hemisphere (79.56 ± 0.5259 vs. 81.01 ± 0.9270, P < 0.01; Figure 1D), the right hemisphere (79.21 ± 0.7091 vs. 80.22 ± 0.5824, P < 0.05; Figure 1D), and the cerebellum (79.65 ± 0.7151 vs. 80.61 ± 0.6580, P < 0.05; Figure 1D). Statistical Analysis Brain edema was assessed intravitally by MR imaging (Figure 2). SAH rats receiving vehicle showed a significant increase in brain edema compared with sham rats (Figure 2). Both low and high doses of melatonin appeared to alleviate brain edema compared with the amount in SAH rats receiving vehicle, but no statistical analysis was performed to test for this effect owing to the low resolution of 3-tesla MR scans (Figure 2). In addition, SAH rats, whether they received vehicle or melatonin, showed enlarged lateral ventricles compared to sham rats (Figure 2). All data were expressed as the mean ± standard deviation (SD). One-way ANOVA of the mean values followed by a Holm-Sidak test was performed for multiple groups. A Mann-Whitney U test was used for Garcia scores and SAH grading scores. The analyses were performed using SPSS version 22.0 (SPSS Inc.). Statistical significance was defined as P < 0.05. Melatonin Treatment Alleviated Brain Edema and Improved Neurological Function Two doses of melatonin (5 mg/kg and 10 mg/kg) were administered via intraperitoneal injection 2 h after SAH induction. No significant difference was observed in SAH scores among the SAH+Vehicle group (13.72 ± 2.244), the SAH+Melatonin low-dose group (13.61 ± 2.355), and the SAH+Melatonin high-dose group (14.11 ± 2.447) at 24 h following SAH induction (P > 0.05, Figures 1A,B). SAH rats receiving vehicle showed significantly worse neurological function than sham rats 24 h after SAH induction (Garcia scores: 12.67 ± 2.114 and 17.28 ± 1.074, respectively; P < 0.01; Figure 1C). Both low and high doses of melatonin therapy were associated with significant improvements in neurological function (Garcia score: 13.94 ± 1.626 for low-dose melatonin, 14.22 ± 2.290 for high-dose melatonin, vs. 12.67 ± 2.114 for SAH rats; P < 0.05; Figure 1C). FIGURE 3 \| Both low and high doses of exogenous melatonin reduced SAH-induced reactive oxygen species (ROS) levels in brain tissues. Brain ROS content showed a signiﬁcant increase in SAH rats receiving vehicle compared with sham rats. SAH rats receiving either low- or high-dose melatonin showed a signiﬁcant inhibition of brain ROS accumulation (n = 6 for each group). Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. The degree of edema in each excised brain was measured as brain water content. SAH rats receiving vehicle showed elevated proportions of brain water content in the left hemisphere (81.01 ± 0.9270 vs. 79.11 ± 0.5128, P < 0.01; Figure 1D), the right hemisphere (80.22 ± 0.5824 vs. 79.12 ± 0.6885, P < 0.05; Figure 1D), and the cerebellum (80.61 ± 0.6580 vs.79.53 ± 0.9520, P < 0.05; Figure 1D) than sham rats. Low-dose melatonin showed no effect on reducing brain edema in any part of the rat brain (P > 0.05, FIGURE 3 \| Both low and high doses of exogenous melatonin reduced SAH-induced reactive oxygen species (ROS) levels in brain tissues. Brain ROS content showed a signiﬁcant increase in SAH rats receiving vehicle compared with sham rats. SAH rats receiving either low- or high-dose melatonin showed a signiﬁcant inhibition of brain ROS accumulation (n = 6 for each group). Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. Frontiers in Molecular Neuroscience \| www.frontiersin.org Melatonin Treatment Alleviated Brain Edema and Improved Neurological Function Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehi oup. (B) Representative images of immunoﬂuorescent staining show the TUNEL-positive neuronal cells (n = 6 for each group). Scale bar: 100 µm. FIGURE 4 \| Both low and high doses of exogenous melatonin decreased neuronal cell apoptosis. (A) Neuronal cell apoptosis (n = 6 for each group): a signiﬁcant increase in neuronal cell apoptosis was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell apoptosis compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. (B) Representative images of immunoﬂuorescent staining show the TUNEL-positive neuronal cells (n = 6 for each group). Scale bar: 100 µm. Melatonin Treatment Alleviated Brain Edema and Improved Neurological Function FIGURE 3 \| Both low and high doses of exogenous melatonin reduced FIGURE 3 \| Both low and high doses of exogenous melatonin reduced SAH-induced reactive oxygen species (ROS) levels in brain tissues. Brain ROS content showed a signiﬁcant increase in SAH rats receiving vehicle compared with sham rats. SAH rats receiving either low- or high-dose melatonin showed a signiﬁcant inhibition of brain ROS accumulation (n = 6 for each group). Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. Frontiers in Molecular Neuroscience \| www.frontiersin.org March 2018 \| Volume 11 \| Article 93 5 Shi et al. Shi et al. al. Melatonin Regulates Apoptosis and Autopha GURE 4 \| Both low and high doses of exogenous melatonin decreased neuronal cell apoptosis. (A) Neuronal cell apoptosis (n = 6 for each group): a signiﬁcant ease in neuronal cell apoptosis was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell optosis compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle up. (B) Representative images of immunoﬂuorescent staining show the TUNEL-positive neuronal cells (n = 6 for each group). Scale bar: 100 µm. Melatonin Regulates Apoptosis and Autophagy IGURE 4 \| Both low and high doses of exogenous melatonin decreased neuronal cell apoptosis. (A) Neuronal cell apoptosis (n = 6 for each group): a signiﬁcant crease in neuronal cell apoptosis was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell poptosis compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle roup. (B) Representative images of immunoﬂuorescent staining show the TUNEL-positive neuronal cells (n = 6 for each group). Scale bar: 100 µm. gh-dose melatonin showed significant inhibition of neuronal Chelerythrine Abolished the Inhibitory GURE 4 \| Both low and high doses of exogenous melatonin decreased neuronal cell apoptosis. (A) Neuronal cell apoptosis (n = 6 for each group): a signiﬁcant crease in neuronal cell apoptosis was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell poptosis compared with the level in the SAH+Vehicle group. Chelerythrine Abolished the Inhibitory Effects of Melatonin on SAH-Induced Apoptosis and Autophagy high-dose melatonin showed significant inhibition of neuronal apoptosis (% of TUNEL-positive neurons: 17.95 ± 5.221% for low-dose melatonin, 13.74 ± 3.690% for high-dose melatonin, and 32.54 ± 4.877% for SAH rats receiving vehicle; P < 0.05; Figure 4) and neuronal autophagy (% of LC3-positive neurons: 20.92 ± 3.660% for low-dose melatonin, 14.97 ± 2.691% for high-dose melatonin, and 26.36 ± 4.357% for SAH rats, P < 0.05; Figure 5). high-dose melatonin showed significant inhibition of neuronal apoptosis (% of TUNEL-positive neurons: 17.95 ± 5.221% for low-dose melatonin, 13.74 ± 3.690% for high-dose melatonin, and 32.54 ± 4.877% for SAH rats receiving vehicle; P < 0.05; Figure 4) and neuronal autophagy (% of LC3-positive neurons: 20.92 ± 3.660% for low-dose melatonin, 14.97 ± 2.691% for high-dose melatonin, and 26.36 ± 4.357% for SAH rats, P < 0.05; Figure 5). Chelerythrine is an MST1 agonist that promotes neuronal apoptosis by increasing caspase-dependent cleavage of MST1. No significant difference was observed in SAH grade scores among SAH rats receiving vehicle (14.25 ± 2.179), SAH rats March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 6 Shi et al. Shi et al. Melatonin Regulates Apoptosis and Autophagy GURE 5 \| Both low and high doses of exogenous melatonin decreased neuronal cell autophagy. (A) Neuronal cell autophagy (n = 6 for each group): a signiﬁcant rease in neuronal cell autophagy was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell ophagy compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the H+Vehicle group. (B) Representative images of immunoﬂuorescent staining show the LC3-positive neuronal cells (n = 6 for each group). Scale bar: 50 µm. FIGURE 5 \| Both low and high doses of exogenous melatonin decreased neuronal cell autophagy. (A) Neuronal cell autophagy (n = 6 for each group): a signiﬁcant ncrease in neuronal cell autophagy was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell autophagy compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. (B) Representative images of immunoﬂuorescent staining show the LC3-positive neuronal cells (n = 6 for each group). Scale bar: 50 µm. Chelerythrine Abolished the Inhibitory Effects of Melatonin on SAH-Induced Apoptosis and Autophagy GURE 5 \| Both low and high doses of exogenous melatonin decreased neuronal cell autophagy. (A) Neuronal cell autophagy (n = 6 for each group): a signiﬁca rease in neuronal cell autophagy was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal c ophagy compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the FIGURE 5 \| Both low and high doses of exogenous melatonin decreased neuronal cell autophagy. (A) Neuronal cell autophagy (n = 6 for each group): a signiﬁcant increase in neuronal cell autophagy was observed in the SAH+Vehicle group. Both low and high doses of melatonin treatment signiﬁcantly decreased neuronal cell autophagy compared with the level in the SAH+Vehicle group. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. (B) Representative images of immunoﬂuorescent staining show the LC3-positive neuronal cells (n = 6 for each group). Scale bar: 50 µm. Figure 6C). In addition, SAH rats receiving vehicle showed a significant increase in the percentages of TUNEL-positive (32.49 ± 5.601% vs. 4.623 ± 1.485%, P < 0.01; Figure 6D) and LC3-positive (24.13 ± 2.430% vs. 4.187 ± 0.9042%, P < 0.01; Figure 6D) neurons compared with sham rats; melatonin treatment inhibited this SAH-induced neuronal apoptosis (17.81 ± 3.304% vs. 32.49 ± 5.601%, P < 0.01; Figure 6D) and autophagy (17.41 ± 1.522% vs. 24.13 ± 2.430%, P < 0.01; Figure 6D); and chelerythrine reversed this receiving melatonin (13.67 ± 1.875), and SAH rats receiving melatonin plus chelerythrine (14.08 ± 2.539) at 24 h after SAH induction (P > 0.05, Figures 6A,B). SAH rats receiving vehicle had worse neurological function than sham rats (12.75 ± 2.261 vs. 16.83 ± 1.337, P < 0.01; Figure 6C); melatonin improved the SAH-induced neurologic deficit (14.58 ± 2.234 vs. 12.75 ± 2.261, P < 0.05; Figure 6C); and chelerythrine reversed this neuroprotective effect of melatonin (12.67 ± 1.826 vs. 14.58 ± 2.234, P < 0.05; March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 7 Shi et al. Melatonin Regulates Apoptosis and Autophagy FIGURE 6 \| The effects of melatonin on SAH-induced brain injury were reversed by chelerythrine. Chelerythrine Abolished the Inhibitory Effects of Melatonin on SAH-Induced Apoptosis and Autophagy (A) The rats were randomly divided into four groups: the Sham +Vehicle (1% ethanol in 1 mL of saline, intraperitoneal administration; dimethyl sulfoxide, intraventricular injection) group, the SAH+Vehicle (1% ethanol in 1 mL of saline, intraperitoneal administration; dimethyl sulfoxide, intraventricular injection) group, the SAH+Melatonin (10 mg/kg, intraperitoneal administration) + Vehicle (dimethyl sulfoxide, intraventricular injection) group, and the SAH+Melatonin (10 mg/kg, intraperitoneal administration) + Chelerythrine (1 mmol/L, 10 µL, intraventricular injection) group (n = 12 for each group). (B) SAH grade (n = 12 for each group): similar SAH grades were observed among SAH rats. (C) Garcia test (n = 12 for each group): Garcia scores were decreased 24 h after SAH induction. Melatonin could increase Garcia scores, and this effect was reversed by chelerythrine. (D) Percentage of TUNEL- or LC3-positive neurons (n = 12 for each group): SAH rats receiving vehicle showed a signiﬁcant increase in TUNEL- and LC3-positive neurons compared with sham rats. Melatonin treatment could inhibit this SAH-induced neuronal apoptosis and autophagy; chelerythrine reversed this neuroprotective effect of melatonin on both apoptosis and autophagy. Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. &P < 0.05 vs. the SAH+Melatonin group. FIGURE 6 \| The effects of melatonin on SAH-induced brain injury were reversed by chelerythrine. (A) The rats were randomly divided into four groups: the Sham +Vehicle (1% ethanol in 1 mL of saline, intraperitoneal administration; dimethyl sulfoxide, intraventricular injection) group, the SAH+Vehicle (1% ethanol in 1 mL of saline, intraperitoneal administration; dimethyl sulfoxide, intraventricular injection) group, the SAH+Melatonin (10 mg/kg, intraperitoneal administration) + Vehicle (dimethyl sulfoxide, intraventricular injection) group, and the SAH+Melatonin (10 mg/kg, intraperitoneal administration) + Chelerythrine (1 mmol/L, 10 µL, intraventricular injection) group (n = 12 for each group). (B) SAH grade (n = 12 for each group): similar SAH grades were observed among SAH rats. (C) Garcia test (n = 12 for each group): Garcia scores were decreased 24 h after SAH induction. Melatonin could increase Garcia scores, and this effect was reversed by chelerythrine. (D) Percentage of TUNEL- or LC3-positive neurons (n = 12 for each group): SAH rats receiving vehicle showed a signiﬁcant increase in TUNEL- and LC3-positive neurons compared with sham rats. Melatonin treatment could inhibit this SAH-induced neuronal apoptosis and autophagy; chelerythrine reversed this neuroprotective effect of melatonin on both apoptosis and autophagy. Chelerythrine Abolished the Inhibitory Effects of Melatonin on SAH-Induced Apoptosis and Autophagy 0.7237 ± 0.05824, P < 0.01; Figure 7B) and Bcl-2 (0.681 ± 0.1484 vs. 1.170 ± 0.2773, P < 0.01; Figure 7E). g Western blotting was performed to measure the expression of MST1, cl-MST1, Bcl-2, Bax, cleaved-Caspase 3, Beclin 1, and LC3B-I/II (Figure 7). SAH rats receiving vehicle showed a 77.69% decrease in the expression of MST1 (P < 0.01, Figure 7B), a 262.1% increase in the expression of cl-MST1 (P < 0.01, Figure 7C), a 255.9% increase in the expression of cleaved-Caspase 3 (P < 0.01, Figure 7D), an 84.86% decrease in the expression of Bcl-2 (P < 0.01, Figure 7E), a 249.6% increase in the expression of Bax (P < 0.01, Figure 7F), a 197.5% increase in the expression of Beclin 1 (P < 0.01, Figure 7G), and a 326.5% increase in the expression of LC3B-II (P < 0.01, Figure 7H). Melatonin treatment decreased the expression of MST1 (0.7237 ± 0.05824 vs. 0.2231 ± 0.05328, P < 0.01; Figure 7B) while increasing the expression of cl-MST1 (1.7832 ± 0.3882 vs. 2.6212 ± 0.3957, P < 0.01; Figure 7C) compared with SAH rats receiving vehicle. In addition, melatonin therapy inhibited apoptosis-related proteins including cleaved-Caspase 3 (1.632 ± 0.2888 vs. 2.559 ± 0.2470, P < 0.01; Figure 7D) and Bax (1.535 ± 0.8086 vs. 2.496 ± 0.4967, P < 0.05; Chelerythrine Abolished the Inhibitory Effects of Melatonin on SAH-Induced Apoptosis and Autophagy Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. &P < 0.05 vs. the SAH+Melatonin group. neuroprotective effect of melatonin on both apoptosis (23.16 ± 3.425% vs. 17.81 ± 3.304%, P < 0.05; Figure 6D) and autophagy (22.06 ± 3.727% vs. 17.41 ± 1.522%, P < 0.05; Figure 6D). Figure 7F) while up-regulating the anti-apoptotic protein Bcl-2 (1.170 ± 0.2773 vs. 0.3514 ± 0.1897, P < 0.01; Figure 7E). Moreover, melatonin treatment inhibited autophagy-related parameters including Beclin 1 protein (1.2358 ± 0.1900 vs. 1.975 ± 0.3259, P < 0.01; Figure 7G) and the expression of LC3B-II (3.165 ± 0.3465 vs. 7.029 ± 06811, P < 0.05; Figure 7H). These effects of melatonin on regulating apoptosis and autophagy were reversed by chelerythrine, showing a significant increase in the expression of cl-MST1 (2.342 ± 0.3552 vs. 1.7832 ± 0.3882, P < 0.05; Figure 7C), cleaved-Caspase 3 (2.182 ± 0.1672 vs. 1.632 ± 0.2888, P < 0.01; Figure 7D), Belin 1 (1.544 ± 0.2099 vs. 1.2358 ± 0.1900, P < 0.05; Figure 7G), and the expression of LC3B-II (5.800 ± 0.7068 vs. 3.165 ± 0.3465, P < 0.05; Figure 7H), while there was a significant decrease in the expression of MST1 (0.3874 ± 0.1183 vs. 0.7237 ± 0.05824, P < 0.01; Figure 7B) and Bcl-2 (0.681 ± 0.1484 vs. 1.170 ± 0.2773, P < 0.01; Figure 7E). Figure 7F) while up-regulating the anti-apoptotic protein Bcl-2 (1.170 ± 0.2773 vs. 0.3514 ± 0.1897, P < 0.01; Figure 7E). Moreover, melatonin treatment inhibited autophagy-related parameters including Beclin 1 protein (1.2358 ± 0.1900 vs. 1.975 ± 0.3259, P < 0.01; Figure 7G) and the expression of LC3B-II (3.165 ± 0.3465 vs. 7.029 ± 06811, P < 0.05; Figure 7H). These effects of melatonin on regulating apoptosis and autophagy were reversed by chelerythrine, showing a significant increase in the expression of cl-MST1 (2.342 ± 0.3552 vs. 1.7832 ± 0.3882, P < 0.05; Figure 7C), cleaved-Caspase 3 (2.182 ± 0.1672 vs. 1.632 ± 0.2888, P < 0.01; Figure 7D), Belin 1 (1.544 ± 0.2099 vs. 1.2358 ± 0.1900, P < 0.05; Figure 7G), and the expression of LC3B-II (5.800 ± 0.7068 vs. 3.165 ± 0.3465, P < 0.05; Figure 7H), while there was a significant decrease in the expression of MST1 (0.3874 ± 0.1183 vs. DISCUSSION The present study provided the first evidence that melatonin can regulate the homeostasis of apoptosis and autophagy. March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 8 Shi et al. Shi et al. Melatonin Regulates Apoptosis and Autophagy FIGURE 7 \| Western blots show the expression levels of mammalian sterile 20-like kinase 1 (MST1), cl-MST1, apoptosis-related proteins and autophagy-related proteins in response to melatonin treatment and chelerythrine administration. (A) Rats were randomly divided into four groups: the Sham group, the SAH+Vehicle group, the SAH+Melatonin group and the SAH+Melatonin+Chelerythrine group. SAH rats receiving vehicle showed the changes in the expression of MST1 (B), cl-MST1 (C), cleaved-Caspase 3 (D), Bcl-2 (E), Bax (F), Beclin 1 (G) and LC3B-II (H). Melatonin treatment attenuated the SAH-induced expressional changes in all these proteins (B,H), and this effect of melatonin could be reversed by chelerythrine (B,H). Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. &P < 0.05 vs. the SAH+Melatonin group. FIGURE 7 \| Western blots show the expression levels of mammalian sterile 20-like kinase 1 (MST1), cl-MST1, apoptosis-related proteins and autophagy-related proteins in response to melatonin treatment and chelerythrine administration. (A) Rats were randomly divided into four groups: the Sham group, the SAH+Vehicle group, the SAH+Melatonin group and the SAH+Melatonin+Chelerythrine group. SAH rats receiving vehicle showed the changes in the expression of MST1 (B), cl-MST1 (C), cleaved-Caspase 3 (D), Bcl-2 (E), Bax (F), Beclin 1 (G) and LC3B-II (H). Melatonin treatment attenuated the SAH-induced expressional changes in all these proteins (B,H), and this effect of melatonin could be reversed by chelerythrine (B,H). Error bars represented the mean ± SD. ∗P < 0.05 vs. the Sham group; #P < 0.05 vs. the SAH+Vehicle group. &P < 0.05 vs. the SAH+Melatonin group. (Martinez-Cruz et al., 2002; Ersahin et al., 2009; Wang et al., 2012a), inhibit neuroinflammation (Fang et al., 2009), and reduce cell death (Wang et al., 2012a), resulting in reduction of mortality (Ersahin et al., 2009), alleviation of brain edema (Wang et al., 2012a), and amelioration of functional defects (Ersahin et al., 2009; Fang et al., 2009) in several different SAH models. Oxidative stress, largely generated by SAH-induced oxyhemoglobin stimulation, is the direct cause of cell death in the pathogenesis of EBI (Caner et al., 2012). Frontiers in Molecular Neuroscience \| www.frontiersin.org DISCUSSION Melatonin Regulates Apoptosis and Autophagy FIGURE 8 \| The molecular mechanism potentially involved in the role of melatonin in regulating the balance between apoptosis and autophagy. (A) Under normal conditions, Bcl-2 binds with Bax to inhibit the Bax-induced formation of a channel that is permeable to cytochrome c to activate caspase-dependent apoptosis. In addition, MST1 present in the cell at baseline helps maintain cell survival by phosphorylating Beclin 1 to prevent the formation of Beclin 1-Atg14L-Vps34, which would inhibit autophagy and activate apoptosis. (B) When SAH occurs, the increased brain ROS content activates the cleavage of MST1 to produce cl-MST1, which is transferred into the nucleus and phosphorylates several histones, inducing neuronal cell apoptosis. In addition, SAH stress reduces the expression of Bcl-2 protein, resulting in the displacement of Bax from Bcl-2 to increase apoptosis. Meanwhile, both the decrease in Beclin 1 phosphorylation by MST1 and the reduced expression of Bcl-2 cause the dissociation of the Bcl-2/Beclin 1 complex, enhancing cell autophagy. FIGURE 8 \| The molecular mechanism potentially involved in the role of melatonin in regulating the balance between apoptosis and autophagy. (A) Under normal conditions, Bcl-2 binds with Bax to inhibit the Bax-induced formation of a channel that is permeable to cytochrome c to activate caspase-dependent apoptosis. In addition, MST1 present in the cell at baseline helps maintain cell survival by phosphorylating Beclin 1 to prevent the formation of Beclin 1-Atg14L-Vps34, which would inhibit autophagy and activate apoptosis. (B) When SAH occurs, the increased brain ROS content activates the cleavage of MST1 to produce cl-MST1, which is transferred into the nucleus and phosphorylates several histones, inducing neuronal cell apoptosis. In addition, SAH stress reduces the expression of Bcl-2 protein, resulting in the displacement of Bax from Bcl-2 to increase apoptosis. Meanwhile, both the decrease in Beclin 1 phosphorylation by MST1 and the reduced expression of Bcl-2 cause the dissociation of the Bcl-2/Beclin 1 complex, enhancing cell autophagy. 2002; Ersahin et al., 2009; Wang et al., 2012a), inhibiting neuroinflammation (Fang et al., 2009), and reducing neuronal apoptosis (Wang et al., 2012a). Thus, melatonin down-regulated autophagy in response to the improvement of the intercellular environment. chain to promote mitochondrial oxidative phosphorylation, inhibiting intrinsic apoptosis in hepatic cells (Reiter et al., 2010). However, the underlying mechanism whereby melatonin regulates autophagy remains largely unknown. DISCUSSION Compelling evidence has indicated that melatonin, as a powerful free radical scavenger, may regulate several cell death pathways, including apoptosis and autophagy (Chen et al., 2014). Many studies have confirmed that melatonin can modulate the expression of the Bcl-2 family genes, producing up-regulation of the anti-apoptotic Bcl-2 and down-regulation of the pro-apoptotic Bax (Wu et al., 2017). These previous findings were confirmed by our present study, showing significant up-regulation of Bcl-2 and down-regulation of Bax in SAH rats receiving melatonin compared to SAH rats receiving vehicle. In addition, melatonin acts at complexes I and IV in the respiratory Melatonin showed neuroprotective effects by alleviating brain edema and improving neurological function. These properties were associated with the suppression of SAH-induced neuronal apoptosis and autophagy. The most inspiring finding in the present study was the potential mechanism in which melatonin might reduce brain ROS content to inhibit the cleavage of MST1 to produce cl-MST1, resulting in suppression of caspase-dependent apoptosis and Beclin 1-induced autophagy. In addition, the effect of melatonin on the expression of Bcl-2 and Bax might also be involved in its regulatory control over the balance between apoptosis and autophagy. Melatonin showed neuroprotective effects by alleviating brain edema and improving neurological function. These properties were associated with the suppression of SAH-induced neuronal apoptosis and autophagy. The most inspiring finding in the present study was the potential mechanism in which melatonin might reduce brain ROS content to inhibit the cleavage of MST1 to produce cl-MST1, resulting in suppression of caspase-dependent apoptosis and Beclin 1-induced autophagy. In addition, the effect of melatonin on the expression of Bcl-2 and Bax might also be involved in its regulatory control over the balance between apoptosis and autophagy. Melatonin provided strong protection for experimental SAH rats against EBI in the present study, alleviating brain edema, reducing ROS damage, and improving neurological function. These findings were consistent with previous studies reporting that either a low dose (5 or 10 mg/kg; Ersahin et al., 2009; Fang et al., 2009) or high dose melatonin (150 mg/kg; Wang et al., 2012a) via subarachnoid (Martinez- Cruz et al., 2002) or intraperitoneal injection (Ersahin et al., 2009; Wang et al., 2012a) could attenuate oxidative stress March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org 9 Shi et al. Shi et al. Frontiers in Molecular Neuroscience \| www.frontiersin.org REFERENCES Dong, Y., Fan, C., Hu, W., Jiang, S., Ma, Z., Yan, X., et al. (2016). Melatonin attenuated early brain injury induced by subarachnoid hemorrhage via regulating NLRP3 inflammasome and apoptosis signaling. J. Pineal Res. 60, 253–262. doi: 10.1111/jpi.12300 Cahill, J., Calvert, J. W., Marcantonio, S., and Zhang, J. H. (2007). p53 may play an orchestrating role in apoptotic cell death after experimental subarachnoid hemorrhage. Neurosurgery 60, 531–545; discussion 545. doi: 10.1227/01.neu. 0000249287.99878.9b Ersahin, M., Toklu, H. Z., Cetinel, S., Yüksel, M., Yegen, B. C., and Sener, G. (2009). Melatonin reduces experimental subarachnoid hemorrhage-induced oxidative brain damage and neurological symptoms. J. Pineal Res. 46, 324–332. doi: 10.1111/j.1600-079x.2009.00664.x Cahill, J., Calvert, J. W., and Zhang, J. H. (2006). Mechanisms of early brain injury after subarachnoid hemorrhage. J. Cereb. Blood Flow Metab. 26, 1341–1353. doi: 10.1038/sj.jcbfm.9600283 Es,refogˇlu, M., Gül, M., Ates,, B., and Selimogˇlu, M. A. (2006). Ultrastructural clues for the protective effect of melatonin against oxidative damage in cerulein- induced pancreatitis. J. Pineal Res. 40, 92–97. doi: 10.1111/j.1600-079x.2005. 00288.x Caner, B., Hou, J., Altay, O., Fujii, M., and Zhang, J. H. (2012). Transition of research focus from vasospasm to early brain injury after subarachnoid hemorrhage. J. Neurochem. 123, 12–21. doi: 10.1111/j.1471-4159.2012. 07939.x Fang, Q., Chen, G., Zhu, W., Dong, W., and Wang, Z. (2009). Influence of melatonin on cerebrovascular proinflammatory mediators expression and oxidative stress following subarachnoid hemorrhage in rabbits. Mediators Inflamm. 2009:426346. doi: 10.1155/2009/426346 Carloni, S., Favrais, G., Saliba, E., Albertini, M. C., Chalon, S., Longini, M., et al. (2016). Melatonin modulates neonatal brain inflammation through endoplasmic reticulum stress, autophagy, and miR-34a/silent information regulator 1 pathway. J. Pineal Res. 61, 370–380. doi: 10.1111/jpi.12354 Fernández, A., Ordóñez, R., Reiter, R. J., González-Gallego, J., and Mauriz, J. L. (2015). Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis. J. Pineal Res. 59, 292–307. doi: 10.1111/jpi.12264 Chan, S. W., Lim, C. J., Chen, L., Chong, Y. F., Huang, C., Song, H., et al. (2011). The Hippo pathway in biological control and cancer development. J. Cell. Physiol. 226, 928–939. doi: 10.1002/jcp.22435 Galluzzi, L., Bravo-San Pedro, J. M., Blomgren, K., and Kroemer, G. (2016). Autophagy in acute brain injury. Nat. Rev. Neurosci. 17, 467–484. doi: 10.1038/nrn.2016.51 Chen, J., Wang, L., Wu, C., Hu, Q., Gu, C., Yan, F., et al. (2014). Melatonin- enhanced autophagy protects against neural apoptosis via a mitochondrial pathway in early brain injury following a subarachnoid hemorrhage. J. Pineal Res. 56, 12–19. DISCUSSION Consistent with this theory, we found that the expression of cl-MST1, Bax, cleaved-Caspase 3, Beclin 1 and LC3B-II were dramatically increased, while the expression of MST1 and Bcl-2 was significantly decreased in rats after SAH. Melatonin showed its powerful free-radical scavenging capacity by reducing SAH-induced brain ROS accumulation, leading to suppression of MST1 to maintain the balance between apoptosis and autophagy. direct evidence to distinguish these two stages, previous studies indicated that formation of autophagosomes is predominated rather than degradation in the first 24 h after SAH (Lee et al., 2009). In conclusion, the present study provides the first evidence that melatonin alleviates SAH-induced brain injury including brain edema and functional defects by suppressing excessive neuronal apoptosis and autophagy. The underlying mechanism may, at least in part, involve the ROS-MST1 pathway. Further studies must explore the long-term effect of melatonin on SAH-induced brain injury by other potential pathways. Several limitations should be noted in the present study. First, we did not examine the interactions among Bcl-2, Bax, and Beclin 1, although the combinations of Bcl-2/Bax and Bcl- 2/Beclin 1 were examined in previous studies (Youle and Strasser, 2008; Maejima et al., 2013). Second, the present study focused only on the role of melatonin in the ROS-MST1 pathway. Although the effects of melatonin on regulating apoptosis and autophagy can be attenuated by chelerythrine, other molecular pathways may also participate in this regulatory action of melatonin. Finally, autophagy is a dynamic and complex process including three sequential steps: formation of autophagosomes, fusion of autophagosomes with lysosomes, and degradation (Galluzzi et al., 2016). The increased protein levels of LC3II can be observed in both formation and degradation stages (Galluzzi et al., 2016). Although the present study didn’t provide AUTHOR CONTRIBUTIONS JianminZ was the principal investigator. LS and SC designed the study and developed the analysis plan. LS, FL, JingweiZ and JY completed this experiment. FL and JingweiZ analyzed the data. LS and FL contributed to writing the article. KZ revised the manuscript and SC polished the language. FUNDING This study was funded by the Natural Science Foundation of Zhejiang Province (Y15H090022). DISCUSSION Dual regulatory effects of melatonin on autophagy have been reported in previous studies, with both inhibitory and stimulatory roles for melatonin in autophagy (Es,refogˇlu et al., 2006; Motilva et al., 2011; Coto-Montes et al., 2012). Our present study indicated that melatonin (10 mg/kg via intraperitoneal injection) suppressed SAH-induced autophagy at 2 h after SAH induction. However, Chen et al. (2014) found that melatonin treatment (150 mg/kg, intraperitoneal injection) enhanced autophagy and decreased apoptotic cell death in the filament perforation model of SAH. These contradictory findings reflect the complexity of autophagy. Autophagy is a highly dynamic processes in which damaged cellular components are degraded (Levine and Klionsky, 2004). Enhanced autophagy can accelerate the clearance of damaged organelles or cytosol, thus promoting cell survival (Graef and Nunnari, 2011). However, it should be noted that autophagy is a stress- responsive process that is highly activated under extreme conditions (Wang et al., 2012b). We believed that melatonin improved the intercellular environment through several actions, including attenuating oxidative stress (Martinez-Cruz et al., The most inspiring aspect of the present study was that it explored the regulatory mechanism controlling apoptosis vs. autophagy. The cellular kinase MST1 may be a critical molecule that regulates apoptosis and autophagy by altering the expression or association of Bcl-2, Bax, and Beclin 1 (Dhingra and Kirshenbaum, 2013). Under normal conditions (Figure 8A), Bcl-2 binds with Bax to inhibit the Bax-induced formation of a channel that is permeable to cytochrome c to activate caspase-dependent apoptosis (Cahill et al., 2007). In addition, MST1 present in the cell at baseline helps maintain cell survival by phosphorylating Beclin 1 to prevent the formation of Beclin 1-Atg14L-Vps34, which would inhibit autophagy and activate apoptosis (Maejima et al., 2013). When SAH occurs (Figure 8B), the increased brain ROS content activates the cleavage of MST1 to produce cl-MST1, which is transferred into the nucleus and phosphorylates several histones, leading to neuronal cell apoptosis (Cheung et al., 2003). In addition, SAH stress reduced the expression of Bcl-2 protein, with the result that Bax was displaced from Bcl-2 to increase apoptosis. Meanwhile, both the decreased phosphorylation of Beclin 1 by March 2018 \| Volume 11 \| Article 93 Frontiers in Molecular Neuroscience \| www.frontiersin.org Frontiers in Molecular Neuroscience \| www.frontiersin.org 10 Shi et al. Melatonin Regulates Apoptosis and Autophagy MST1 and the reduced expression of Bcl-2 caused dissociation of the Bcl-2/Beclin 1 complex, enhancing cell autophagy (Dhingra and Kirshenbaum, 2013). REFERENCES Activated autophagy pathway in experimental subarachnoid hemorrhage. Brain Res. 1287, 126–135. doi: 10.1016/j.brainres.2009.06.028 autophagy pathway in experimental subarachnoid hemorrhage. Brain Res. 1287, 126–135. doi: 10.1016/j.brainres.2009.06.028 Wang, Z., Shi, X. Y., Yin, J., Zuo, G., Zhang, J., and Chen, G. (2012b). Role of autophagy in early brain injury after experimental subarachnoid hemorrhage. J. Mol. Neurosci. 46, 192–202. doi: 10.1007/s12031-011-9575-6 Lehtinen, M. K., Yuan, Z., Boag, P. R., Yang, Y., Villén, J., Becker, E. B., et al. (2006). 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Pineal Res. 60, 55–64. doi: 10.1111/jpi.12288 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Reiter, R. J., Manchester, L. C., and Tan, D. X. (2010). Neurotoxins: free radical mechanisms and melatonin protection. Curr. Neuropharmacol. 8, 194–210. doi: 10.2174/157015910792246236 Copyright © 2018 Shi, Liang, Zheng, Zhou, Chen, Yu and Zhang. 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 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. Ura, S., Masuyama, N., Graves, J. D., and Gotoh, Y. 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https://openalex.org/W2161125276	https://europepmc.org/articles/pmc3759854?pdf=render	English	null	How relevant is social interaction in second language learning?	Frontiers in human neuroscience	2,013	cc-by	8,078	INTRODUCTION The complexity of this code is further increased by the fact that human communication entails much more than the simple cod- ing or decoding of linguistic utterances: For a communicative act to be effective, it is necessary for both the sender and receiver to understand the intentional state of a partner (Newman-Norlund et al., 2009; De Ruiter et al., 2010), an ability termed Theory of Mind (ToM) or mentalizing (Frith and Frith, 2006). The processes subtending ToM can be triggered by different contextual cues as long as they come from an agent (Frith and Frith, 2006); their function is to facilitate predictions about the others’ behavior via both verbal (Carruthers, 2002) and non-verbal (Noordzij et al., 2009; Willems et al., 2010) communication. An example of the latter case is reported in severe aphasic patients: although virtually unable to express themselves verbally, these patients are able to pass tests intended to speciﬁcally tackle their residual communicative abilities; for example, they are able to engage in intention recognition with a partner in a non verbal game requiring to signal the position of a speciﬁc target on a checker- board (Willems and Varley, 2010; Willems et al., 2011). Another example comes from normally developing infants: although they have not yet developed verbal language, they are able to use the caregiver’s gaze direction as a cue to orient attention; this behav- ior requires a proto-mentalizing ability to infer the caregiver’s intention and represents one of the ﬁrst communicative acts in children (Tomasello, 1995; Tomasello and Carpenter, 2007; Csibra and Gergely, 2009; see below). In adults mentalizing processes In his book “Pragmatics of human communication” (1967), the psychologist and philosopher Paul Watzlawic stated that it is impossible not to communicate (Watzlawick et al., 1967). Indeed, in his view, every behavior is a form of communication intended to convey a message from a sender to a receiver (Shannon and Weaver, 1963). The interaction between partners crucially deﬁnes a communicative intention: while a sender and a receiver do not have to be present at the same time or in the same place, there is no communication without one of the two partners. The interactive nature of this process is reﬂected by the word “communication”, meaning “share with someone”, “let someone know” (from the Latin cum—with—and munire—to bind together). Keywords: language, learning, social interaction, communication, joint attention INTRODUCTION However, the study of the most widespread vehicle of human communication, language, has so far suffered from an individualistic approach. Here, we review recent ﬁndings bridging social cognition and communication by highlighting evidence that points towards the necessity to consider the impact of social interaction when investigating second language (L2) learning. Laura Verga1* and Sonja A. Kotz1,2 Verbal language is the most widespread mode of human communication, and an intrinsically social activity. This claim is strengthened by evidence emerging from different ﬁelds, which clearly indicates that social interaction inﬂuences human communication, and more speciﬁcally, language learning. Indeed, research conducted with infants and children shows that interaction with a caregiver is necessary to acquire language. Further evidence on the inﬂuence of sociality on language comes from social and linguistic pathologies, in which deﬁcits in social and linguistic abilities are tightly intertwined, as is the case for Autism, for example. However, studies on adult second language (L2) learning have been mostly focused on individualistic approaches, partly because of methodological constraints, especially of imaging methods. The question as to whether social interaction should be considered as a critical factor impacting upon adult language learning still remains underspeciﬁed. Here, we review evidence in support of the view that sociality plays a signiﬁcant role in communication and language learning, in an attempt to emphasize factors that could facilitate this process in adult language learning. We suggest that sociality should be considered as a potentially inﬂuential factor in adult language learning and that future studies in this domain should explicitly target this factor. HUMAN NEUROSCIENCE REVIEW ARTICLE published: 03 September 201 doi: 10.3389/fnhum.2013.0055 How relevant is social interaction in second language learning? published: 03 September 2013 doi: 10.3389/fnhum.2013.00550 Frontiers in Human Neuroscience REVIEW ARTICLE published: 03 September 2013 doi: 10.3389/fnhum.2013.00550 REVIEW ARTICLE published: 03 September 2013 doi: 10.3389/fnhum.2013.00550 Correspondence: Correspondence: Laura Verga, Department of Neuropsychology, Research group Subcortical Contributions to Comprehension, Max Planck Institute for Human Cognitive and Brain Sciences, Stephanstrasse 1a, 04103 Leipzig, Germany e-mail: verga@cbs.mpg.de How relevant is social interaction in second language learning? Laura Verga1* and Sonja A. Kotz1,2 1 Department of Neuropsychology, Research group Subcortical Contributions to Comprehension, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany 2 School of Psychological Sciences, The University of Manchester, Manchester, UK Edited by: Edited by: Srikantan S. Nagarajan, University of California, San Francisco, USA Reviewed by: Mikko E. Sams, Helsinki University of Technology, Finland Caroline A. Niziolek, University of California, San Francisco, USA *Correspondence: Laura Verga, Department of Neuropsychology, Research group Subcortical Contributions to Comprehension, Max Planck Institute for Human Cognitive and Brain Sciences, Stephanstrasse 1a, 04103 Leipzig, Germany e-mail: verga@cbs.mpg.de Srikantan S. Nagarajan, University of California, San Francisco, USA Srikantan S. Nagarajan, University of California, San Francisco, USA Reviewed by: Mikko E. Sams, Helsinki University of Technology, Finland Caroline A. Niziolek, University of California, San Francisco, USA Reviewed by: Mikko E. Sams, Helsinki University of Technology, Finland Caroline A. Niziolek, University of California, San Francisco, USA HUMAN COMMUNICATION AND THE ROLE OF SOCIAL INTERACTION Important for the topic of this review, these “mirror” neurons deal with the decoding of an action goal not only when one is performing an action, but also when observing the same action being performed by someone else (Rizzolatti and Fabbri-Destro, 2008; Keysers and Gazzola, 2009). These neurons thus provide an interface between one’s own motor repertoires and others’ (Knoblich and Sebanz, 2006). This “goal-sharing” property sup- ports the hypothesis that brain areas exhibiting mirror-like prop- erties should be more active during joint action than during solitary actions (Newman-Norlund et al., 2007). Although these “fake” social interactive tasks allow this hypothesis to be indirectly tested, recent developments in neuroimaging have allowed the creation of new techniques to be applied to fMRI (Montague et al., 2002), EEG (Astolﬁet al., 2010, 2011), and NIRS (Cui et al., 2012), enabling two (and sometimes more) people to be tested at the same time. These “hyper-scanning” techniques (Dumas et al., 2011) allow ecologically valid interactions to be studied in a number of tasks, which could then also be applied to interactive learning paradigms. The clear advantage is that they allow a direct comparison of processes happening in two brains at the same time, a comparison which could otherwise only be inferred. Thus, one could potentially observe both the effects of mentalizing (King-Casas et al., 2005; Astolﬁet al., 2010; Saito are activated by cues such as the identity of the person they are interacting with. In a recent study, Newmann-Norlund and col- leagues demonstrated that in a non-verbal communicative task, adult participants adapted their communicative behavior to the presumed cognitive abilities of the partner. In the employed task participants had to communicate to a partner the spatial location of a target on a checkerboard by moving a token to the position of the target; they were told that the partner could either be an adult or a child. When they were prone to believe they were interacting with a child, participants spent more time moving the cursor, thus emphasizing a crucial element of communication such as the target location (Newman-Norlund et al., 2009). When the partner is a peer, adults still adapt their behavior; in most of the cases, this adaptation is reciprocal and results in behavioral resemblance between the partners. HUMAN COMMUNICATION AND THE ROLE OF SOCIAL INTERACTION AN COMMUNICATION AND THE ROLE OF SOCIAL Human language is one of the most complex codes used to communicate between individuals. In its verbal form it is based on a small subset of sounds that can be combined in a potentially inﬁnite number of bigger elements (words, phrases, sentences). September 2013 \| Volume 7 \| Article 550 \| 1 Frontiers in Human Neuroscience www.frontiersin.org Social interaction in second language learning Verga and Kotz the limitation that dual settings pose to imaging set-ups. Luckily, the inﬂuence of an interactive social approach has increased exponentially over the last decade (Knoblich and Sebanz, 2006, 2008; Galantucci and Sebanz, 2009; Schilbach et al., 2013), leading to an attempt to ﬁnd new techniques and to create experimental situations tailored towards real-life situations often involving more than one person (Montague et al., 2002; Hasson et al., 2012). This effort has lead to the development of paradigms intended to speciﬁcally tackle social situations (Schippers et al., 2010; Anders et al., 2011), in which participants are often made to believe that they are interacting with someone. For example, pairs of participants may be required to take turns in the fMRI scanner while observing a video recording of the partner during meaningful gestural (Schippers et al., 2009, 2010; Redcay et al., 2010) or affective (Anders et al., 2011) communication, while they believe this interaction is happening in real time. These kinds of “fake” communicative situations have allowed researchers to observe in-vivo activations in brain areas involved in the ToM system. This is supported by a network encompassing the medial prefrontal cortex (mPFC), the posterior superior temporal sulcus (pSTS), the temporo-parietal junction (TPJ), and the temporal poles (TP) (Amodio and Frith, 2006; Frith and Frith, 2006; Saxe, 2006; Decety and Lamm, 2007; Newman-Norlund et al., 2007; Noordzij et al., 2009). Another system usually involved in “social” tasks is the human Mirror Neuron System (MNS). This system encompasses a fronto-parietal network of the ventral premotor cortex (vPMC), the inferior frontal gyrus (IFG), and the inferior parietal lobule (iPL) in its rostral portion (Rizzolatti and Craighero, 2004), and possibly other regions, including the dorsal premotor cortex (dPMC), the supplementary motor cortex (SMA), and the temporal lobe (Keysers and Gazzola, 2009). HUMAN COMMUNICATION AND THE ROLE OF SOCIAL INTERACTION For example, pairs of adults tend to coor- dinate their body postures and gaze patterns during conversation, even without being aware of it (Shockley et al., 2007, 2009), and reduce the variability of their actions to better synchronize with each other (Vesper et al., 2011, 2012). Another example is the tendency to share feelings and emotions of others, often leading to the mimicry of an observed emotion (de Vignemont and Singer, 2006; Singer, 2006). An immediate evolutionary advantage of these phenomena is to facilitate learning mechanisms based on observation and imitation (Frith and Frith, 2012). However, how do these coordinative and imitative phenomena inﬂuence language? First of all, effective communication is based on the ability to know when it is the right moment to speak. This turn taking ability relies on general coordinative rules, both on the side of motor coordination (Shockley et al., 2009), and on the side of conversation. For example, you do not want your partner to wait forever for an answer, but you also do not want to speak while he is still speaking (“minimal gap, minimal overlap” rule, Stivers et al., 2009). Furthermore, aspects of a conversation, such as the speaking rate and the similarity of words spoken in a dyad, also inﬂuence the coordinative pattern as demonstrated by Shockley et al. (2007). The authors showed that pairs of participants were maximally synchronized in their bodily movements when they were uttering the same words at the same time (Shockley et al., 2007). Even more importantly, imitative motor phenomena are inﬂuenced by the conceptual level of the conversation: for exam- ple, hand gestures in a conversation are likely to be imitated and repeated by the partners, but only if they make sense in the context of the speech (Mol et al., 2012). Taken together, this evidence suggests that there is a two- way inﬂuence between social interaction and communication Taken together, this evidence suggests that there is a two- way inﬂuence between social interaction and communication. However, the role played by social interaction has been greatly undervalued so far, especially in studies on language learning, even though this context represents a prototypical interactive communicative situation. In the following sections, we will ﬁrst describe technical limitations that may have been responsible for such paucity in research; then we highlight evidence on the impact of social interaction on learning in clinical and non- clinical populations. Frontiers in Human Neuroscience BRAIN IMAGING IN INTERACTING INDIVIDUALS: ISSUES AND SOLUTIONS Probably one of the reasons why social interaction has not been considered as a factor in language learning studies until recently is September 2013 \| Volume 7 \| Article 550 \| 2 Frontiers in Human Neuroscience www.frontiersin.org Social interaction in second language learning Verga and Kotz Hari and Kujala, 2009; Syal and Finlay, 2011). This asymmetrical learning setting, in which knowledge is passed from parents to offspring, is not limited to humans and can be found, for example, in many bird species that use complex vocal codes to communicate (Kuhl, 2007; Hari and Kujala, 2009; Frith and Frith, 2012). However, ToM abilities underlying human communication seem to represent a unicum in nature. Indeed, even our closer animal relatives, the chimpanzees, do not have the human ability to really “share” intentionality: as an example, chimpanzees are perfectly able to follow the gaze of an interacting human, but they do not try to start joint attention, nor do they try to infer the referent of the gaze as human children do (Tomasello and Carpenter, 2007). This human ability to share intentionality and acquired knowledge with other humans has been proposed to be at the core of the evolution of verbal language (Tomasello, 1995; Pinker, 2010). A series of experiments conducted by Kuhl and colleagues aimed to investigate this possibility and to test the impact of social interaction on phonetic discrimination in children (Kuhl et al., 2003; Kuhl, 2007). Cohorts of American infants were exposed to native speakers of Mandarin Chinese and subsequently performed a phonetic discrimination task; the exposure either occurred via direct interaction or via pre-recorded video tapes. Interestingly, infants were able to learn different Mandarin phonemes when they were exposed to them by a real person, but not when the exposure was merely via a recording (Kuhl et al., 2003). There are two plausible explanations for this effect; ﬁrst, a live human may attract more attention and increase motivation, as compared to a recording. Second, a real person can provide referential information, crucial for linking words and concepts (Waxman and Gelman, 2009). In particular, Kuhl and colleagues pointed out that joint attention towards an object being named can facilitate a child’s capacity for word segmentation (Kuhl et al., 2003). Similarly, results from Hirotani et al. (2009) suggest that joint attention helps to strengthen the association between a word and its referent, thus facilitating learning. BRAIN IMAGING IN INTERACTING INDIVIDUALS: ISSUES AND SOLUTIONS These authors found that semantic integration, reﬂected in the N400 effect, seemed to be present when children learn new words in a joint attention condition but not in a non-joint-attention context. Although infant learning represents a particular case, vocabulary learning poses similar demands to both children—learning their ﬁrst language (L1)—and to adults—when learning a new lan- guage. Thus, factors facilitating word learning in children could potentially impact adult learners in a similar way. is inﬂuenced by social interaction, but also their brain activation patterns. Indeed, synchronized EEG activity in frontal and central regions has been found in theta and delta oscillations of pairs of guitarists playing a melody together (Lindenberger et al., 2009); similarly, when pairs of participants are required to spontaneously imitate each others, their brain activity becomes synchronized in the alpha-mu band over right-centro-parietal regions (Dumas et al., 2010). Activity in this frequency band has been proposed to represent a neuromarker of human social coordination and, more speciﬁcally, has been linked to the human MNS (Tognoli et al., 2007). Saito et al. (2010) used fMRI hyper-scanning to scan two people at the same time while they were engaged in a real-time gaze exchange; that is to say, the pair were asked to direct one anothers’ attention to an object via eye movements. The authors found that the exchange of attention via eye gaze resulted in an inter-subject synchronization of the neural activity in the right IFG (Saito et al., 2010). Mentalizing and mirror systems thus seem to be recruited in social tasks (Uddin et al., 2007; Van Overwalle, 2008; Van Overwalle and Baetens, 2009; Ciaramidaro et al., 2013), but their activity is inﬂuenced by the presence of a partner. Thus, the question arises: what happens in the case of learning a new language? A ﬁrst attempt to answer this question arises from a recent study by Jeong and colleagues; the authors suggest that when words in a novel language are learnt in a social situation (but not when they are learnt from a text), elicited brain activity (in the right supramarginal gyrus) is similar to the activity elicited by words in one’s mother tongue (Jeong et al., 2010). However, the social situation depicted in this study was represented by movie clips of a dialogue. Thus, the question remains: what happens in a natural (social) learning situation? Frontiers in Human Neuroscience LANGUAGE LEARNING AND SOCIAL INTERACTION IN CHILDREN As previously pointed out, the ability to socially interact emerges very early in life (Grossmann and Johnson, 2007), and is repre- sented by a number of basic interactions that children in the ﬁrst year of life are able to master, such as following the caregivers’ gaze, attracting her/his attention, and responding to her/his atten- tional requests. This set of abilities is usually grouped under the name “joint attention”, entailing an interaction between a child, the caregiver, and the focus of attention (an object) (Carpenter et al., 1998; Mundy et al., 2003; Mundy and Sigman, 2006; Mundy and Newell, 2007; Mundy and Jarrold, 2010). From a psycho- logical point of view, the role of triadic attention ability during childhood is to create a common psychological ground shared between the infant and the caregiver, and relies on the formation of ToM in children (Tomasello, 1995). In this common space, adults act as experts and guide the children toward the relevant information that should be learnt, by using an effective signal such as eye gaze (Csibra and Gergely, 2009; De Jaegher et al., 2010). In this asymmetrical learning setting, children behavior is further facilitated by the fact that adults tend to adapt their communica- tive behavior by emphasizing crucial aspects of communication (for example, by spending more time on them; Newman-Norlund et al., 2009). Moreover, the interaction with the caregiver increases motivation, thus reinforcing a given behavior (Vrtiˇcka et al., 2008; Frontiers in Human Neuroscience THE ROLE OF SOCIALITY IN SECOND LANGUAGE LEARNING Evidence thus accumulates to favor the view that the development of verbal language is, at least, supported by establishing common ground between a sender and a receiver. In turn, the events that take place in such common space are mostly dependent on the interaction between the partners (Mundy and Jarrold, 2010). However, a note of caution needs to be used when comparing language learning in children and in adults. Indeed, learning of a L2 can occur largely independent of the presence of another person, and is usually learnt via explicit formal training as com- pared to a L1, which is acquired effortlessly without explicit instructions (Abutalebi, 2008). Nevertheless, the case of word learning represents a link between language learning in infants September 2013 \| Volume 7 \| Article 550 \| 3 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Social interaction in second language learning Verga and Kotz and in adults. Indeed, words in a new language can be acquired incidentally (Nagy et al., 1987; Swanborn and De Glopper, 1999; Laufer and Hulstijn, 2001; Rodríguez-Fornells et al., 2009); new words encountered while reading a text can be easily learnt. In this situation an adult learner has to face the same problems as an infant, namely the indetermination of the referents: there are multiple words in a language and multiple possible referents in terms of meaning. However, how can the correct meaning be assigned to an unknown word? The easiest way to go about this problem is exempliﬁed by associative learning, a procedure that concentrates on the statistical learning of the co-occurrence of data from speech and its context (Breitenstein et al., 2004; Whiting et al., 2007, 2008). The advantage of this procedure is that it poses low cognitive demands during training (Pulvermüller, 1999; Dobel et al., 2010) and is resistant to errors made during a phase of guessing (Carpenter et al., 2012). The underlying rationale is that once a word is heard in an utterance or seen in a sentence, a set of potential meanings can be inferred from the context, thus reducing the number of possible referents (Adelman et al., 2006). This way, novel word forms can be acquired and integrated in the lexicon relatively quickly and successfully. For instance, neural responses evoked after training are indistinguishable from those obtained in response to “old” words, as demonstrated in the disappearance or reduction of a N400 response (Mestres- Missé et al., 2007). THE ROLE OF SOCIALITY IN SECOND LANGUAGE LEARNING The N400 component is a negative deﬂection starting 200–300 ms after the presentation of a word, and has been associated with semantic processing (Lau et al., 2008). Its disappearance in a learning paradigm thus possibly corresponds to establishing a link between a novel lexeme and conceptual information (Mestres-Missé et al., 2007; Dobel et al., 2010). The neural network supporting word learning involves regions of the semantic circuitry such as the left IFG (BA 45), the mid- dle temporal gyrus (MTG, BA 21), the parahippocampal gyrus, and several subcortical structures (Mestres-Missé et al., 2008). Although, in adults, new vocabulary can be learnt independently of the presence of a partner, social interaction may increase the number of cues and referential information in much the same way as it does in infant learning (Kuhl, 2004, 2007, 2010). Indeed, the interaction between partners in conversation could lead L2 learn- ers to focus on certain aspects of the context and certain words in speech (Yu and Ballard, 2007). The coordinative phenomena we describe above could play a role in this process, maximizing the efﬁciency of the conversation and consequently facilitating the focusing of attention: this proposal has been made for word learning in toddlers. Indeed, it has been shown that in toddler- adult dyads, the number of new words learnt by the toddlers is proportional to the quality of the synchronization during the interaction with the caregiver (Pereira et al., 2008). Again, it is important to note that the case of word learning is not dissimilar in adults and infants, and so one may expect facilitating factors (such as the focusing of attention driven by synchronization) to play a role in word learning for both adults and children Indeed the necessity to consider sociality as a factor in L2 studies seems striking, as further suggested by the evidence that when new words are encoded in a social context, but not when they are learnt by translation, the pattern of activation in the retrieval phase is similar to the one observed for L1 words (Jeong et al., 2010). and in adults. Indeed, words in a new language can be acquired incidentally (Nagy et al., 1987; Swanborn and De Glopper, 1999; Laufer and Hulstijn, 2001; Rodríguez-Fornells et al., 2009); new words encountered while reading a text can be easily learnt. THE ROLE OF SOCIALITY IN SECOND LANGUAGE LEARNING In this situation an adult learner has to face the same problems as an infant, namely the indetermination of the referents: there are multiple words in a language and multiple possible referents in terms of meaning. However, how can the correct meaning be assigned to an unknown word? The easiest way to go about this problem is exempliﬁed by associative learning, a procedure that concentrates on the statistical learning of the co-occurrence of data from speech and its context (Breitenstein et al., 2004; Whiting et al., 2007, 2008). The advantage of this procedure is that it poses low cognitive demands during training (Pulvermüller, 1999; Dobel et al., 2010) and is resistant to errors made during a phase of guessing (Carpenter et al., 2012). The underlying rationale is that once a word is heard in an utterance or seen in a sentence, a set of potential meanings can be inferred from the context, thus reducing the number of possible referents (Adelman et al., 2006). This way, novel word forms can be acquired and integrated in the lexicon relatively quickly and successfully. For instance, neural responses evoked after training are indistinguishable from those obtained in response to “old” words, as demonstrated in the disappearance or reduction of a N400 response (Mestres- Missé et al., 2007). The N400 component is a negative deﬂection starting 200–300 ms after the presentation of a word, and has been associated with semantic processing (Lau et al., 2008). Its disappearance in a learning paradigm thus possibly corresponds to establishing a link between a novel lexeme and conceptual information (Mestres-Missé et al., 2007; Dobel et al., 2010). The neural network supporting word learning involves regions of the semantic circuitry such as the left IFG (BA 45), the mid- dle temporal gyrus (MTG, BA 21), the parahippocampal gyrus, and several subcortical structures (Mestres-Missé et al., 2008). Although, in adults, new vocabulary can be learnt independently of the presence of a partner, social interaction may increase the number of cues and referential information in much the same way as it does in infant learning (Kuhl, 2004, 2007, 2010). Indeed, the interaction between partners in conversation could lead L2 learn- ers to focus on certain aspects of the context and certain words in speech (Yu and Ballard, 2007). THE ROLE OF SOCIALITY IN SECOND LANGUAGE LEARNING The coordinative phenomena we describe above could play a role in this process, maximizing the efﬁciency of the conversation and consequently facilitating the focusing of attention: this proposal has been made for word learning in toddlers. Indeed, it has been shown that in toddler- adult dyads, the number of new words learnt by the toddlers is proportional to the quality of the synchronization during the interaction with the caregiver (Pereira et al., 2008). Again, it is important to note that the case of word learning is not dissimilar in adults and infants, and so one may expect facilitating factors (such as the focusing of attention driven by synchronization) to play a role in word learning for both adults and children. Indeed, although it is possible to learn a new language alone, adults often learn a new language in social contexts, most commonly in a teacher—learner setting, a setting which requires interaction with a partner as well as sophisticated reading of a speaker’s intentions (Bloom, 2002; Mestres-Missé et al., 2007, 2008). Thus, LEARNING AND SOCIAL COGNITION IN PATHOLOGIES Learning new words, or re-learning words that have been forgot- ten, is the goal not only for infants and L2 learners, but also for pathological populations, including, for example people suffering from autism, dementias, or aphasia. In these pathologies, the role of social interaction is becoming increasingly acknowledged as a crucial variable for therapeutic outcome success. Communica- tive deﬁcits in autism spectrum disorders have been frequently attributed to higher cognitive processing impairments, and espe- cially to ToM deﬁcits (Baron-Cohen et al., 1985). However, more recent evidence indicates that lower level processes may also be affected. For instance, recent ﬁndings suggest that autistic chil- dren display low-level difﬁculties in temporal processing, includ- ing impaired timing and deﬁcits in the perceived duration of an event, which can in turn inﬂuence the perception of relevant social cues such as eye gaze (Allman, 2011; Allman et al., 2011; Falter and Noreika, 2011; Falter et al., 2012). The fact that ToM and timing abilities may be crucial for language, even in a popu- lation who display impaired ToM, comes from the discovery that autistic children improve their language abilities after a treatment focusing on the optimization of their joint attention capacities (Kasari et al., 2008). Similarly, social interaction plays a role in language re-learning in aphasia. A paradigmatic example of this claim comes from a speciﬁc form of therapy for severe aphasic patients based on music, namely Melodic Intonation Therapy (Norton et al., 2009). This approach uses musical and sensory stimulation in order to improve the speech production of the aphasic patient and is centered on the role of the therapist. Although the beneﬁcial effect of the therapy has been traditionally attributed to the effect of music tout-court, recent evidence challenges this perspective and suggests that rhythm (and not necessarily melody) holds the key to understanding the impact of music therapy (Stahl et al., 2011). Considering that music therapy is therapist-centered, this result well ﬁts a joint-action explanation: rhythm is deﬁned by the coordinated action between a therapist and a patient. This strongly inﬂuences timing and its variability of the single individual in the interaction. 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Neuroim- age 59, 2430–2437. doi: 10.1016/j. neuroimage.2011.09.003 Keysers, C., and Gazzola, V. (2009). CONCLUDING REMARKS l h l f In conclusion, the role of social interaction in language learning has, thus far, been widely overlooked, partly because of technical constraints posed by interactive settings in imaging studies. We propose that further studies on language learning in adults should further explore the powerful impact of social interaction. This necessity comes from at least four lines of research: ﬁrst, language use intended as communication is an interactive phenomenon, relying on the ability of the partners to infer the others’ mental states and to coordinate with each other in successful turn- September 2013 \| Volume 7 \| Article 550 \| 4 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Social interaction in second language learning Verga and Kotz and a student. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). September 2013 \| Volume 7 \| Article 550 \| 7 Frontiers in Human Neuroscience REFERENCES The use, distribution or reproduction in other forums is permitted, provided the origi- nal author(s) or licensor are credited and that the original publication in this jour- nal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Yu, C., and Ballard, D. H. (2007). A uniﬁed model of early word learn- ing: integrating statistical and social cues. Neurocomputing 70, 2149– 2165. doi: 10.1016/j.neucom.2006. 01.034 Whiting, E., Chenery, H. J., Chalk, J., Darnell, R., and Copland, D. A. (2008). The explicit learning of new names for known objects is improved by dexamphetamine. 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https://openalex.org/W1506867680	https://www.frontiersin.org/articles/10.3389/fnbeh.2015.00195/pdf	English	null	Social defeat-induced anhedonia: effects on operant sucrose-seeking behavior	Frontiers in behavioral neuroscience	2,015	cc-by	11,965	Social defeat-induced anhedonia: effects on operant sucrose-seeking behavior Danai Riga 1, J. Trisna Theijs 1, Taco J. De Vries 1, 2, August B. Smit 1 and Sabine Spijker 1* 1 Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, Netherlands, 2 Department of Anatomy and Neurosciences, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, Netherlands Reduced capacity to experience pleasure, also known as anhedonia, is a key feature of the depressive state and is associated with poor disease prognosis and treatment outcome. Various behavioral readouts (e.g., reduced sucrose intake) have been employed in animal models of depression as a measure of anhedonia. However, several aspects of anhedonia are poorly represented within the repertoire of current preclinical assessments. We recently adopted the social defeat-induced persistent stress (SDPS) paradigm that models a maintained depressive-like state in the rat, including social withdrawal and deﬁcits in short-term spatial memory. Here we investigated whether SDPS elicited persistent deﬁcits in natural reward evaluation, as part of anhedonia. We examined cue-paired operant sucrose self-administration, enabling us to study acquisition, motivation, extinction, and relapse to sucrose seeking following SDPS. Furthermore, we addressed whether guanfacine, an α2-adrenergic agonist that reduces stress-triggered maladaptive behavioral responses to drugs of abuse, could relief from SDPS-induced anhedonia. SDPS, consisting of ﬁve social defeat episodes followed by prolonged (≥8 weeks) social isolation, did not affect sucrose consumption during acquisition of self-administration. However, it strongly enhanced the motivational drive to acquire a sucrose reward in progressive ratio training. Moreover, SDPS induced initial resilience to extinction and rendered animals more sensitive to cue-induced reinstatement of sucrose-seeking. Guanfacine treatment attenuated SDPS-induced motivational overdrive and limited reinstatement of sucrose seeking, normalizing behavior to control levels. Together, our data indicate that long after the termination of stress exposure, SDPS induces guanfacine-reversible deﬁcits in evaluation of a natural reward. Importantly, the SDPS-triggered anhedonia reﬂects many aspects of the human phenotype, including impaired motivation and goal-directed conduct. ORIGINAL RESEARCH published: 07 August 2015 doi: 10.3389/fnbeh.2015.00195 ORIGINAL RESEARCH published: 07 August 2015 doi: 10.3389/fnbeh.2015.00195 Keywords: social defeat-induced persistent stress (SDPS), anhedonia, depression, sucrose self-administration, guanfacine Edited by: George W. Huntley, Icahn School of Medicine at Mount Sinai, USA Reviewed by: A. J. Robison, Michigan State University, USA Dan Christoffel, Stanford University, USA Correspondence: Sabine Spijker, Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, De Boelelaan 1085, 1081 HV Amsterdam, Netherlands s.spijker@vu.nl Reviewed by: A. J. Robison, Michigan State University, USA Dan Christoffel, Stanford University, USA Reviewed by: A. J. Robison, Michigan State University, USA Dan Christoffel, Stanford University, USA Correspondence: Sabine Spijker, Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, De Boelelaan 1085, 1081 HV Amsterdam, Netherlands s.spijker@vu.nl ORIGINAL RESEARCH published: 07 August 2015 doi: 10.3389/fnbeh.2015.00195 Introduction As a result, depressed patients show defective decision-making and inappropriate behavioral adjustments in face of emotionally charged events (Cella et al., 2010). Conventionally, these deﬁcits are linked to dysfunctional brain circuitry responsible for decision-making, attribution of incentive salience, behavioral reinforcement and expression of motivated behavior (e.g., via the prefrontal cortex and striatum) (Russo and Nestler, 2013). One of the most prominent characteristics of depressive disorders is a diminished ability to experience pleasure, a mental state traditionally termed as anhedonia. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM, 5th Edition), anhedonia, or reduced interest in engagement to otherwise rewarding activities, such as socialization and sexual intercourse, is a key feature of Major Depressive Disorder (MDD) diagnosis (American Psychiatric Association, 2013). The anhedonic phenotype can occur in presence or in absence of mood-related depressive symptoms, e.g., feelings of sadness or helplessness, and it can persist beyond their recovery (Schrader, 1997). Furthermore, in MDD, anhedonia is strongly associated with predicted severity and persistence of the disorder, as well as poor treatment outcomes (Spijker et al., 2001; Vrieze et al., 2013). Anhedonia falls in the category of pathologies of the aﬀective domain, and might be largely explained by eﬀects of dysfunctional processing of reinforcing information on mood and cognition. Indeed, depressed individuals suﬀer cognitive deﬁcits predominantly related to the aﬀective domain (Eshel and Roiser, 2010). In particular in MDD, information processing associated with reward is aberrant, with negative stimuli triggering maladaptive responses, and positive information making only small impact. As a result, depressed patients show defective decision-making and inappropriate behavioral adjustments in face of emotionally charged events (Cella et al., 2010). Conventionally, these deﬁcits are linked to dysfunctional brain circuitry responsible for decision-making, attribution of incentive salience, behavioral reinforcement and expression of motivated behavior (e.g., via the prefrontal cortex and striatum) (Russo and Nestler, 2013). With respect to maladaptive processing of reward-associated information, we recently developed a ﬁrst preclinical model of comorbidity between primary depression and secondary alcohol abuse disorder. SDPS resulted in increased motivation for alcohol and elevated susceptibility to relapse to alcohol- seeking (Riga et al., 2014). Following up on these studies, we questioned here whether the eﬀects of SDPS on reward evaluation are speciﬁc to alcohol- and alcohol-signifying cues, or whether they reﬂect global alterations in the reward-processing system. Citation: Riga D, Theijs JT, De Vries TJ, Smit AB and Spijker S (2015) Social defeat-induced anhedonia: effects on operant sucrose-seeking behavior. Front. Behav. Neurosci. 9:195. doi: 10.3389/fnbeh.2015.00195 August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 1 Depression and sucrose-associated anhedonia Riga et al. Introduction To this end, we investigated whether SDPS induces an anhedonia-like phenotype by examining its consequences not only regarding consumption, but also with respect to motivation, extinction, and reinstatement toward a non-drug reward, i.e., sweetened water. Furthermore, by employing a pharmacological intervention in the form of a systemic guanfacine administration, previously shown to reverse SDPS-eﬀects on alcohol-seeking (Riga et al., 2014), we aimed to disentangle possible neuronal pathways of depression-induced impairments in motivation and reinforcement. Emerging revisited concepts of anhedonia (Der-Avakian and Markou, 2012) pose an important question for preclinical models of depression in terms of construct validity, beyond simplistic assessments of consummatory approach, e.g., sucrose intake. Similarly, in light of gaining insight into aﬀected brain circuitry and underlying molecular mechanisms, it is imperative to extend the current anhedonia-parameters’ applicability to theories conceptualizing human anhedonia. Previous assessments of the magnitude of anhedonia-like behavior in rodents were based on measurements of preference for a given reward, such as sweetened solutions, without taking into account the contributions of motivational aspects. In particular, sucrose preference-based behavioral readouts fail to dissect reward-related learning, subsequent retention of pleasure- coding information and reward-based decision-making (Ho and Sommers, 2013), unlike operant reward paradigms (Nielsen et al., 2000; Donahue et al., 2014). Introduction the maintenance of the depressive-like state. However, by itself, isolation during adulthood does not induce behavioral or physiological hallmarks of depression (Ruis et al., 1999; Fone and Porkess, 2008; Riga et al., 2014). Using the SDPS model, we and others have established a maintained depressive state in the rat, in which antidepressant-reversible behavioral and neurobiological hallmarks of the human disorder, such as aﬀective and cognitive deﬁcits, reduced neurogenesis, and aberrant physiology of the hippocampus, are present several months after social defeat (Reijmers et al., 2001; Artola et al., 2006; van Bokhoven et al., 2011; Riga et al., 2014). Focusing on such a maintained depressive state, rather than the short-lasting eﬀects of initial stress exposure, has the advantage of modeling the enduring characteristics of human depression. Whereas indicators of acute stress, such as elevated corticosterone levels (van Bokhoven et al., 2011) are absent in this model, in the weeks following social defeat rats gradually develop a sensitization to heterotypic stressors (Buwalda et al., 2005). This is exempliﬁed by increased responsiveness of the HPA axis (Buwalda et al., 1999) and impaired social approach-avoidance lasting up to 6 months (Riga et al., 2014). One of the most prominent characteristics of depressive disorders is a diminished ability to experience pleasure, a mental state traditionally termed as anhedonia. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM, 5th Edition), anhedonia, or reduced interest in engagement to otherwise rewarding activities, such as socialization and sexual intercourse, is a key feature of Major Depressive Disorder (MDD) diagnosis (American Psychiatric Association, 2013). The anhedonic phenotype can occur in presence or in absence of mood-related depressive symptoms, e.g., feelings of sadness or helplessness, and it can persist beyond their recovery (Schrader, 1997). Furthermore, in MDD, anhedonia is strongly associated with predicted severity and persistence of the disorder, as well as poor treatment outcomes (Spijker et al., 2001; Vrieze et al., 2013). Anhedonia falls in the category of pathologies of the aﬀective domain, and might be largely explained by eﬀects of dysfunctional processing of reinforcing information on mood and cognition. Indeed, depressed individuals suﬀer cognitive deﬁcits predominantly related to the aﬀective domain (Eshel and Roiser, 2010). In particular in MDD, information processing associated with reward is aberrant, with negative stimuli triggering maladaptive responses, and positive information making only small impact. Frontiers in Behavioral Neuroscience \| www.frontiersin.org Animals and Social Defeat-induced Persistent Stress (SDPS) Paired-housed male Wistar rats (Harlan CPB, Horst, Netherlands) 6–7 weeks old, weighing <200 g upon arrival were habituated to the facility (2 weeks), and then were exposed to SDPS (Riga et al., 2014) followed by operant sucrose self- administration (SA) (Figure 1A). During the SDPS paradigm, residents (male Long-Evans, Charles River, UK) were paired- housed with age-matched tube-ligated females (Wistar, Harlan) in order to promote territorial behavior and aggression. The female Wistar and all cage enrichment were removed from the residents’ cage before defeat commenced. During the SDPS paradigm, Wistar rats (n = 8) were exposed to ﬁve daily defeat sessions with the resident, according to the resident-intruder Over the years, we adopted an animal paradigm that mimics a sustained depressive-like state in rats, the so-called social defeat- induced persistent stress (SDPS) model. The SDPS paradigm employs an etiologically valid stressor, i.e., social stress in the form of acute social defeat, followed by long-term social isolation (2–3 months) in the absence of chronic sensory interaction with the stressor (Von Frijtag et al., 2000). Social isolation is a necessary component of the SDPS paradigm (De Jong et al., 2005), serving as a sub-threshold stressor that supports August 2015 \| Volume 9 \| Article 195 2 Depression and sucrose-associated anhedonia Riga et al. the test arena (plastic, opaque, 79×57 × 42 cm) for at least 10 min during 3 consecutive days. Animals were subjected to the Social Approach-Avoidance (SAA) and the Object Place Recognition (OPR) tasks 8 weeks following the last defeat session (Figure 1A). FIGURE 1 \| SDPS induces deﬁcits in the cognitive and the affective domain. (A) SDPS animals were exposed to 5 daily defeat sessions, immediately followed by social isolation (single-housing). Eight weeks following the last defeat episode, Social Approach Avoidance (SAA), and Object Place Recognition tests were employed to assess the effects of SDSP on the affective and cognitive domain. Animals were then subjected to a 7-weeks long operant sucrose self-administration paradigm. (B) Whereas both control and SDPS rats spent the majority of time interacting with the unfamiliar social target at the SAA test, SDPS triggered avoidance behavior, indicated by the signiﬁcantly reduced interaction index as compared to controls. (C) SPDS rats failed to retain the position of the displaced object in the short-term spatial memory OPR test, in contrast to controls, which spent the majority of time exploring the object in its new location. Cue-paired Operant Sucrose Self-administration Cue-paired Operant Sucrose Self-administration Following assessment of the depressive-like state, training toward Cue-paired Operant Sucrose Self-administration Following assessment of the depressive-like state, training toward the acquisition of cue-paired operant sucrose self-administration (SA) commenced (Figure 2A). Based on previous studies with alcohol (Riga et al., 2014), a slightly modiﬁed protocol for sucrose SA was used, omitting home-cage taste familiarization. Social Approach-Avoidance (SAA) Social Approach Avoidance (SAA) SDPS-induced deﬁcits in social behavior were determined by the SAA test, using an unfamiliar Long-Evans adult male rat (resident) (as adopted by Golden et al., 2011). During test day, Wistar rats were habituated to the testing arena (5 min). A sample phase followed, in which two empty target boxes (TBs, perforated metal, 16 × 7 × 8 cm) were placed on opposite arena walls, and general activity and explorative behavior was measured (5 min). The sample phase was immediately followed by the test phase, in which an unfamiliar resident was introduced to one TB; Wistar rats were then allowed to freely explore and approach either of the TBs (5 min). Approach-avoidance behavior (interaction index) was calculated as time spent in active zone (resident zone)/total exploration time (resident + neutral zone). Active and inactive zones were randomly assigned, to avoid development of preference. FIGURE 1 \| SDPS induces deﬁcits in the cognitive and the affective domain. (A) SDPS animals were exposed to 5 daily defeat sessions, FIGURE 1 \| SDPS induces deﬁcits in the cognitive and the affective domain. (A) SDPS animals were exposed to 5 daily defeat sessions, immediately followed by social isolation (single-housing). Eight weeks following the last defeat episode, Social Approach Avoidance (SAA), and Object Place Recognition tests were employed to assess the effects of SDSP on the affective and cognitive domain. Animals were then subjected to a 7-weeks long operant sucrose self-administration paradigm. (B) Whereas both control and SDPS rats spent the majority of time interacting with the unfamiliar social target at the SAA test, SDPS triggered avoidance behavior, indicated by the signiﬁcantly reduced interaction index as compared to controls. (C) SPDS rats failed to retain the position of the displaced object in the short-term spatial memory OPR test, in contrast to controls, which spent the majority of time exploring the object in its new location. P < 0.050 for Student’s t-test (gray); dotted gray line indicates the 50% preference index. Animals and Social Defeat-induced Persistent Stress (SDPS) P < 0.050 for Student’s t-test (gray); dotted gray line indicates the 50% preference index. Fixed Ratio (FR) Rats were trained to nose-poke for a 0.20 mL 12% sucrose reward in 1-h sessions provided every day. Sucrose delivery (US) was accompanied by discrete audiovisual stimuli (CS, 4- s active hole illumination and tone presentation). Initially, a continuous reinforcement (ﬁxed ratio 1, FR1) schedule was implemented, in which each reward delivery was followed by a 15-s time-out period, during which nose poking has no Assessment of Depressive Symptomatology Before participation in any behavioral measurement, all animals were transferred to the video-recording room and habituated to Object Place Recognition (OPR) j g ( ) Hippocampal-dependent short-term memory was determined with an object place recognition test (Dere et al., 2007) using a 15-min retention interval. Animals were habituated to the test arena as described above. During the sample phase, two identical objects (cylinders or cubes, metal, 8 × 8 × 35 cm) were placed in two opposite corners of the arena, and animals were allowed to explore (5 min). In the test-phase, the previously presented objects were replaced with two identical ones, and one of the objects was displaced to a diﬀerent corner. Discrimination between the spatial locations of the two objects was used as measurement for spatial memory [exploration index = time spent in novel location/total exploration time (novel + familiar location)] in a 4-min test. The position of novel and familiar locations and object shapes were randomly assigned to avoid development of preference. immediately followed by social isolation (single-housing). Eight weeks following the last defeat episode, Social Approach Avoidance (SAA), and Object Place Recognition tests were employed to assess the effects of SDSP on the affective and cognitive domain. Animals were then subjected to a 7-weeks long operant sucrose self-administration paradigm. (B) Whereas both control and SDPS rats spent the majority of time interacting with the unfamiliar social target at the SAA test, SDPS triggered avoidance behavior, indicated by the signiﬁcantly reduced interaction index as compared to controls. (C) SPDS rats failed to retain the position of the displaced object in the short-term spatial memory OPR test, in contrast to controls, which spent the majority of time exploring the object in its new location. P < 0.050 for Student’s t-test (gray); dotted gray line indicates the 50% preference index. protocol. During the pre- and post-phases of each defeat session (5 min each), the Wistar rat was positioned in the Long Evans home-cage, however, the resident had no access to the intruder due to placement of a transparent, perforated plastic partition- wall. The ﬁght-phase (5 min) was initiated and terminated by respectively, removing or replacing the partition wall. For each defeat session, intruders were matched to a diﬀerent resident. At defeat days, control animals (n = 7) were transferred to the residents’ holding room and allowed to explore an empty defeat cage for 15 min. Object Place Recognition (OPR) From the ﬁrst defeat session/cage exposure onwards, all animals were single-housed and remained in social isolation for the rest of the experimental conditions, in absence of further sensory interaction with the stressor (residents). All animals were housed in humidity/temperature-controlled rooms (50%/21 ± 1◦C). Food and water were available ad libitum for the whole experimental period. All experimental manipulations were conducted during the dark phase of a reversed 12-h light-dark cycle (lights on at 19.00 h). All experiments were approved by the VU University Amsterdam Animal Users Care Committee. Assessment of Depressive Symptomatology Before participation in any behavioral measurement, all animals were transferred to the video-recording room and habituated to Frontiers in Behavioral Neuroscience \| www.frontiersin.org 3 protocol. During the pre- and post-phases of each defeat session (5 min each), the Wistar rat was positioned in the Long Evans home-cage, however, the resident had no access to the intruder due to placement of a transparent, perforated plastic partition- wall. The ﬁght-phase (5 min) was initiated and terminated by respectively, removing or replacing the partition wall. For each defeat session, intruders were matched to a diﬀerent resident. At defeat days, control animals (n = 7) were transferred to the residents’ holding room and allowed to explore an empty defeat cage for 15 min. From the ﬁrst defeat session/cage exposure onwards, all animals were single-housed and remained in social isolation for the rest of the experimental conditions, in absence of further sensory interaction with the stressor (residents). All animals were housed in humidity/temperature-controlled rooms (50%/21 ± 1◦C). Food and water were available ad libitum for the whole experimental period. All experimental manipulations were conducted during the dark phase of a reversed 12-h light-dark cycle (lights on at 19.00 h). All experiments were approved by the VU University Amsterdam Animal Users Care Committee. All video recordings were analyzed with Viewer2 software (BiObserve GmbH, Bonn, Germany). Approach/avoidance behavior (SAA) and retention of spatial memory (OPR) were based on animals’ performance at the ﬁrst minute of each test. Statistical Analyses y All statistics were performed using SPSS (version 15.0, IBM) and data are presented as mean ± SEM. All behavioral data collected from SAA and OPR tests were analyzed using One-Way analysis of variance (ANOVA) with defeat as the between-subjects factor. Similarly, data collected during sucrose SA, including PR, relapse and guanfacine administration, were analyzed using mixed ANOVAs with defeat as the between-subjects factor and session (or, if applicable, treatment) as the within-subjects factor. When P-values reached level of signiﬁcance (P < 0.05), further analysis was performed using One-Way ANOVA, paired or unpaired (post-hoc) Student’s t-test. Homogeneity and equality of variance were estimated and Hyunh-Feldt or Levene’s test corrections were implemented in case of assumption violation. All interaction or exploration indexes (SAA, OPR) were calculated based on animals’ performance during the 1st minute of the test phase of each task. For the aforementioned tasks, preference indices (interaction or exploration) were based on a ﬁctive group showing no discrimination, while retaining the variation of the tested sample (Akkerman et al., 2012). programmed consequences. Responding on the inactive hole was monitored, but had no consequences. When FR1 performance reached stable levels (Criteria: (1) >50 rewards per 1-h session; (2) no statistically signiﬁcant diﬀerences between the last 2 FR sessions, as assessed using repeated measures ANOVA), animals were introduced to FR2 and subsequently FR3 training schedules. Consolidation of sucrose SA was estimated by peak performance at FR3 (491 ± 39 active responses/1-h session for controls and 496 ± 40 for SDPS). Animals were trained under FR schedules for a total of 3 weeks. SDPS Induces Deﬁcits in the Affective and Cognitive Domains Eight weeks following the last defeat episode we assessed the development of the depressive-like state on both aﬀective and cognitive domains (Figure 1A). To examine the eﬀects of SDPS on social behavior, the social approach-avoidance (SAA) test was Assessment of Depressive Symptomatology Before participation in any behavioral measurement, all animals were transferred to the video-recording room and habituated to August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 3 Depression and sucrose-associated anhedonia Riga et al. subsequent analysis of extinction performance. Animals were provided with 2 daily 1-h FR1 training sessions before the onset of extinction training. Extinction consisted of 1-h exposure to the training context in absence of sucrose and sucrose-associated cues. Following nine daily sessions, operant responding was successfully extinguished as reﬂected in rats reaching <5% of group-average FR1 responding by the last extinction session. All animals participated in a 30-min cue-induced reinstatement session, at the start of which a single 0.20 mL sucrose reward was delivered. Nose poking during the session resulted in presentation of the discrete compound audiovisual cues (but no sucrose reward) on an FR1 schedule. Reinstatement of sucrose seeking was calculated based on animals’ performance during the last extinction session. FIGURE 2 \| SDPS does not affect acquisition of sucrose SA nor sucrose consumption. (A) Animals participated at the operant sucrose self-administration (SA) paradigm, including 3 weeks of FR1–3 training schedules, 1 week of PR, 2 weeks of extinction and ﬁnally the relapse tests; FR1–3 training (B,C) is highlighted. (B) All animals increased responding to the sucrose delivery-associated hole, as function of the FR schedules examined, indicating consolidation of the task. SDPS had no effect on acquisition of sucrose SA, since SDPS and controls performed identical under all three ratios assessed. (C) No difference in the number of rewards obtained during acquisition of sucrose SA between SDPS and control rats were observed. Notably, both groups stabilized their sucrose intake after the introduction of FR2 training schedule, with a group average of ∼100 rewards/session, corresponding to ∼5 g/kg of sucrose. Progressive Ratio (PR) Animals were subjected to eight daily 2-h progressive ratio (PR) sessions, during which the eﬀort (number of nose-pokes) to obtain a reward was progressively increased according to: response ratio = (5e(0.2 ∗reward number)) −5, rounded to the nearest integer (Richardson and Roberts, 1996). Each session automatically ended when no reward was delivered (no FR was reached) within an hour. All animals completed the 2-h training sessions, independently of group. Guanfacine Administration Guanfacine-HCl [N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride] was tested during PR sessions ﬁve and eight, given 3 days apart, as well as on cue-induced reinstatement in two separate tests, given at a 4-day interval without additional extinction training. Saline (1 mL/kg) or guanfacine (0.5 mg/kg dissolved in saline) were systemically (i.p.) administered 1 h before the session/test in a cross-over design. FIGURE 2 \| SDPS does not affect acquisition of sucrose SA nor sucrose consumption. (A) Animals participated at the operant sucrose self-administration (SA) paradigm, including 3 weeks of FR1–3 training schedules, 1 week of PR, 2 weeks of extinction and ﬁnally the relapse tests; FR1–3 training (B,C) is highlighted. (B) All animals increased responding to the sucrose delivery-associated hole, as function of the FR schedules examined, indicating consolidation of the task. SDPS had no effect on acquisition of sucrose SA, since SDPS and controls performed identical under all three ratios assessed. (C) No difference in the number of rewards obtained during acquisition of sucrose SA between SDPS and control rats were observed. Notably, both groups stabilized their sucrose intake after the introduction of FR2 training schedule, with a group average of ∼100 rewards/session, corresponding to ∼5 g/kg of sucrose. All self-administration procedures took place in MED Associates INC R⃝(St. Albans, VT, USA) operant behavior chambers, surrounded by sound-attenuating cubicles. Data were collected using the MED-PC software package. SDPS Increases Motivation for a Natural Reward Acquisition of Operant Sucrose Self-administration SDPS Increases Motivation for a Natural Reward Acquisition of Operant Sucrose Self-administration Animals were subjected to seven FR1, three FR2 and ﬁve FR3 training sessions (Figure 2A). Overall, SDPS did not aﬀect responding for a sucrose reward under any of the schedules investigated (Figure 2B). All animals learned to discriminate between the active and the inactive hole from the ﬁrst training session onwards [Con, t(6) = 2.80, P = 0.031 at FR1 session 4; SDPS, t(7) = 3.27, P = 0.014 at FR1 session 3]. In FR1 schedule, repeated measures ANOVA revealed a signiﬁcant eﬀect of training on active responding [F(3.67, 47.75) = 17.36, P < 0.001], as all animals increased performance over time. No eﬀect of group × training was observed, indicating similar FR1 acquisition in both controls and SDPS: F(3.67, 47.75) = 0.88, P = 0.474. Indeed, no between-group diﬀerences in responding were found: F(1, 13) = 0.75, P = 0.402. Similarly during FR2, an overall increase in the number of active responses was observed [F(2, 26) = 8.97, P = 0.001], which was independent of group: group × training, F(2, 26) = 0.04, P = 0.958. As with FR1, no between-group diﬀerences in acquisition were observed in FR2: F(1, 13) = 0.04, P = 0.850. FR3 further increased animals responding on the active hole over time [F(4, 52) = 7.03, P < 0.001], independently of group: group × training, F(4, 52) = 1.88, P = 0.128. As with preceding training schedules, no signiﬁcant diﬀerences between controls and defeated animals were found: F(1,13) = 0.03, P = 0.858. As guanfacine, an α2-adrenergic agonist, has been shown to reverse SDPS eﬀects on PR-responding for alcohol (Riga et al., 2014), we questioned whether it would also be beneﬁcial against the increased motivation for sucrose intake. Guanfacine was administered in two separate PR sessions (PR sessions 5 and 8), in a cross-over design (Figure 3D). Repeated measures ANOVA revealed that pretreatment with guanfacine (GUA), 1 h before the session, reduced active responding for sucrose independently of defeat, compared with saline (SAL) treated animals (Figure 3E, Supplementary Figure 2): treatment, F(1, 13) = 11.44, P = 0.005; group × treatment, F(1, 13) = 2.26, P = 0.157. Importantly, a signiﬁcant group eﬀect was observed [F(1, 13) = 12.96, P = 0.003], as SDPS rats showed enhanced performance at both PR sessions. Extinction and Reinstatement The day after the last PR session, all animals were re-trained to FR1 to minimize between-group diﬀerences that could aﬀect August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 4 Depression and sucrose-associated anhedonia Riga et al. between-group diﬀerences were observed (Supplementary Figure 1B). Taken together, the FR data showed that SDPS did not alter acquisition of sucrose self-administration nor sucrose intake as compared with controls. employed (Figure 1B). All animals showed willingness to interact with the social target: Con, t(6) = 14.42, P < 0.001; SDPS, t(7) = 4.23, P = 0.004 vs. ﬁctional). However, defeated rats exhibited a signiﬁcant decrease in interaction time as compared with controls: t(12.39) = 2.61, P = 0.022. Similarly, cognitive performance was impaired in the SDPS group, as assessed using the object place recognition (OPR) test (Figure 1C). In particular, SDPS animals displayed deﬁcits in retention of an object’s spatial location when compared with controls: t(13) = 2.54, P = 0.025. Taken together, these results pointed to the establishment of a depressive-like state that persists over time and that mimics core phenotypic manifestations of the human disorder (Austin et al., 2001; Millan et al., 2012). SDPS Increases Motivation for a Natural Reward Acquisition of Operant Sucrose Self-administration Guanfacine eﬀects did not carry-over in- between treatment days, as a similar group eﬀect observed prior to guanfacine treatment on PR responding was present when analyzing sessions after guanfacine treatment [PR6–7: F(1, 13) = 7.57, P = 0.017]. Progressive Ratio (PR) and Guanfacine Treatment Progressive Ratio (PR) and Guanfacine Treatment The PR training schedule was introduced to examine SDPS- triggered diﬀerences in motivation towards sucrose acquisition (Figure 3A). Repeated measures ANOVA over the last 2 PR sessions before the ﬁrst guanfacine challenge (PR3–4) showed no training eﬀect [F(1, 13) = 0.05, P = 0.831], nor a training × group interaction [F(1, 13) = 0.65, P = 0.433], indicating stabilization of PR performance. Notably, a signiﬁcant group eﬀect was observed [F(1, 13) = 6.68, P = 0.023], as SDPS animals showed enhanced responding for sucrose when compared with controls (Figure 3B). Similar to active responses, break points, calculated based on the highest FR completed (average of treatment- free sessions PR3–4 and PR6–7), were signiﬁcantly increased following SDPS [F(1, 14) = 6.15, P = 0.028] pointing toward heightened motivation to acquire sucrose (Figure 3C). Extinction of Operant Sucrose Self-administration (A) The motivation to acquire a sucrose reward was examined in 6 treatment-free PR training sessions (highlighted; B,C). (B) SDPS increased responding during both the pre- (PR3–4) and post- (PR6–7) treatment sessions, and no carry-over effects of guanfacine treatment were detected (see E). (C) Break points, depicted as the maximum FR completed when averaging over the four treatment-free sessions, conﬁrmed the SDPS-triggered exaggeration of motivational drive in defeated animals. (D) The effect of guanfacine on PR responding was assessed at PR sessions ﬁve and eight using a cross-over treatment administration design (highlighted, E). (E) Guanfacine administration reduced overall PR responding. Independently of treatment regime, the SDPS group showed signiﬁcantly increased number of responses as compared with controls. P < 0.050; *P < 0.010; (B): repeated ANOVA (group effect); (C): One-Way ANOVA (group effect); (E): repeated ANOVA (treatment effect). FIGURE 3 \| SDPS triggers motivational overdrive toward a sucrose reward; guanfacine attenuates enhanced motivation. (A) The motivation to acquire a sucrose reward was examined in 6 treatment-free PR training sessions (highlighted; B,C). (B) SDPS increased responding during both the pre- (PR3–4) and post- (PR6–7) treatment sessions, and no carry-over effects of guanfacine treatment were detected (see E). (C) Break points, depicted as the maximum FR completed when averaging over the four treatment-free sessions, conﬁrmed the SDPS-triggered exaggeration of FIGURE 3 \| SDPS triggers motivational overdrive toward a sucrose reward; guanfacine attenuates enhanced motivation. (A) The motivational drive in defeated animals. (D) The effect of guanfacine on PR responding was assessed at PR sessions ﬁve and eight using a cross-over treatment administration design (highlighted, E). (E) Guanfacine administration reduced overall PR responding. Independently of treatment regime, the SDPS group showed signiﬁcantly increased number of responses as compared with controls. P < 0.050; P < 0.010; (B): repeated ANOVA (group effect); (C): One-Way ANOVA (group effect); (E): repeated ANOVA (treatment effect). motivation to acquire a sucrose reward was examined in 6 treatment-free PR training sessions (highlighted; B,C). (B) SDPS increased responding during both the pre- (PR3–4) and post- (PR6–7) treatment sessions, and no carry-over effects of guanfacine treatment were detected (see E). (C) Break points, depicted as the maximum FR completed when averaging over the four treatment-free sessions, conﬁrmed the SDPS-triggered exaggeration of carry-over effects of guanfacine treatment were detected (see E). Extinction of Operant Sucrose Self-administration Extinction of Operant Sucrose Self-administration Following PR, all animals were re-trained under FR1 schedule (reFR1), in order to restore similar between-group performance before proceeding with extinction training (Figure 4A). Already from the ﬁrst reFR1 session, SDPS and control rats showed identical responding, at a similar level as during FR acquisition (Supplementary Figure 3). This was repeated at the second reFR1 session provided: repeated measures ANOVA; reFR1 active responses: training, F(1, 13) = 0.16, P = 0.693; group × training, F(1, 13) = 1.92, P = 0.189; group F(1, 13) = 0.06, P = 0.803; reFR1 rewards: training, F(1, 13) = 0.00, P = 0.977; group × training, F(1, 13) = 0.19, P = 0.666; group F(1, 13) = 0.51, P = 0.489]. Together reFR1 data conﬁrmed that SDPS eﬀects on PR- responding were not carried over to subsequent reinforcement schedules, and that PR training did not inﬂuence consummatory approach toward a sucrose solution. ( , ) With respect to the number of rewards gained per session, controls and SDPS animals showed similar performance in each of the FR training schedules employed (Figure 2C): FR1: training, F(3.38, 43.93) = 29.67, P < 0.001; group × training, F(3.38, 43.93) = 1.11, P = 0.358; group F(1, 13) = 0.35, P = 0.566; FR2: training, F(1.65, 21.49) = 2.73, P = 0.096; group × training, F(1.65, 21.49) = 0.17, P = 0.807; group F(1, 13) = 0.00, P = 0.993; FR3: training, F(4, 52) = 3.37, P = 0.016; group × training, F(4, 52) = 1.20, P = 0.321; group F(1, 13) = 0.04, P = 0.949. Accordingly, at the end of the FR training period, animals consumed considerable, but equivalent, amounts of the sweetened water solution: Con, 19.7 ± 1.4 mL (∼4.7 g/kg); SDPS, 20.2 ± 1.4 mL (∼4.9 g/kg) per hour. SDPS and control rats performed identical for inactive responses, as both groups reduced responding over time in a similar rate (Supplementary Figure 1A). Time-out responses increased over time during all training schedules, but no Animals were then subjected to extinction of the context of reward delivery in nine daily sessions (Figure 4A). Analysis of the August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 5 Depression and sucrose-associated anhedonia Riga et al. FIGURE 3 \| SDPS triggers motivational overdrive toward a sucrose reward; guanfacine attenuates enhanced motivation. Extinction of Operant Sucrose Self-administration (C) Break points, depicted as the maximum FR completed when averaging over the four treatment-free sessions, conﬁrmed the SDPS-triggered exaggeration of F(1, 13) = 3.93, P = 0.069. By extinction session nine, both groups responded at <5% of their initial (reFR1) performance: Con, 4.3 ± 0.8 and SDPS, 3.4 ± 0.8 responses per hour. These data indicate that, although SDPS rats showed an initial resistance to extinction learning, they could be extinguished to control levels following multiple extinction sessions. FIGURE 4 \| SDPS hinders extinction of sucrose SA. (A) Following retraining to FR1 (see Supplementary Figure 3), all animals were subjected to extinction of the sucrose-delivery context, in nine daily sessions (highlighted). (B) Responding to the active hole stabilized in controls following the ﬁrst two sessions, indicating extinction of the context conveying sucrose availability. A signiﬁcant training × group (tr × gr) interaction and a trend for group (gr) effect indicate initial resistance to extinction displayed by the SDSP animals. P < 0.001; repeated ANOVA (training effect). Reinstatement and the Effect of Guanfacine Treatment Cue-induced reinstatement of sucrose seeking was assessed in two separate 30-min relapse tests (REL). Using a cross-over design, rats were systemically administered either guanfacine (GUA) or saline (SAL) 1 h before the test (Figure 5A). During the saline session, the presentation of cues that were previously associated with the delivery of sucrose was suﬃcient to reinstate responding in both groups, as compared with their own extinction performance: repeated measures ANOVA for relapse SAL: F(1, 13) = 83.13, P < 0.001. A signiﬁcant relapse x group interaction, [F(1, 13) = 4.87, P = 0.046] and a trend for a group eﬀect [F(1, 13) = 2.92, P = 0.111] indicated that SDPS moderately increased reinstatement of sucrose seeking. Post-hoc analysis conﬁrmed that both groups showed a signiﬁcant eﬀect of reinstatement as compared with their own extinction performance [Con, t(6) = −4.52, P = 0.004; SDPS, t(7) = −9.25, P < 0.001]. Yet, in the reinstatement session, SDPS animals showed a trend for higher seeking behavior than controls [Relapse SAL, F(1, 14) = 3.93, P = 0.069; Supplementary Figure 4]. FIGURE 4 \| SDPS hinders extinction of sucrose SA. (A) Following FIGURE 4 \| SDPS hinders extinction of sucrose SA. (A) Following retraining to FR1 (see Supplementary Figure 3), all animals were subjected to extinction of the sucrose-delivery context, in nine daily sessions (highlighted). (B) Responding to the active hole stabilized in controls following the ﬁrst two sessions, indicating extinction of the context conveying sucrose availability. A signiﬁcant training × group (tr × gr) interaction and a trend for group (gr) effect indicate initial resistance to extinction displayed by the SDSP animals. P < 0.001; repeated ANOVA (training effect). FIGURE 4 \| SDPS hinders extinction of sucrose SA. (A) Following retraining to FR1 (see Supplementary Figure 3), all animals were subjected to extinction of the sucrose-delivery context, in nine daily sessions (highlighted). (B) Responding to the active hole stabilized in controls following the ﬁrst two sessions, indicating extinction of the context conveying sucrose availability. A signiﬁcant training × group (tr × gr) interaction and a trend for group (gr) effect indicate initial resistance to extinction displayed by the SDSP animals. P < 0.001; repeated ANOVA (training effect). SDPS-induced Impairments in the Affective and Cognitive Domains FIGURE 5 \| SDPS mildly affects reinstatement of sucrose SA; guanfacine limits relapse. (A) Following extinction, the ability of the sucrose-coupled cues to reinstate active responding was assessed in two relapse tests, given at 4 days interval, using a cross-over treatment administration design (highlighted). (B) SDPS mildly affected cue-induced relapse (left); after guanfacine treatment, relapse to sucrose seeking was limited in both groups (right), as shown by a signiﬁcant treatment effect. A trend for group differences was observed, as SDSP animals responded at a relatively higher rate when compared with controls during the SAL session; this difference was no longer detected following treatment (GUA). #P = 0.069; post-hoc (group effect); P < 0.001; repeated ANOVA (treatment effect). In line with literature (Blanchard et al., 2001; Nestler and Hyman, 2010) a signiﬁcant and sustained decline in approach behavior develops following SDPS (Riga et al., 2014). Social withdrawal is considered to be one of the most robust behavioral readouts in the ﬁeld. It is employed to assess the magnitude of the depressive state, e.g., susceptibility vs. resilience (Krishnan et al., 2007) and to identify depression-mediating brain circuitries, as well as novel therapeutic approaches (Bruchas et al., 2011; Fanous et al., 2011). In our study, the duration of avoidance of the social target, long after the last exposure to a resident, is indicative of an established depressive state that persists well-beyond the actual stress incidence, mimicking clinical observations of MDD. Apart from impairments in the aﬀective domain, cognitive dysfunction, including poor working memory, mnemonic deﬁcits, and impaired concentration [DSM 5th edition, (American Psychiatric Association, 2013)] is a hallmark of depression. Almost half of the depressed population exhibits deﬁcits in at least one cognitive domain, with working memory and attention-related deﬁcits most consistently reported (McIntyre et al., 2013). These deﬁcits complement the depressive (endo)phenotype, exaggerating diﬃculties in decision-making and other aspects of executive control (Hasler et al., 2004; Murrough et al., 2011; Millan et al., 2012). In this study, we conﬁrmed previous observations that SDPS triggered lasting impairments in short-term spatial memory (Riga et al., 2014) analogous to depression-induced cognitive impairments that characterize the enduring state of depression (Femenia et al., 2012). FIGURE 5 \| SDPS mildly affects reinstatement of sucrose SA; guanfacine limits relapse. Reinstatement and the Effect of Guanfacine Treatment ﬁrst two extinction sessions, during which sucrose unavailability was consolidated in controls, revealed a signiﬁcant training eﬀect [F(1, 13) = 106.60, P < 0.001], as both groups reduced responding to the active hole over time (Figure 4B). Importantly, a signiﬁcant training × group interaction was observed [F(1, 13) = 6.54, P = 0.024], as the initial rate of responding in SDPS rats was higher in comparison with controls. This was partially conﬁrmed by a trend for group eﬀect: During the guanfacine session, both groups showed a signiﬁcant increase in responding vs. their own extinction performance: repeated measures ANOVA for relapse GUA: Frontiers in Behavioral Neuroscience \| www.frontiersin.org August 2015 \| Volume 9 \| Article 195 6 Depression and sucrose-associated anhedonia Riga et al. SDPS Promotes the Development of an Anhedonia-like Phenotype Blunted responses to aﬀective stimuli have been demonstrated consistently in depressed patients (Eshel and Roiser, 2010; Disner et al., 2011), including dysfunctional reward-related learning (Vrieze et al., 2013), reduced primary hedonic responses (Pizzagalli et al., 2008; McCabe et al., 2009), untenable connectivity between reward structures (Heller et al., 2009), decreases in reward anticipation and poorly integrated positive feedback (Smoski et al., 2009). These ﬁndings implicate impairments of aﬀective cognition and reward processing in the development of depressive disorders. Similarly, direct links between behavioral anhedonia and the activity of reward- mediating systems (Keedwell et al., 2005; Schlaepfer et al., 2008) exemplify the strong association between dysfunctional reward processing and the magnitude of the depressive state. SDPS-induced Impairments in the Affective and Cognitive Domains (A) Following extinction, the ability of the sucrose-coupled cues to reinstate active responding was assessed in two relapse tests, given at 4 days interval, using a cross-over treatment administration design (highlighted). (B) SDPS mildly affected cue-induced relapse (left); after guanfacine treatment, relapse to sucrose seeking was limited in both groups (right), as shown by a signiﬁcant treatment effect. A trend for group differences was observed, as SDSP animals responded at a relatively higher rate when compared with controls during the SAL session; this difference was no longer detected following treatment (GUA). #P = 0.069; post-hoc (group effect); *P < 0.001; repeated ANOVA (treatment effect). F(1, 13) = 32.20, P < 0.001. No relapse × group interaction [F(1, 13) = 0.00, P = 0.955], nor group eﬀects [F(1, 13) = 1.15, P = 0.303] were observed conﬁrming that guanfacine did not completely prevent reinstatement of sucrose seeking in either group. Importantly, a signiﬁcant treatment eﬀect was shown between the 2 relapse days (Figure 5B): repeated measures ANOVA for treatment: F(1, 13) = 16.85, P = 0.001. This was accompanied by a non-signiﬁcant treatment × group interaction [F(1, 13) = 2.01, P = 0.179] and a trend for a signiﬁcant group eﬀect: F(1, 13) = 3.99, P = 0.067]. Taken together, our data indicate that guanfacine pretreatment signiﬁcantly reduced reinstatement of sucrose seeking, and that this reduction in responding seemed more prominent in the SDSP group. Guanfacine Attenuates the SDPS-induced Anhedonia-like Phenotype Guanfacine is a selective α2-adrenergic agonist recently FDA- approved for the treatment of attention deﬁcit hyperactivity disorder (ADHD) (Muir and Perry, 2010). Owing to its cognitive enhancing properties (Sofuoglu et al., 2013), guanfacine has been used in both clinical and pre-clinical settings to limit stress- and cue-induced drug-seeking and craving (Lee et al., 2004; Smith and Aston-Jones, 2011; Fox et al., 2012, 2015; Fredriksson et al., 2015; McKee et al., 2015). In addition, we recently showed that a comparable dose of guanfacine prevents SDPS-induced motivational overdrive and susceptibility to cue- induced relapse in an alcohol self-administration paradigm (Riga et al., 2014). In naïve rats, the eﬀects of guanfacine against stress-facilitated reinstatement of food pellets showed large inter-individual variability (Le et al., 2011), making it of great interest to examine whether it can selectively reverse the anhedonia-like phenotype as seen after SDPS. In the present study, guanfacine was used against SDPS-triggered increases in PR-responding and in cue-paired reinstatement of sucrose seeking. Guanfacine pretreatment was suﬃcient to reduce overall PR responding, but these eﬀects were independent of the depressive phenotype, as they were observed in both control and SDPS rats. Although it did not completely abolish the increase in break points, SDPS PR responding decreased considerably after guanfacine, indicating a beneﬁcial eﬀect against depression-related pathological manifestations, such as maladaptive motivational drive. Furthermore, whereas guanfacine pretreatment reduced cue-induced reinstatement of sucrose-seeking in both groups, it limited the magnitude of SDPS relapse to control levels. Together, these results pinpoint to SDPS-speciﬁc eﬀects of guanfacine, and identify a novel property of the drug, as being beneﬁcial against the depression-induced anhedonia-like phenotype. In accordance with this updated conceptualization of anhedonia, the main aspect described in the present study concerns the ability of SDPS animals to evaluate a given reward and thus to modulate their behavior as function of reward- signaling information in the long-term. This is reﬂected in altered progressive ratio responding, extinction and reinstatement of sucrose seeking. SDPS increased break points under PR training schedules, a putative measurement of motivational drive (Hodos, 1961) and promoted inelastic demand (Diergaarde et al., 2012), as depicted in a clear shift in demand curve (Supplementary Figure 5). Additionally, SDPS aﬀected extinction learning as SDPS rats showed a reduced capacity to incorporate new information signaling sucrose unavailability. Discussion The depressive state is characterized by multifaceted behavioral manifestations that span from negative mood and suicidality to indecisiveness, cognitive confusion, and blunted emotional reactivity (Leistedt and Linkowski, 2013). Animal models aiming to mimic depressive disorders are frequently impeded by such phenotypic complexity. In this study we showed that SDPS, an animal paradigm that mimics a sustained depressive state, is able to induce deﬁcits in processing of aﬀective information and reward evaluation, similar to that observed in patients. Furthermore, we showed that guanfacine, an FDA-approved agent that reverses SDPS-induced excessive alcohol seeking (Riga et al., 2014), rescues the anhedonia-like phenotype. Preclinically, the concept of anhedonia was, for many years, conﬁned to ﬁndings of reduced appetitive interest or reduced preference for naturally rewarding items (e.g., sucrose). This has been consistently portrayed as representative of depression- induced deﬁcits in behavioral reinforcement, an animal- equivalent of anhedonia. Clinically, anhedonia is nowadays not valued as a steady-state depressive symptom linked to an absolute “hedonic capacity,” but rather encompassing a much broader spectrum of consummatory and motivational deﬁcits, involving reward-related decision-making and goal-directed Frontiers in Behavioral Neuroscience \| www.frontiersin.org Frontiers in Behavioral Neuroscience \| www.frontiersin.org August 2015 \| Volume 9 \| Article 195 7 Depression and sucrose-associated anhedonia Riga et al. motivation for natural vs. non-natural rewards (Supplementary Figure 6). behavior (Treadway and Zald, 2011; Der-Avakian et al., 2014). As reviewed by Der-Avakian and Markou (2012), anhedonia extends beyond the loss of feeling pleasure and conveys failure in (i) reward anticipation or prediction; (ii) estimation of the value-to-cost ratio for a given reward; (iii) assessment of reward accessibility as function of the eﬀort required; (iv) integration of this information to justify (or oppose to) reward acquisition; and ﬁnally (v) motivation to act toward reward acquisition. Thus, anhedonia is a progressively developing phenotype that cannot be solely based on measurements of preference for a given reward and should include the contributions of motivational approach and reward-related learning and decision-making (Ho and Sommers, 2013). Guanfacine Attenuates the SDPS-induced Anhedonia-like Phenotype Finally, SDPS rats showed a relative increase in reinstatement of sucrose seeking as compared with controls, reﬂecting maladaptive processing of reward-related information, and excessive reactivity to reward- paired cues. These deﬁcits do not likely result from a general decline in learning capacity following SDPS, as SDPS rats and controls showed similar discriminative ability at the start of SA training (active vs. inactive hole) and similar SA acquisition rates at FR1–3. Moreover, SDPS rats did not show slower extinction of the alcohol-associated context (Riga et al., 2014), indicative of speciﬁcity for the type of reward. Previously, we observed a similar, even exaggerated, phenotype for SDPS-induced motivation and relapse of operant alcohol intake (Riga et al., 2014). In accordance, intermittent social defeat stress increases PR operant responding for cocaine and cocaine binging (Covington and Miczek, 2005; Covington et al., 2005). In contrast, chronic defeat stress (∼5 weeks of daily defeat sessions) induces a remarkable decrease in both PR responding and cumulative cocaine intake for up to 5 weeks following the last defeat session (Miczek et al., 2011). It is noteworthy that diﬀerent types of social stress persistently alter the incentive value of a given reward, although the direction of changes in motivational drive depends on the stress-status of the animal. Indeed, and in contrast to the lasting eﬀect of SDPS, acute defeat results in decreased motivational drive, as expressed in reduced PR saccharin-reinforced responding (Miczek et al., 2011) and decreased operant alcohol self-administration (Funk et al., 2005). We previously reported that SDPS-induced social withdrawal can predict higher motivation to seek and consume alcohol (Riga et al., 2014). In the current study, such correlations between approach-avoidance behavior and motivational deﬁcits were not observed, suggesting a diﬀerential eﬀect of SDPS on Methodological Considerations Although SDPS animals and controls showed similar consummatory behavior toward a presumably enticing sucrose solution, during PR training SDPS rats failed to appreciate the relative eﬀort-to-outcome relationship and displayed inelastic demand when reward was delivered at higher costs. During extinction training, depressed animals exhibited delayed uncoupling of the context of sucrose delivery from the reward itself, indicative of dysfunctional processing of novel reward-associated information. Finally, SDPS-induced proneness to reinstatement of sucrose seeking conﬁrmed an excessive increase in the incentive salience of reward-signifying cues. Taken together, these eﬀects are in accordance with the vast majority of literature on the human depression-associated anhedonia, describing maladaptive integration of learned and retrieved reward-coding information and, consequently, misguided behavioral adaptations in response to reward-related stimuli (Pizzagalli et al., 2008; Eshel and Roiser, 2010; Vrieze et al., 2013). Similarly our results reﬂect a large share of clinical observations linking anhedonia and depression with motivational deﬁcits that correspond better to impaired reward- associated anticipation, appraisal, and decision-making rather than diminished consummatory and/or experiential hedonic responses (Chentsova-Dutton and Hanley, 2010; Padrao et al., 2013). Together, a large body of evidence indicates that diﬀerences in sucrose preference can be used to assess acute eﬀects of social defeat or short-lasting eﬀects after chronic defeat stress exposure in rodents. In addition, these ﬁndings illustrate that assessment of sucrose intake is not suitable to estimate the anhedonia- like phenotype during the maintenance phase of a depressive state (Von Frijtag et al., 2000; Kamal et al., 2010). This is in accordance with our current data, since SDPS rats, although in a stable, sustained depressive-like state reﬂected by maladaptive motivational drive, did not display any alteration in sucrose intake during acquisition or re-training at FR1, as compared with controls. To our knowledge, this is the ﬁrst study reporting long- lasting anhedonia-like behaviors as assessed by instrumental responding for a natural reward. Importantly, in our study, by incorporating motivation, extinction and reinstatement into the assessed behavioral repertoire, we identiﬁed novel readouts aﬀected by the depressive state and we advanced our understanding on what is, and how we can measure, anhedonia at the preclinical level. p Highly palatable food, such as items containing sugar or fat, induces stable preference and elevated consumption in rodents (Hone-Blanchet and Fecteau, 2014). In our study, the intake during FR training schedules was identical between control and defeated animals, implying that both groups estimated the caloric value of sucrose equally. Methodological Considerations Given that anhedonia is a key feature of clinical depression, animal models aiming to explore this complex disorder should employ some kind of assessment of reward deﬁcits and (diminished) reinforcement (Anisman and Matheson, 2005). To this end, several diﬀerent behavioral readouts have been established over the years to emulate depression- induced anhedonia. Amongst them, sucrose-based (preference or anticipation) paradigms have been most extensively used, although yielding contradictory results (Der-Avakian et al., 2014). In particular, varying ﬁndings, i.e., increases, decreases or unaltered behavior, of the eﬀects of stress on sucrose consumption or preference have been reported depending on the models applied, including diﬀerences in species, strain (Nielsen et al., 2000; Pothion et al., 2004; Henningsen et al., 2009; Razzoli et al., 2011) and gender (Bai et al., 2014), type of stressors (non- social or social), timing and duration of stress (Meerlo et al., 1996; Rygula et al., 2005; Krishnan et al., 2007; Miczek et al., August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 8 Depression and sucrose-associated anhedonia Riga et al. 2011; Muto et al., 2014), food availability (Forbes et al., 1996; Barr and Phillips, 1998), and sucrose concentrations employed (Bondar et al., 2009; Hollis et al., 2011). In the majority of studies, the most robust changes in sucrose intake are observed during or acutely following chronic stressors, whereas these changes quickly recover following termination of stress exposure. Few studies examined the after-eﬀects of social stress on anhedonia, reporting reduced sucrose consumption up to 3 weeks following defeat (Becker et al., 2008; Carnevali et al., 2012) or unaltered intake at 9–11 weeks following the last defeat encounter (Von Frijtag et al., 2000). in both groups at PR training (Figure 3) and at relapse (Figure 5). Similarly, guanfacine did not aﬀect PR inactive responding, further excluding non-speciﬁc eﬀects on motor function (Supplementary Figure 8). It should be noted that guanfacine-induced suppression of responding was more robust in the defeated animals at both PR training and relapse tests, whereas in the latter, guanfacine was suﬃcient to normalize the enhanced SDPS responding back to control levels. In the present study, the established depressive state led to maladaptive motivational drive and vulnerability to sucrose- associated context and cues. Methodological Considerations Thus, the increased responding observed in PR training in SDPS animals cannot be attributed to diﬀerences in “liking” or taste. Similarly, diﬀerences in total ﬂuid intake cannot account for the SDPS eﬀects on sucrose seeking, as SDPS animals display similar water intake as controls, from the week following the defeat week onwards (Supplementary Figure 7). It is noteworthy that, contradictory ﬁndings have been reported when employing a diﬀerent animal model of depression, i.e., the chronic mild stress (CMS) paradigm, depending on the concentration of sucrose. In particular, CMS leads to enhanced sucrose solution intake in higher concentrations (30–40%), whereas it suppresses intake of lower sucrose concentrations (1–2%) (Willner, 1997). 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E., van Den Bos, R., and Spruijt, B. M. (2000). Defeat followed by individual housing results in long- term impaired reward- and cognition-related behaviours in rats. Behav. Brain Res. 117, 137–146. doi: 10.1016/S0166-4328(00)00300-4 Willner, P. (1997). Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology 134, 319–329. doi: 10.1007/s002130050456 Vrieze, E., Pizzagalli, D. A., Demyttenaere, K., Hompes, T., Sienaert, P., De Boer, P., et al. (2013). Reduced reward learning predicts outcome in major depressive disorder. Biol. Psychiatry 73, 639–645. doi: 10.1016/j.biopsych.2012.10.014 Frontiers in Behavioral Neuroscience \| www.frontiersin.org Von Frijtag, J. C., Reijmers, L. G., van Der Harst, J. E., Leus, I. E., van Den Bos, R., and Spruijt, B. M. (2000). Defeat followed by individual housing results in long- term impaired reward- and cognition-related behaviours in rats. Behav. Brain Res. 117, 137–146. doi: 10.1016/S0166-4328(00)00300-4 Vrieze, E., Pizzagalli, D. A., Demyttenaere, K., Hompes, T., Sienaert, P., De Boer, P., et al. (2013). Reduced reward learning predicts outcome in major depressive disorder. Biol. Psychiatry 73, 639–645. doi: 10.1016/j.biopsych.2012.10.014 Willner, P. (1997). Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology 134, 319–329. doi: 10.1007/s002130050456 References Cognitive dysfunction in depression: neurocircuitry and new therapeutic strategies. Neurobiol. Learn. Mem. 96, 553–563. doi: 10.1016/j.nlm.2011.06.006 Treadway, M. T., and Zald, D. H. (2011). Reconsidering anhedonia in depression: lessons from translational neuroscience. Neurosci. Biobehav. Rev. 35, 537–555. doi: 10.1016/j.neubiorev.2010.06.006 Muto, J., Lee, H., Uwaya, A., Park, J., Nakajima, S., Nagata, K., et al. (2014). Oral administration of inosine produces antidepressant-like eﬀects in mice. Sci. Rep. 4:4199. doi: 10.1038/srep04199 van Bokhoven, P., Oomen, C. A., Hoogendijk, W. J., Smit, A. B., Lucassen, P. J., and Spijker, S. (2011). Reduction in hippocampal neurogenesis after social defeat is long-lasting and responsive to late antidepressant treatment. Eur. J. Neurosci. 33, 1833–1840. doi: 10.1111/j.1460-9568.2011.07668.x Nestler, E. J., and Hyman, S. E. (2010). Animal models of neuropsychiatric disorders. Nat. Neurosci. 13, 1161–1169. doi: 10.1038/nn.2647 August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 11 Depression and sucrose-associated anhedonia Riga et al. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Copyright © 2015 Riga, Theijs, De Vries, Smit and Spijker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. August 2015 \| Volume 9 \| Article 195 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 12
https://openalex.org/W2750153854	https://research.tue.nl/files/74568411/hornplas2017.pdf	English	null	Plasma-assisted and thermal atomic layer deposition of electrochemically active Li<sub>2</sub>CO<sub>3</sub>	RSC advances	2,017	cc-by	9,883	Document status and date: Published: 24/08/2017 Document status and date: Published: 24/08/2017 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: Plasma-assisted and thermal atomic layer deposition of electrochemically active Li2CO3 Citation for published version (APA): Hornsveld, N., Put, B. H. F., Kessels, W. M. M., Vereecken, P. M., & Creatore, M. (2017). Plasma-assisted and thermal atomic layer deposition of electrochemically active Li2CO3. RSC Advances, 7(66), 41359-41368. https://doi.org/10.1039/c7ra07722j DOI: 10.1039/c7ra07722j DOI: 10.1039/c7ra07722j Download date: 24. Oct. 2024 Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. eld, a B. Put,ab W. M. M. Kessels,a P. M. Vereeckenbc and M. Creatorea Thin-ﬁlm lithium carbonate (Li2CO3) has applications in various electrochemical devices, like Li-ion batteries, gas sensors and fuel cells. ALD of Li2CO3 is of interest for these applications as it allows for uniform and conformal coating of high-aspect ratio structures and particles with very precise thickness control. However, there are few studies that focus on its fabrication and characterization. In this work, plasma-assisted and thermal ALD were adopted to grow ultra-thin, conformal Li2CO3 ﬁlms between 50 and 300 C using lithium tert-butoxide as a precursor and O2 plasma or H2O/CO2 as co-reactants. More speciﬁcally, we focus on the plasma-assisted process by ﬁlm growth, stability and conductivity studies and emphasize the diﬀerences from its more extensively adopted thermal counterpart. Plasma-assisted ALD allows for higher growth per cycle values (0.82 vs. 0.60 ˚A), lower substrate temperatures and shorter cycle times. The stoichiometry of the ﬁlms, ranging from Li2CO3 to Li2O, can be controlled by substrate temperature and O2 plasma exposure time. The ionic conductivity for both plasma-assisted and thermal ALD is measured for the ﬁrst time and is in the order of 1010 S cm1 after normalizing to the diﬀerent eﬀective surface areas. The Li-ion conductivities found here are in line with literature values predicted by simulation studies. Received 13th July 2017 Accepted 10th August 2017 DOI: 10.1039/c7ra07722j rsc.li/rsc-advances power and energy density.6–8 Key to nanostructured energy storage systems is thin lms processing of battery active and passive materials compatible with complex battery design. Conventional deposition techniques, such as physical vapor deposition (PVD) and plasma-enhanced chemical vapor deposi- tion (PE-CVD), do not generally lead to conformal thin layers on high aspect ratio structures. aDepartment of Applied Physics, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands. E-mail: n.hornsveld@tue.nl; m.creatore@tue.nl bImec, Kapeldreef 75, Leuven 3001, Belgium cDepartment of Microbial and Molecular Systems, KU Leuven, 3001 Leuven, Belgium † Electronic supplementary information (ESI) available: Additional in situ SE thickness measurements (thermal ALD of Li2CO3 at 50, 150, 250 and 300 C and plasma ALD between 50 and 200 C), hydrogen concentration in the lms measured by ERD, XPS measurements before and aer Ar+ sputtering showing chemical decomposition of Li2CO3, a table of XPS stoichiometry results and SEM images of Li2CO3 deposited using thermal ALD at diﬀerent temperatures. See DOI: 10.1039/c7ra07722j RSC Advances RSC Advances View Article Online View Journal \| View Issue Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To enable fast charging thin-lm batteries, conformality and uniform material composition in extreme aspect ratios are required.9 Therefore, novel deposition techniques leading to enhanced control in thin lm properties and conformality are being presently introduced in the eld of Li- ion batteries.10–12 Atomic layer deposition (ALD), which is based on sequential and self-limiting half-reactions between precursors (co-reactants) and surface, has emerged as a powerful tool since it shows potential towards exceptional conformality on high-aspect ratio structures, thickness control at sub-nanometer level, and tunable lm properties.13 Promising applications of ALD for Li-ion batteries include (surface modication of) particle-based electrodes, 3D-structured electrodes, and 3D all-solid-state microbatteries.10–12,14,15 Due to the excellent conformality and thickness control of ALD, is suggested that ALD of lithium carbonate (Li2CO3) is especially benecial when it is employed as electrode passivating lm or as additive to improve electrode performance. This journal is © The Royal Society of Chemistry 2017 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 Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. counterpart. This is expected to be generally helpful to assess the opportunities of ALD for Li-ion materials. With plasma- assisted ALD a high reactivity is delivered to the deposition surface by the plasma species and therefore less thermal budget is required at the substrate to drive the ALD surface chemistry than for thermal ALD. The application of plasmas in combi- nation with ALD generally leads to a wider range in substrate temperature and processing conditions and thus enables a larger variety of material properties.21 The process details of ALD Li2CO3 as collected from literature are shown in Table 1. Although the plasma-assisted ALD process for Li2CO3 has been already reported in literature, the understanding of the ALD lm growth (primarily in terms of developed chemistry during and aer deposition) is so far insuﬃcient. In addition, the eﬀect of the process parameters on the layer properties was not re- ported. To the best of our knowledge we provide the rst proper conductivity measurement of thin lm Li2CO3. counterpart. This is expected to be generally helpful to assess the opportunities of ALD for Li-ion materials. With plasma- assisted ALD a high reactivity is delivered to the deposition surface by the plasma species and therefore less thermal budget is required at the substrate to drive the ALD surface chemistry than for thermal ALD. The application of plasmas in combi- nation with ALD generally leads to a wider range in substrate temperature and processing conditions and thus enables a larger variety of material properties.21 The process details of ALD Li2CO3 as collected from literature are shown in Table 1. Although the plasma-assisted ALD process for Li2CO3 has been already reported in literature, the understanding of the ALD lm growth (primarily in terms of developed chemistry during and aer deposition) is so far insuﬃcient. In addition, the eﬀect of the process parameters on the layer properties was not re- ported. To the best of our knowledge we provide the rst proper conductivity measurement of thin lm Li2CO3. As substrate, 60 nm titanium nitride (TiN) sputtered on silicon was used (Endura PVD). TiN is a barrier for Li-ion diﬀusion, and also functions as current collector for electro- chemical testing of the layers. The lm thickness and dielectric function were monitored in situ by spectroscopic ellipsometry (SE) with a J.A. Woollam, Inc. M2000U (0.75–5.0 eV) tool. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The dielectric function of Li2CO3 was parameterized using a math- ematical description in the form of B-splines, since this method requires no prior knowledge about the dielectric function of the deposited lms. Thickness values were conrmed by high resolution SEM (Zeiss Sigma) operated at 2 kV acceleration voltage. The entire chemical composition of the samples has been characterized with ion beam analysis methods by Detect99. The hydrogen proles were determined by Elastic Recoil Detection (ERD) using a 1.9 MeV He+ beam impinging on the sample under 15 glancing incidence, and 30 recoil angle. All other elements could be determined in a single run by Elastic Back- scattering Spectrometry (EBS) with 2.8 MeV protons at perpen- dicular incidence. This technique is similar to Rutherford Backscattering Spectrometry (RBS) but has the advantage that the cross sections for light elements is enhanced by the use of high energy protons as projectiles instead of He. Spectra have been registered at 170 and 150 scattering angle. EBS was in this case necessary for the determination of the Li and C contents, for which the sensitivity of RBS is rather poor. Finally, the O, N and Ti contents have been conrmed by channeling RBS using 1900 keV He ions in the same conguration as for the EBS measurements. Moreover, Li2CO3 ALD represents an attractive model process for incorporating Li into multicomponent (ternary) ALD materials, since it is a highly stable compound with respect to process stability and ease of postdeposition analysis of the lms. Li2O and LiOH oen develop simultaneously during processing due to the reversible reaction with H2O and CO2 and are being deposited by ALD as well.16–20,22,23 These materials are also widely used as building block for the fabrication of various other Li-ion battery active materials.24–26 41360 \| RSC Adv., 2017, 7, 41359–41368 This journal is © The Royal Society of Chemistry 2017 Introduction Lithium carbonate is considered a potential electrode passiv- ating lm in Li-ion batteries1 and electrolyte material or sensing layer in electrochemical devices like fuel cells or chemical sensors.2,3 This is attributed to its purely ionically conductive behavior and good electrochemical stability.4 In addition, it is widely used as building block for the fabrication of electrodes and electrolytes or as an additive to improve electrode perfor- mance.5 Despite its widespread usage, there are only few studies in the literature focusing on its characterization. With the trend of manufacturing devices that have smaller size, weight and lower power consumption, thin lm fabrication techniques are becoming preferable. This is especially applicable in the eld of Li-ion batteries, where thin-lm 3D battery archi- tectures are currently being investigated.6 Nanostructured energy storage systems have demonstrated a substantial increase in In the present work we focus on the ALD characterization of Li2CO3. More specically, we focus on the plasma-assisted ALD process of Li2CO3 by (in situ) growth studies and emphasize the diﬀerences from its more extensively adopted thermal RSC Adv., 2017, 7, 41359–41368 \| 41359 This journal is © The Royal Society of Chemistry 2017 View Article Online RSC Advances RSC Advances Paper Paper the fabricated layers was minimized by transporting the samples in an evacuated transport tube. Table 1 Li2CO3 ALD process details collected from earlier publications Precursor Co-reactant(s) Tsub (C) GPC (˚A) Ref. LiOtBu H2O, CO2 225 0.8 16 LiHMDS H2O, CO2 89–332 0.41–0.14 17 LiTMSO H2O, CO2 200–300 0.5–0.3 18 LiOtBu O2 plasma 225, 300 0.4 19 Li(thd) O3 185–225 0.3–0.07 20 The LiOtBu precursor was bubbled from the precursor pod with Ar at a reactor pressure of 20 mTorr and the precursor and precursor supply line were heated to 140 C and 150 C respectively to avoid precursor condensation. The reactor pressure during the O2 plasma generation was 13 mTorr. The process table was heated to 50–300 C, since it is reported that precursor decomposition takes place from 350 C.27 The wafers were placed on an aluminum substrate holder. The reactor wall is maintained at a temperature of 120 C (which is the maximum wall temperature), except for the deposition at 50 or 100 C, where the reactor wall is kept at 50 or 100 C, respectively. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:1 This article is licensed under a Creative Commons Attribution 3.0 Unporte valve, dosing of 0.5 s or less did not deliver any CO2 in the chamber (no pressure increase was observed). Purge steps aer LiOtBu and CO2 dose of 2 s are suﬃcient to remove the reaction products and unreacted species from the processing chamber, although 5 s purge was necessary for H2O. A growth per cycle of 0.60 ˚A was found for the thermal ALD process. three-electrode Teon cell was clamped on top of the sample using a Kalrez O-ring. A Luggin capillary was used to connect the cell with the reference-electrode compartment. One molar LiClO4 (battery-grade, Sigma-Aldrich) in propylene carbonate was used as the electrolyte. The experiments were carried out in an Ar glovebox (O2, H2O < 1 ppm). Impedance spectroscopy results were analyzed by tting an equivalent circuit to the data, using MEISP soware (Kumho Chemical Laboratories). The obtained ts had a c2 value in the range from 103 to 105. The relative standard deviation for all tted values remained below 5%. The saturation behavior of the surface reactions during the two half cycles of the plasma-assisted ALD process, i.e. the exposure to the lithium precursor and O2 plasma, is shown in Fig. 2b: self-limiting growth was veried for both the precursor and the plasma step. For both half-cycles purge steps of 2 s are suﬃcient to remove the reaction products and unreacted species. A growth per cycle of 0.82 ˚A was obtained for a deposi- tion temperature of 150 C. This journal is © The Royal Society of Chemistry 2017 Experimental All depositions presented in this work were carried out using the thermal and remote plasma ALD reactor FlexAL (Oxford Instruments). The reactor consists of a rotary and turbo molecular pump such that it can reach a base pressure of <106 Torr by overnight pumping. The pump unit as well as the inductively coupled plasma (ICP) source are connected to the deposition chamber trough gate valves. X-ray photoelectron spectroscopy (XPS) spectra were recor- ded by a Thermo Scientic K-alpha+ system using mono- chromatic Al Ka X-rays. The spot size of the beam was 400 mm and the base pressure of the system was 108 mbar. The depth prole was obtained by an Ar ion gun of 500 eV during 5 s for each sputter step. The crystal structure of the Li2CO3 layers was determined using a Philips X'Pert MPD diﬀractometer equipped with a Cu Ka source (1.54 ˚A radiation). The morphology of the deposited layers was investigated by the SEM which was described before. The ALD processes developed in this work are shown in Fig. 1. For thermal ALD (Fig. 1a), the cycle consists of the exposure of the substrate to LiOtBu (97%, Sigma Aldrich) lithium precursor, H2O gas and CO2 gas respectively, alternated by argon purges. The plasma-assisted ALD process (Fig. 1b) consists of a combination of the substrate exposure to the same lithium precursor and to an O2-fed plasma with a plasma power of 100 W, alternated by argon purging steps. To ensure the gas ow and pressure have stabilized, the reactor was lled with oxygen prior to switching on the plasma source. Air exposure of For electrochemical impedance spectroscopy (EIS) testing an Autolab (Metrohm) potentiostat with a frequency-response analyzer module (frequency range from 0.01 Hz to 1 MHz) was used, operated by Nova soware (Metrohm Autolab). A This journal is © The Royal Society of Chemistry 2017 41360 \| RSC Adv., 2017, 7, 41359–41368 RSC Advances View Article Online Fig. 1 Schematic of the pulsing sequence of the ALD cycle for depositions of Li2CO3 by (a) thermal and (b) plasma-assisted ALD. The H2O dose during the thermal ALD process was only 50 ms, after which all valves were closed for 4 s to allow for the molecules to react. Therefore, this is referred to as “H2O reaction”. Paper RSC Advances View Article Online RSC Advances View Article Online Paper Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Schematic of the pulsing sequence of the ALD cycle for depositions of Li2CO3 by (a) thermal and (b) plasma-assisted ALD. The H2O dose during the thermal ALD process was only 50 ms, after which all valves were closed for 4 s to allow for the molecules to react. Therefore, this is referred to as “H2O reaction”. ALD process characterization This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 41359–41368 \| 41361 View Article Online RSC Advances Paper oen have a higher growth per cycle when compared to thermal processes which is in agreement with our results.21 as function of the number of ALD cycles, which can be explained by crystallization of the lms (in the ESI† more growth curves are shown for thermal ALD Li2CO3). p g Next, the process window of ALD lms was extended to deposition temperatures between 50 and 300 C. Fig. 3a shows the thickness evolution as a function of the number of ALD cycles for various deposition temperatures. For plasma-assisted ALD and Tsub # 200 C, the thickness of the lms develops linearly with the number of cycles. For temperatures higher than 250 C, the lm thickness develops according to two slopes, which correlates with a transition in lm chemical composition, as discussed in the next section. Due to high temperature sensitivity of the reactions and minor temperature uctuations of the process table, the trends of the thickness as a function of the number of cycles at 250 C varied from occa- sion to occasion: in some cases they tend to be similar to the growth behavior for T # 200 C and in other instances to T > 250 C. Moreover, lms deposited at 250 C showed large height diﬀerences (see SEM images in the next section), which complicated the extraction of the lm thickness. For the thermal process, no transition in lm chemical composition was observed. However, the growth per cycle increases slightly In Fig. 3b, both the temperature dependency of the growth per cycle for the plasma-assisted, as well as the thermal ALD process is shown. It can be noticed that the growth per cycle of the thermal ALD process strongly decreases at 50 C, indicating the lower limit of the temperature window. The ALD process window for the plasma-assisted process is extended towards lower temperatures, as it has a similar growth per cycle at 50 C as at higher temperatures. The overall growth per cycle is higher for the plasma-assisted process as compared to the thermal process. ALD process characterization The previously reported growth per cycle of 0.8 ˚A for thermal ALD of Li2CO3,16 diﬀers from our values. The reason for this diﬀerence is unclear, yet it is noted that the main diﬀerence with our process is a higher reactor pressure. Plasma-assisted ALD using LiOtBu on the other hand was reported to have a growth per cycle of 0.4 ˚A,19 but the obtained stoichiometry was a mixture of Li2CO3 and Li2O. Our lms grown at 150 C are found to be pure Li2CO3, as shown in the next section. More- over, no saturation curves were shown in literature to support the data for this particular case. In general, plasma processes A deposition temperature of 150 C was adopted to investigate the saturation behavior of the surface reactions during the sub- cycles of both thermal and plasma ALD Li2CO3. Film thickness was monitored every 10 cycles by in situ SE measurements while changing precursor or co-reactant dose times. Saturation curves for the thermal ALD process of Li2CO3 are shown in Fig. 2a. A LiOtBu dose of 6 s, a water dose of 50 ms and a CO2 dose of 2 s were chosen as standard conditions for thermal ALD of Li2CO3. Without H2O dosing no lm growth occurred and without CO2, LiOH was formed. Due to a slower response speed of the CO2 Fig. 2 Saturation curves for (a) thermal and (b) plasma-assisted ALD of Li2CO3 obtained at 150 C showing the self-limiting growth behavior of the ALD process. The solid line serves as a guide to the eye. Dotted lines indicate the chosen standard values and the corresponding growth per cycle. Note that the reaction time of H2O after dosing was kept at 4 s. Fig. 2 Saturation curves for (a) thermal and (b) plasma-assisted ALD of Li2CO3 obtained at 150 C showing the self-limiting growth behavior of the ALD process. The solid line serves as a guide to the eye. Dotted lines indicate the chosen standard values and the corresponding growth per cycle. Note that the reaction time of H2O after dosing was kept at 4 s. ALD process characterization As mentioned before, this is more oen observed for plasma processes due to the relatively high reactivity of the plasma species which could create a higher density of reactive surface sites.21 The diﬀerences in growth per cycle between thermal and plasma ALD, but also between diﬀerent deposition temperatures for plasma-assisted ALD are conrmed by EBS data (see Fig. 3b). A slight decrease in growth per cycle with increasing temperature was observed for the thermal ALD process. This decrease might be caused by desorption of part of the absorbed precursor monolayer from the surface, by higher purity lm growth or by an increase in lm crystallinity. The crystallinity of the lms will be further discussed in the next paragraph. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 (a) In situ thickness measurements by SE for plasma-assisted ALD of Li2CO3 at 50, 200, 275 and 300 C and thermal ALD of Li2CO3 at 150 C. The ﬁlm growth between 50 and 200 C with plasma ALD was very similar (see ESI†). (b) Growth per cycle as a function of process table temperature for both plasma-assisted (squares) and thermal (circles) ALD processes. Open Access Article. Published on 24 August This article is licensed under a C To summarize, it is shown that both plasma-assisted and thermal ALD of Li2CO3 show typical ALD growth behavior within a wide temperature window. Plasma-assisted ALD allows for lower temperature depositions, which results in a wider choice of (substrate) materials that can be used. This is partic- ularly useful when temperature-sensitive materials are adopted, such as polymers.21 In addition, the plasma-assisted process allows for higher growth per cycle values and shorter cycle times compared to thermal ALD, without compromising on the quality of the layer. This will enable higher throughputs for ALD reactors. This journal is © The Royal Society of Chemistry 2017 Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. for temperatures from 200 to 400 C only occurs in the top few nanometers of the Li2O and then a self-limiting shell of Li2CO3 develops.29 This in contrast to LiOH, which would be extremely reactive towards CO2. Hence, the lm at 300 C most likely consists of Li2O as aer the rst CO2 pulse D only slightly changes suggesting that only a surface reaction is occurring. During subsequent exposure steps, the layer does not further react with CO2, implying the absence of LiOH. Aer the CO2 pulses, the sample was subjected to H2O pulses. As observed in Fig. 4, the 300 C lm consisting of Li2O is reacting with the H2O forming LiOH. Next, CO2 is pulsed again, and the layer is again reacting to form Li2CO3. Unlike the lm deposited at 300 C, the as-deposited 275 C lm was very reactive towards the rst CO2 pulses and therefore (partly) consisted of LiOH. For all other lms, which include plasma ALD lms deposited at 50–250 C and thermal ALD lms deposited at 50–300 C, the change in D was negligibly small, indicating that almost no reaction with H2O or CO2 occurred. These stable Li2CO3 lms were used for ex situ XPS analysis which will be discussed later in this section. the ESI†). The relatively high carbon content suggests that the lm reacted from LiOH towards Li2CO3 upon air exposure. The reactivity of plasma ALD lms deposited at 300 C will be dis- cussed in more detail later in this paragraph. For all samples, the amount of deposited lithium atoms scales with the growth per cycle obtained by SE (see Fig. 3b). Assuming the mass density of stoichiometric Li2CO3 to be equal to the bulk density of 2.11 g cm3, one monolayer of lithium carbonate contains roughly 10.58 Li atoms per nm2.‡ Based on the EBS growth per cycle of 2.09 Li atom per nm2 for the thermal ALD process, in each cycle approximately 1/5th of a monolayer of Li2CO3 is deposited. For the plasma-assisted process, about 1/4th to 1/3rd of a monolayer is deposited every cycle. For plasma ALD at 300 C, the amount of deposited Li atoms deviates strongly from the lower temperature processes. At the same time the SE growth per cycle for this sample, but also for the one prepared at 275 C, deviates from the lower temperature depositions. ‡ The surface density in atoms per cm2 can be calculated via 2 r N m 2=3 , where r is the mass density, m the molecular mass of Li2CO3 and N Avogadros number. Chemical composition The stoichiometry of 50 nm thick lms was probed using a combination of ERD and EBS. The atomic percentage of all the elements in the lm is reported in Table 2. For thermal and plasma-assisted ALD lms grown up to 250 C, the results point out a stoichiometry very similar to Li2CO3. The lms are slightly sub-stoichiometric, since the carbon content in the lms is relatively low (stoichiometric Li2CO3 contains 33.3 at% Li, 16.7 at% C and 50.0 at% O). Furthermore, the calculated mass density for these samples is slightly lower than the bulk density of 2.11 g cm3. Only a few at% of H is found in the lms. Plasma ALD at 300 C on the other hand shows a high hydrogen content, suggesting the co-presence of LiOH. As mentioned before, air exposure of the fabricated layers was minimized by transporting the samples in an evacuated trans- port tube, although some degradation of the layers cannot be excluded. The hydrogen content is not constant through the lm thickness, but is mainly present in the lm's depth indi- cating air reactivity aer deposition (a depth prole shown in Fig. 3 (a) In situ thickness measurements by SE for plasma-assisted ALD of Li2CO3 at 50, 200, 275 and 300 C and thermal ALD of Li2CO3 at 150 C. The ﬁlm growth between 50 and 200 C with plasma ALD was very similar (see ESI†). (b) Growth per cycle as a function of process table temperature for both plasma-assisted (squares) and thermal (circles) ALD processes. This journal is © The Royal Society of Chemistry 2017 41362 \| RSC Adv., 2017, 7, 41359–41368 Paper Table 2 Properties of 50 nm ﬁlms fabricated using the standard thermal ALD process at 150 C and plasma ALD process at 150, 250 and 300 C. The growth per cycle in terms of atoms per nm2 and the atomic percentages where determined by EBS and ERD. The mass density was obtained by combining EBS and SE results. In the ﬁrst row the typical error is given for a certain parameter Sample GPC (at. This journal is © The Royal Society of Chemistry 2017 Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The lms deposited at higher temperatures changed when they were removed from the reactor as could be observed visually. Furthermore, the ellipsometric data obtained ex situ could not be tted using the same model as during in situ measurements. The instability, deviating growth per cycle and increased hydrogen content of these layers suggests that the formed layers were not Li2CO3 but rather a mixture of LiOH and Li2O. The increased growth per cycle can be explained by the fact that LiOH is hygroscopic and easily absorbs H2O and forms a hydrate.16,23,28 It is reported for thermal ALD of LiOH at 225 C that long purge times of 2 hours lead to a stable mass measured by QCM as a result of H2O desorption.16 The in situ SE measurements presented in Fig. 4 show the formation of LiOH/Li2O for plasma-assisted ALD at processing temperatures > 250 C. For these temperatures it was also Fig. 4 Phase shift of the incident light (D) from in situ SE measure- ments of the reaction of CO2 and H2O with 50 nm plasma ALD ﬁlms grown at 250, 275 and 300 C. First, CO2 was dosed into the reaction chamber 20 times with pulses of 2 s, next H2O was pulsed 20 times for 50 ms and afterwards CO2 was again pulsed 20 times. SE data was recorded after every pulse and D is plotted at 589 nm. To further investigate the lm instability for growth temperatures above 250 C, 50 nm thick lms were fabricated at diﬀerent deposition temperatures. These were then subse- quently exposed to CO2 and H2O pulses inside the ALD reactor. The lms were measured by in situ SE and the phase change (D) was monitored at 589 nm aer every pulse as shown in Fig. 4. For plasma depositions at 250 C, D was unaﬀected by CO2 and H2O exposure, pointing out the stability of the lm. However, the lm deposited at a slightly higher temperature of 275 C strongly reacted with CO2, whereas for the lm deposited at 300 C D only slightly changes during the rst pulse. Both LiOH and Li2O could react with CO2 to form Li2CO3. However, it is reported in literature that the reaction between Li2O and CO2 Fig. Chemical composition per nm2) [Li] (at%) [C] (at%) [O] (at%) [H] (at%) Mass density (g cm3) Thermal 150 C 2.09 0.1 33.4 1.7 14.6 0.8 50.7 2.0 1.3 0.2 1.95 0.20 Plasma 150 C 3.08 32.7 15.1 49.6 2.6 2.06 Plasma 250 C 2.76 30.5 15.8 50.9 2.8 1.97 Plasma 300 C 6.83 30.9 13.3 44.7 11.1 n.a. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 In situ SE thickness measurement for plasma-assisted ALD of Li2CO3 at 300 C using diﬀerent O2 plasma exposure times. shown that the lm thickness versus number of ALD cycles develops diﬀerently. In the rst 100 cycles the lm growth was signicantly diﬀerent from the rest of the deposition which is correlated with the transition in lm chemical composition. Since the use of plasma at processing temperatures > 250 C has a signicant eﬀect on the lm growth and composition, also the eﬀect of the plasma duration was investigated. Up to this point, a plasma exposure of 3 s was adopted. Fig. 5 shows the thick- ness evolution with number of ALD cycles for various plasma exposure times. As observed from the graph, the bulk growth per cycle for diﬀerent plasma exposure times is similar. However, for longer plasma exposure times, the growth per cycle requires a lower number of ALD cycles to become linear, compared to shorter plasma exposure times. The formation of LiOH/Li2O can thus be further stimulated by prolonging the plasma exposure time. Li2CO3 lms which are only slightly sub-stoichiometric can thus be deposited by thermal as well as by plasma-assisted ALD. In addition, it is possible to deposited Li2O at temperatures > 250 C using the plasma-assisted process. It has already been shown in literature that this is also possible using thermal ALD for temperatures > 250 C, when the CO2 step was omitted from the process.19 Deposition of Li2O can be benecial when pres- ence of carbon in the lms is undesirable. On the other hand, with thermal ALD it is possible to deposit Li2CO3 lms also at high temperatures of 300 C. If high temperature Li2CO3 depositions are preferred, for example when the process is combined in super-cycles (to make more complex material structures) employing also precursors or co-reactants which require higher deposition temperatures, the thermal process could be preferred. The chemical composition of the deposited lms was further studied by ex situ XPS. No sputtering procedure was adopted, as it was found that the stoichiometry of the lms changed due to interaction with Ar ions. Upon chemical decomposition during sputtering, Li2CO3 experiences a transition to Li2O (ESI†). In Fig. 6, the XPS core level spectra of C 1s, Li 1s and O 1s are shown for thermal ALD at 150 C. The binding energy scale was Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 4 Phase shift of the incident light (D) from in situ SE measure- ments of the reaction of CO2 and H2O with 50 nm plasma ALD ﬁlms grown at 250, 275 and 300 C. First, CO2 was dosed into the reaction chamber 20 times with pulses of 2 s, next H2O was pulsed 20 times for 50 ms and afterwards CO2 was again pulsed 20 times. SE data was recorded after every pulse and D is plotted at 589 nm. Fig. 4 Phase shift of the incident light (D) from in situ SE measure- ments of the reaction of CO2 and H2O with 50 nm plasma ALD ﬁlms grown at 250, 275 and 300 C. First, CO2 was dosed into the reaction chamber 20 times with pulses of 2 s, next H2O was pulsed 20 times for 50 ms and afterwards CO2 was again pulsed 20 times. SE data was recorded after every pulse and D is plotted at 589 nm. Fig. 4 Phase shift of the incident light (D) from in situ SE measure- ments of the reaction of CO2 and H2O with 50 nm plasma ALD ﬁlms grown at 250, 275 and 300 C. First, CO2 was dosed into the reaction chamber 20 times with pulses of 2 s, next H2O was pulsed 20 times for 50 ms and afterwards CO2 was again pulsed 20 times. SE data was recorded after every pulse and D is plotted at 589 nm. This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 41359–41368 \| 41363 View Article Online RSC Advances Paper Fig. 5 In situ SE thickness measurement for plasma-assisted ALD of Li2CO3 at 300 C using diﬀerent O2 plasma exposure times. calibrated by using the hydrocarbon contamination C 1s peak at 285.0 eV. As expected, the C 1s spectrum shows a main peak at 290.1 eV assigned to the carbonate environment in Li2CO3. The other peaks tted with a red line are associated with adventitious carbon on the sample surface. In the Li 1s spec- trum a peak at 55.4 eV is observed which is associated to Li2CO3. The O 1s spectrum consists of a peak with a maximum at 531.8 eV. The shape of the peak is slightly asymmetric. To visualize the asymmetry a second peak is tted to the data. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The asymmetry can be explained by the crystallographic structure of Li2CO3 in which two oxygen atoms have an identical environ- ment, but the third oxygen has a diﬀerent environment of surrounding atoms.30 The obtained stoichiometry for this lm was: 33.0 at% lithium, 17.7 at% carbon and 49.3 at% oxygen, corresponding to stoichiometric Li2CO3. Proportionate, there is slightly more carbon than obtained by EBS. To some extent, the diﬀerence can be explained by the low penetration depth of XPS (10 nm) and the presence of carbon on the sample surface. All other lms (prepared by plasma as well as thermal ALD) are measured and subsequently tted using the same method and all showed the expected Li2CO3 stoichiometry. The results are summarized in the ESI.† Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Crystallinity and morphology XRD was used to determine the crystallinity of the lms deposited at diﬀerent temperatures. Fig. 7 shows the X-ray dif- fractograms for 50 nm thick lms. Both plasma and thermal Fig. 6 XPS (a) C 1s, (b) Li 1s and (c) O 1s spectra for Li2CO3 grown by thermal ALD at 150 C. The measured spectra (black line) and ﬁtted peaks (blue and red) are plotted together. The obtained Li2CO3 stoichiometry is 33.0 at% Li, 17.7 at% C and 49.3 at% O. Fig. 6 XPS (a) C 1s, (b) Li 1s and (c) O 1s spectra for Li2CO3 grown by thermal ALD at 150 C. The measured spectra (black line) and ﬁtted peaks (blue and red) are plotted together. The obtained Li2CO3 stoichiometry is 33.0 at% Li, 17.7 at% C and 49.3 at% O. This journal is © The Royal Society of Chemistry 2017 41364 \| RSC Adv., 2017, 7, 41359–41368 RSC Advances View Article Online RSC Advances View Article Online Paper Paper Fig. 7 XRD gonio scans of 50 nm thick Li2CO3 ﬁlms grown on TiN using thermal (blue) and plasma (red) ALD at diﬀerent processing temperatures. The bars in the lower panel indicate the expected diﬀraction from a Li2CO3 powder sample. The reﬂections at 42.5 originate from the TiN (200) textured substrate. Paper tive Commons Attribution 3.0 Unported Licence. direction, similar to the thermal process. At higher deposition temperatures, the spectra change signicantly and the crystal- linity seems to be suppressed. Since the samples are measured by gonio XRD, we cannot exclude that there is a crystalline component present in the lms because this technique is less sensitive to 50 nm thick lms. In Fig. 8, SEM images of the surfaces and cross-sections are shown corresponding to the samples measured by XRD. The lm growth, and resulting topography, change drastically when increasing temperature. Although for lower deposition temperatures highly uniform lms are formed, at 250 C, the lms show large thickness variations. We expect that this is related to the combustion of organic ligands and the formation of LiOH/Li2O during depo- sition induced by the O2 plasma. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Conformality Fig. 7 XRD gonio scans of 50 nm thick Li2CO3 ﬁlms grown on TiN using thermal (blue) and plasma (red) ALD at diﬀerent processing temperatures. The bars in the lower panel indicate the expected diﬀraction from a Li2CO3 powder sample. The reﬂections at 42.5 originate from the TiN (200) textured substrate. Fig. 7 XRD gonio scans of 50 nm thick Li2CO3 ﬁlms grown on TiN using thermal (blue) and plasma (red) ALD at diﬀerent processing temperatures. The bars in the lower panel indicate the expected diﬀraction from a Li2CO3 powder sample. The reﬂections at 42.5 originate from the TiN (200) textured substrate. The conformality of the deposition was studied using SEM at diﬀerent positions on high aspect ratio Si pillars. The pillars, which are 50 mm high and have a diameter of 2 mm, are coated with a 30 nm TiN current collector using ALD. On top of the TiN a 50 nm Li2CO3 layer was deposited using a table temperature of 150 C. Fig. 9 shows 3 SEM pictures taken at diﬀerent heights along the pillar for plasma-assisted as well as thermal ALD. Image (a) is taken at the top of the pillars and shows a Li2CO3 layer of 55 nm for the plasma-assisted depo- sition. Image (b) is taken half way the pillar and depicts a 45 nm Li2CO3 layer (82% of thickness at top). At the bottom (c), the plasma-assisted Li2CO3 is 40 nm thick (73%). For thermal ALD on the other hand thicknesses of 60, 52 (87%) and 48 nm (80%) are measured along the pillars. We can conclude that the conformality on high aspect ratio pillars is rather good. Note that only the lithium exposure time was varied. Elongation of the lithium dosing time drastically improves the conformality of thermal ALD. For example, when thermal ALD was adopted using 6 s lithium dosing instead of 10 s, the Li2CO3 layer at the bottom is 20 nm instead of 48 nm. ALD lms show a typical diﬀraction peak originating from crystalline Li2CO3. The gonio measurements, which detect periodicity of planes parallel to the surface, show only the peak at 31.7, meaning the process yields a preferential crystal growth orientation in the <002> direction. For thermal ALD lms, the crystallinity increases slightly with increasing depo- sition temperature. Electrochemical analysis The Li-ion conductivity of plasma and thermal ALD Li2CO3 layers was investigated using impedance spectroscopy. The measurements were performed at open circuit potential (OCP) on 50 nm Li2CO3 lms deposited at 150 C on a TiN current collector. The results are shown in Fig. 10a on a complex plane plot. Both the measured layers show a behavior characteristic of a solid electrolyte: an intercept with the x-axis at high frequency, a semi-circle at medium frequency and a 45 inclined response at low frequency. Such behavior is typically associated with the ionic conductivity through a solid material.31 The high frequency intercept with the x-axis is generally attributed to the resistance of the cables, contacts and liquid electrolyte, the semi-circle originates from the ionic conductivity through the solid material and its capacitive response. The 45 slope is typically associated with a Warburg-like response, here the Li- ion diﬀusion through the liquid electrolyte. Note that in the measurements performed here electronic leakage through the Li2CO3 lms is not probed. From the complex non-linear square tting of the models to the impedance data, diﬀerent component values are extracted. The Rs was found to be around 150 U for both thermal and plasma samples. This value corresponds to the resistance of the cables and the electrochemical cell. The other relevant param- eters are summarized in Table 3. For the thermal sample a resistance (R1) value of 25 kU is extracted, corresponding to a Li-ion conductivity of 4 1010 S cm1, well in accordance values predicted by simulation studies.32 From the constant phase element (CPE1), a capacitance value can be extracted according to the following formula:33,34 Table 3 Summary of the most relevant circuit model parameters Sample R1 (kU) C1 (F) R2 (kU) C2 (F) c2 Thermal 25 2 107 — — 5 104 Plasma 2.5 4 107 3 8 107 2 104 This journal is © The Royal Society of Chemistry 2017 Table 3 Summary of the most relevant circuit model parameters Table 3 Summary of the most relevant circuit model parameters Sample R1 (kU) C1 (F) R2 (kU) C2 (F) c2 Thermal 25 2 107 — — 5 104 Plasma 2.5 4 107 3 8 107 2 104 Further analysis was performed by tting an equivalent circuit to the measured impedance response (shown in Fig. 10a). Conformality Micropillars of 50 mm high and a diameter of 2 mm are used for this purpose. The pillars are coated with a 30 nm TiN layer on top of which a 50 nm layer of Li2CO3 was deposited. Images (a), (b) and (c) show the Li2CO3/TiN layer at respectively the top, middle and bottom of the pillars. Thickness variations from 40–55 nm (plasma) and 48–60 nm (thermal) were observed. For the thermal process a lithium dosing time of 10 s was used, whereas for the plasma process the standard dose time of 6 s was suﬃcient. The scale bar applies to all of the pictures. Fig. 9 Examination of the conformality of the Li2CO3 processes at 150 C. Micropillars of 50 mm high and a diameter of 2 mm are used for this purpose. The pillars are coated with a 30 nm TiN layer on top of which a 50 nm layer of Li2CO3 was deposited. Images (a), (b) and (c) show the Li2CO3/TiN layer at respectively the top, middle and bottom of the pillars. Thickness variations from 40–55 nm (plasma) and 48–60 nm (thermal) were observed. For the thermal process a lithium dosing time of 10 s was used, whereas for the plasma process the standard dose time of 6 s was suﬃcient. The scale bar applies to all of the pictures. For plasma ALD the conformality remains similar when varying the lithium dosing time. It is expected that the conformality can still be improved by further optimization of the precursor and plasma exposure steps. For plasma ALD the conformality remains similar when varying the lithium dosing time. It is expected that the conformality can still be improved by further optimization of the precursor and plasma exposure steps. Fig. 10 (a) Impedance spectroscopy of 50 nm Li2CO3 ﬁlms prepared by thermal (blue) and plasma-assisted (red) ALD at 150 C and (b) equivalent circuit models used to ﬁt the data. which is conrmed by the low c2 value of 5 104. When equivalent circuit model II was tried to t the response of the plasma deposited Li2CO3 layer, no adequate t could be ob- tained. For this reason, model II was used, resulting in the t shown in Fig. 10a. The quality of the t was again conrmed by the low c2 value of 2 104. Conformality A similar trend was observed in literature for thermal ALD of Li2CO3 on Si (100) substrates using a LiN(SiMe3)2 precursor,17 although the crystalline growth started only at 180 C according to their XRD spectra. The onset for crystalline growth could thus be inuenced by the precursor, the substrate or a combination of both. When considering the plasma ALD Li2CO3 lms, the layer deposited at 50 C is highly crystalline and oriented in the <002> Fig. 8 (a) High resolution SEM pictures showing the surface topography and (b) cross-section of Li2CO3 deposited with plasma ALD at 50, 150, and 250 C. The scale bar applies to all of the pictures. Fig. 8 (a) High resolution SEM pictures showing the surface topography and (b) cross-section of Li2CO3 deposited with plasma ALD at 50, 150, and 250 C. The scale bar applies to all of the pictures. This journal is © The Royal Society of Chemistry 2017 This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 41359–41368 \| 41365 Fig. 10 (a) Impedance spectroscopy of 50 nm Li2CO3 ﬁlms prepared by thermal (blue) and plasma-assisted (red) ALD at 150 C and (b) equivalent circuit models used to ﬁt the data. Paper View Article Online View Article Online RSC Advances RSC Advances Fig. 9 Examination of the conformality of the Li2CO3 processes at 150 C. Micropillars of 50 mm high and a diameter of 2 mm are used for this purpose. The pillars are coated with a 30 nm TiN layer on top of which a 50 nm layer of Li2CO3 was deposited. Images (a), (b) and (c) show the Li2CO3/TiN layer at respectively the top, middle and bottom of the pillars. Thickness variations from 40–55 nm (plasma) and 48–60 nm (thermal) were observed. For the thermal process a lithium dosing time of 10 s was used, whereas for the plasma process the standard dose time of 6 s was suﬃcient. The scale bar applies to all of the pictures. RSC Advances ed on 24 August 2017. Downloaded on 11/09/2017 14:37:14. ensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 9 Examination of the conformality of the Li2CO3 processes at 150 C. Open Access Article. Published on 24 August 2017. Downloaded on 11/09/2017 14:37:14. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The impedance response of the plasma-assisted Li2CO3 sample was also probed. Fig. 10a shows that the plasma sample has a lower ionic resistance (as indicated from the intercept with the x-axis). This semi-circle consists of two overlapping semi-circles with slightly diﬀerent time constants which is re- ected in the model choice (model II). The capacitance values were again extracted by using the previously shown formula. The values found were 400 and 800 nF for respectively CPE1 and CPE2. These values correspond to dielectric constants of 40 and 80, so signicantly higher than the one of the thermal sample and too high for typical oxide lms. Both plasma-assisted and thermal ALD of Li2CO3 allow for conformal coating of high aspect ratio substrates as can be important for future Li-ion batteries. To the best of our knowledge this paper provides the rst proper conductivity measurement of Li2CO3 ALD lms. An ion conductivity in the order of 1010 S cm1 was obtained for both plasma and thermal ALD aer normalizing the tted conductivity values to the ratio of the diﬀerent eﬀective surface areas. Studies are ongoing to characterize the electrochemical properties of the Li2CO3 lms in more depth. Such capacity values can be attributed to a change in dielectric constant or diﬀerent geometrical dimensions. However, no diﬀerence in mass density could be detected between the thermal and plasma Li2CO3 layers (see Table 2). Therefore, it is unlikely that a signicant diﬀerence in dielectric constant exists between the two layers. When considering the morphology of the layers (see Fig. 8), a columnar structure can be seen. Therefore, the liquid electrolyte may contact the indi- vidual grains. Clearly this would lead to a signicant increase in contact area and thus an increased capacitance value. The distribution in grain size can in such case lead to a distribution in time constants. The two resistance values extracted from the tting, each correspond to a Li-ion conductivity value in the order of 1 109 S cm1. However, the underestimation of the contact area introduces a signicant uncertainty on this value which, based on the increased surface area, will be over- estimated. When normalizing the conductivity value found here to the ratio of the diﬀerent eﬀective surface areas of the thermal and plasma samples, a conductivity value of 1010 S cm1 is obtained, a value again in line with other literature reports.32 References 1 K. Chung, J.-D. Lee, E.-J. Kim, W.-S. Kim, J.-H. Cho and Y.-K. Choi, Microchem. J., 2003, 75, 71–77. 2 Y. C. Zhang, H. Tagawa, S. Asakura, J. Mizusakib and H. Narita, Solid State Ionics, 1997, 100, 275–281. 3 Y. Shimamoto, T. Okamoto, Y. Itagaki, H. Aono and Y. Sadaoka, Sens. Actuators, B, 2004, 99, 113–117. 4 J. Mizusaki and H. Tagawa, Solid State Ionics, 1992, 53–56, 791–797. 5 S. Bhattacharya, A. R. Riahi and A. T. Alpas, Carbon, 2014, 77, 99–112. 6 J. W. Long, B. Dunn, D. R. Rolison and H. S. White, Chem. Rev., 2004, 104, 4463–4492. 7 J. F. M. Oudenhoven, L. Baggetto and P. H. L. Notten, Adv. Energy Mater., 2011, 1, 10–33. 8 G. W. Rubloﬀ, A. C. Kozen and S. B. Lee, J. Vac. Sci. Technol., A, 2013, 31, 58503. 1 K. Chung, J.-D. Lee, E.-J. Kim, W.-S. Kim, J.-H. Cho and Y.-K. Choi, Microchem. J., 2003, 75, 71–77. 2 Y. C. Zhang, H. Tagawa, S. Asakura, J. Mizusakib and H. Narita, Solid State Ionics, 1997, 100, 275–281. 3 Y. Shimamoto, T. Okamoto, Y. Itagaki, H. Aono and Y. Sadaoka, Sens. Actuators, B, 2004, 99, 113–117. 4 J. Mizusaki and H. Tagawa, Solid State Ionics, 1992, 53–56, 791–797. 4 J. Mizusaki and H. Tagawa, Solid State Ionics, 1992, 53–56, 791–797. Acknowledgements The authors gratefully acknowledge Dr Marcel Verheijen for fruitful discussion and Cristian van Helvoirt and Jeroen van Gerwen for technical assistance. This project is nancially supported by the Dutch program “A green Deal in Energy Materials” ADEM Innovation Lab. This journal is © The Royal Society of Chemistry 2017 Electrochemical analysis The response of the Li2CO3 layer deposited by thermal ALD could be adequately tted using model I shown in Fig. 10b 41366 \| RSC Adv., 2017, 7, 41359–41368 RSC Advances View Article Online RSC Advances View Article Online RSC Ad View Article Online Paper C ¼ Q1/nR1 (1n)/n window. Especially the use of plasma allows for the possibility to deposit at lower process temperatures, which would be benecial for temperature-sensitive substrates. Moreover, plasma-assisted ALD allows for shorter deposition times and higher growth per cycle values leading to an increase in reactor throughput. Usage of this formula assumes a normal distribution of the time constants associated with the constant phase element. It allows an accurate determination of the capacitance value associated with the CPE even with an n value around 0.8.33,34 By using the above formalism, a capacitance value of 200 nF is extracted for CPE1, corresponding to a dielectric constant of 25. This value is higher than what is expected for typical oxide lms. However, the large surface roughness of the thermal lms must be considered (see ESI Fig. S4†). Such roughness increases the eﬀective contact area of the capacitor which is not included when using the geometrical surface area. For this reason also the extracted ionic conductivity value will be slightly over- estimated and can be adjusted for surface roughness down to a value of 1010 S cm1. Stoichiometric Li2CO3 lms can be deposited with both ALD processes. In addition, it is possible to deposited Li2O at temperatures >250 C through combustion of the organic ligands. This could be benecial when presence of carbon in the lms is undesirable. For thermal ALD it is necessary to also omit the CO2 step from the process to obtain Li2O. For plasma- assisted ALD, introduction of Li2O in the lms is accompa- nied with suppression of lm crystallinity and increases lm non-uniformity. The formation of Li2O can be further promoted by prolonging the plasma exposure time. 41368 \| RSC Adv., 2017, 7, 41359–41368 This journal is © The Royal Society of Chemistry 2017 Conclusions 5 S. Bhattacharya, A. R. Riahi and A. T. Alpas, Carbon, 2014, 77, 99–112. 5 S. Bhattacharya, A. R. Riahi and A. T. Alpas, Carbon, 2014, 77, 99–112. A comparison has been made between the remote plasma ALD process and its more extensively adopted thermal counterpart. Self-limiting growth was obtained for both processes within a wide temperature window. 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https://openalex.org/W4281635209	https://zenodo.org/records/6596145/files/ARIMS029.pdf	English	null	METHODS OF DEVELOPING STUDENTS' RESEARCH ACTIVITIES IN THE PROCESS OF BIOLOGY EDUCATION IN PEDAGOGICAL HIGHER EDUCATION INSTITUTIONS	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,465	ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference In educational practice, most of the knowledge is provided ready-made and does not require additional research efforts, but the main challenge for students is to search for information independently, to acquire knowledge. Therefore, one of the most important conditions for increasing the effectiveness of the educational process is the organization of teaching and research activities of students, and its main component is the development of research skills. This not only helps students to meet the requirements of higher education qualifications, but also forms them as mature professionals, creating an internal motivation for academic activity [3] Involvement of students in research activities, modernization and further improvement of the teaching process in the higher education system to increase motivation, increase the quality and effectiveness of teaching, independent learning through the formation of independent and creative activities of students. It is necessary to focus on research work through the proper organization and systematic organization of work in this area, to ensure continuity and to establish control and monitoring. Doing so will allow students to develop independent and creative thinking skills, broaden their scientific horizons, increase their interest in research, and focus on research[1;].. This, in turn, is a serious way to organize the work of scientific and design circles in higher education, where working with gifted students is not limited to the study and analysis of theoretical data, but also experimental. It is necessary to establish an innovative, technological approach to the development of practical measures for the implementation of experiments. To do this, special attention should be paid to the development of independent learning of students [4]. The content of research tasks is to develop students' cognitive independence, to cultivate a careful attitude to nature, to develop special learning skills, to study the diversity of flora and fauna, adaptation to the living environment, the independent study of the biology room. The resulting herbariums are intended to complement the collections [2;]. 1. Learn to use research methods (teacher recommends problem-solving tasks, students solve tasks independently). 1. Learn to use research methods (teacher recommends problem-solving tasks, students solve tasks independently). This method is based on the independent application of the acquired knowledge and skills at a high level. This method provides the experience of creative activity. We used this method to solve creative biological tasks in the classroom. METHODS OF DEVELOPING STUDENTS' RESEARCH ACTIVITIES IN THE PROCESS OF BIOLOGY EDUCATION IN PEDAGOGICAL HIGHER EDUCATION INSTITUTIONS M.N.Ataqulova1 Sh.Q.Nasirova2 1 Senior Lecturer of Navoi State Pedagogical Institute p.f.f.d. PhD 2Senior Lecturer of Navoi State Pedagogical Institute https://doi.org/10.5281/zenodo.6596145 Annotation: The article shows the effectiveness of the use of various educational technologies in the development of student research activities in the process of biological education. In the coverage of research methods, it is important to develop the activities of university students through problem- solving and design technology - complexity and integration. Keywords: scientific research, intellectual map, organizational, content, activity, cognitive map, task, analysis, reflection. research, research-competition, experiment, herbarium, collection, flora, fauna, volera, terrarium, aquarium Orientation of young people to research activities in higher education institutions around the world, training of highly qualified personnel in the field of education, their independent scientific and creative approach to each type of activity, development of competencies to apply the received innovations in practice, science , ensuring the integration of education and production, preparation for scientific and methodological activities as a competent staff is of great importance. Involvement of students in scientific work, independent research activities, the application of experimental results in practice is a solid foundation for the training of independent, potential, creative research and teaching staff. Extensive work is being done in our country on freedom of scientific creativity, research, development and support of research activities. Development of scientific and research activities, "identification of priorities for the systematic reform of science in the future, training of highly qualified personnel with modern knowledge and independent thinking, modernization of scientific infrastructure" to a qualitatively new level, pedagogical higher education Effective organization of methodological and research work in biology in educational institutions, orientation of young people in scientific organizations and higher education institutions to the systematic analysis of research work, appropriate conditions for research activities of young scientists and students special attention is paid to the creation of [3] 126 ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference 2. Express research method. Individual assignments for empirical research include field trips and field trips: 2. Express research method. Individual assignments for empirical research include field trips and field trips: 1. What animals live in an endemic area? 2. Explain the life forms of ornamental plants in endemic areas? such a xplain the life forms of ornamental plants in endemic areas? such as. 3. Theoretical express research should focus on independent and creative thinking. 3. Theoretical express research should focus on independent and creative thinking. The following research topics can be recommended: “Lifestyle of Xerophytic Plants”, “Characteristics of Medicinal Plants”, “Importance of Air Rooted Plants”. The authors will make short presentations. 3. Conduct a learning experience. Learning experience is one of the most effective teaching methods, including practical work in biology and laboratory work. In doing so, the student will be able to work independently and gain new knowledge. In doing so, students develop the skills of analyzing data, observing, recording and recording results, drawing conclusions, and improving basic, personal, and interdisciplinary competencies. 4. Research competition. Competition is also one of the most effective methods. For example, the best "Presentation" contest. The textbook is prepared in advance. This text can be a part of the textbook: "Origin of cultivated plants and centers of diversity", "Selection of microorganisms", "Fundamentals of Genetics" and others. In composing the text of the presentation, students combine basic competencies with logical connections between specific and interdisciplinary competencies. This method teaches students to use scientific literature wisely. 5. Non-traditional lessons (presentation "Natural and artificial ecosystems", lecture "Pest control" - discussion) 5. Non-traditional lessons (presentation "Natural and artificial ecosystems", lecture "Pest control" - discussion) Students prepare research from independent textbooks and media sources. This encourages students to be independent and creative. 6. Research projects. ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference 127 We used this method to solve creative biological tasks in the classroom. 6. Research projects. MN Atakulova., Samarkand 2021 6. Research projects. 6. Research projects. In research projects, students' independent research by making plans based on a topic can be considered the highest level of research activity. By mastering the theoretical express research method, students who have the skills to do practical experimental work are successfully completing the experimental part of the projects implemented according to specially selected methods. However, one lesson is not enough to complete a study project [3;]. Homework can be research: 1. The evolution of plants according to plan ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference Assignment: Describe the evolution of plants according to the following plan. ACADEMIC RESEARCH IN MODERN SCIENCE International scientific-online conference Assignment: Describe the evolution of plants according to the following plan. 1. The evolution of plants on Earth 2. Unicellular and multicellular algae. 3. The growth of algae. 4. An increase in the number of joints 5. Reproduction of crustaceans 6. The development of the open seed world. 7. Closed seeds. development of the world 8. One-seeded and two-seeded class. 9. Album preparation: ("I study the animal world", "Ancient mamm 9. Album preparation: ("I study the animal world", "Ancient mammals")[5;].. The use of educational technologies in the world of biology plays an important role in teaching students to think creatively and logically, in mastering the methods of intellectual activity, in developing their scientific, critical-analytical, logical thinking skills and in directing research activities. Thus, in the process of teaching biology, students use a variety of methods, forms and tools to develop research activities. At the same time, in the process of teaching biology, the technology of developing students' research activities also plays an important role in providing them with sufficient experience in this field. References 1. Site L.A. Development of issledovatelskoy deyatelnosti studentov vuza v usloviyax problemno-konsentrirovannogo obucheniya. Disc. k.p.n. Omsk-2017 2. Tolipova J.O. Innovative technologies in teaching biology / TDPU-2013. −156 b. References 1. Site L.A. Development of issledovatelskoy deyatelnosti studentov vuza v usloviyax problemno-konsentrirovannogo obucheniya. Disc. k.p.n. Omsk-2017 2. Tolipova J.O. Innovative technologies in teaching biology / TDPU-2013. −156 b. 3. Improving the methods of developing research activities in students of pedagogical higher education institutions (on the example of the direction of teaching biology teaching methods). Dissertation work. MN Atakulova., Samarkand-2021. 3. Improving the methods of developing research activities in students of pedagogical higher education institutions (on the example of the direction of teaching biology teaching methods). Dissertation work. 129 129
https://openalex.org/W4256028557	https://www.qeios.com/read/FHUL8V/pdf	English	null	Priapism	Definitions	2,020	cc-by	86	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Priapism National Cancer Institute National Cancer Institute Qeios ID: FHUL8V · https://doi.org/10.32388/FHUL8V Open Peer Review on Qeios Source National Cancer Institute. Priapism. NCI Thesaurus. Code C85022. Persistent and usually painful erection that lasts for at least four hours in the absence of physical or psychological stimulation, which can be caused by hematologic disorders, including sickle cell disease and leukemia, spinal cord injuries, and medications. Qeios ID: FHUL8V · https://doi.org/10.32388/FHUL8V 1/1
https://openalex.org/W12314354	https://hal.inria.fr/hal-01524964/file/978-3-642-32891-6_20_Chapter.pdf	English	null	On the Support of Ad-Hoc Semantic Web Data Sharing	IFIP advances in information and communication technology	2,012	cc-by	4,833	To cite this version: Jing Zhou, Kun Yang, Lei Shi, Zhongzhi Shi. On the Support of Ad-Hoc Semantic Web Data Sharing. 7th International Conference on Intelligent Information Processing (IIP), Oct 2012, Guilin, China. pp.147-156, 10.1007/978-3-642-32891-6_20. hal-01524964 On the Support of Ad-Hoc Semantic Web Data Sharing Jing Zhou, Kun Yang, Lei Shi, Zhongzhi Shi To cite this version: Jing Zhou, Kun Yang, Lei Shi, Zhongzhi Shi. On the Support of Ad-Hoc Semantic Web Data Sharing. 7th International Conference on Intelligent Information Processing (IIP), Oct 2012, Guilin, China. pp.147-156, 10.1007/978-3-642-32891-6_20. hal-01524964 On the Support of Ad-Hoc Semantic Web Data Sharing Jing Zhou, Kun Yang, Lei Shi, Zhongzhi Shi On the Support of Ad-Hoc Semantic Web Data Sharing Jing Zhou, Kun Yang, Lei Shi, Zhongzhi Shi Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01524964 https://inria.hal.science/hal-01524964v1 Submitted on 19 May 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License On the Support of Ad-Hoc Semantic Web Data Sharing Jing Zhou1,2, Kun Yang1, Lei Shi1, and Zhongzhi Shi2 Jing Zhou1,2, Kun Yang1, Lei Shi1, and Zhongzhi Shi2 1 School of Computer Science, Communication University of China, Beijing, 100024, China {zhoujing,yangkun,shilei_cs}@cuc.edu.cn 2 The Key Laboratory of Intelligent Information Processing, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, 100190, China shizz@ics.ict.ac.cn Abstract. Sharing Semantic Web datasets provided by diﬀerent pub- lishers in a decentralized environment calls for eﬃcient support from distributed computing technologies. Moreover, we argue that the highly dynamic ad-hoc settings that would be pervasive for Semantic Web data sharing among personal users in the future pose even more demanding challenges for enabling technologies. We propose an architecture that is based upon the peer-to-peer (P2P) paradigm for ad-hoc Semantic Web data sharing and identify the key technologies that underpin the im- plementation of the architecture. We anticipate that our (current and future) work will oﬀer powerful support for sharing of Semantic Web data in a decentralized manner and becomes an indispensable and com- plementary approach to making the Semantic Web a reality. Keywords: Semantic Web, RDF triples, storage organization, data shar- ing, decentralized query processing 1 The Semantic Web, Data, and Data Storage Jing Zhou et al. 2 Virtually, in the Semantic Web one can create links between any Web data that is identiﬁed by Uniform Resource Identiﬁer (URI) references with the Re- source Description Framework (RDF) [13] being utilised to describe the associa- tion between the data that the link represents. The RDF is a standard model for exchanging data on the Web as well as a language for describing Web resources or anything that is identiﬁable on the Web. The basic RDF data model adopts triples in the form of (subject, predicate, object) to depict the attributes of Web resources. RDF triples are also referred to as RDF statements and stored in data stores known as triplestores. A triplestore may contain millions of triples and is mainly responsible for not only storage but also eﬃcient reasoning over and retrieving triples in it. Typi- cally, triplestores come in three forms: (1) RDF triples are stored in relational database management systems (DBMSs), (2) an XML database is employed to store RDF data, or (3) a proprietary information repository is developed to accommodate RDF triples. Among others, the practice of building triplestores on top of relational DBMSs has gained wide adoption thanks to their power- ful transaction management. The underlying relational DBMSs deal with the storage and organization of RDF triples in one of the following schemes: triple tables, property tables [19], and vertical partitioning [1]. For a more detailed discussion of individual schemes, interested readers may refer to [5]. 2 The Development of Triplestores Triplestores provide storage and management services to Semantic Web data and hence are able to support data sharing in specialized Semantic Web applications including Semantic Web search engines [10], personal information protection products [9], and wikis. In this section, we will present several typical systems and focus on their logical storage, data description, and query processing mechanisms in particular. 3 See http://www.xml.com/pub/a/2004/06/23/kowari.html. 1 The Semantic Web, Data, and Data Storage The Semantic Web was intended to transform heterogeneous and distributed data into the form that machines can directly process and manipulate by pro- viding the data with explicit semantic information, thus facilitating sharing and reusing of data across applications. To make the Semantic Web a reality, one im- portant way is publishing a large amount of data encoded in standard formats on the Web. These data and the associations between them are both essential for supporting large scale data integration and the automated or semi-automated querying of data from disparate sources. In the early days of the Web, documents were connected by means of hyper- links, or links, and data was not provided independently of their representation in the documents. It is obvious that the ultimate objective of the Semantic Web calls for a shift of the associative linking from documents to data [7]–the Seman- tic Web turns into the Web of Linked Data whereby machines can explore and locate related data. 4 At the time of writing of [6], most Semantic Web data were encoded in the XML format and hence Piazza mainly dealt with the issues arising from XML data man- agement. 2.2 Centralised Triplestores YARS2 [10] was a distributed system for managing large amounts of graph- structured RDF data. RDF triples were stored as quadruple (subject, predicate, object, context) with context indicating the URL of the data source for the contained triple. An inverted text index for keyword lookups and the sparse index-based quad indices were established for eﬃcient evaluation of queries. For scalability reason, the quad index placement adopted established distributed hash table substrates that provided directed lookup to machines on which the quadruple of interest could be located. The Index Manager on individual ma- chines provided network access to local indices such as keyword indices and quad indices. The Query Processor was responsible for creating and optimising the log- ical plan for answering queries. It then executed the plan over the network by sending lookup requests to and receiving response data from the remote Index Managers in a multi-threaded fashion. 4store [9] was implemented on a low-cost networked cluster with 9 servers and its hardware platform was built upon a shared-nothing architecture. RDF triples were represented as quads of (model, subject, predicate, object). All the data was divided into a number of non-overlapping segments according to the RIDs (Resource IDentiﬁers) that were calculated for the subject of any given RDF triple. Each Storage Node maintained one or more data segments. To allow the Processing Node to discover the data segments of interest, each Storage Node in a local network advertised its service type, contained dataset with a unique name, and the total number of data segments. Hence, the Processing Node could locate the Storage Nodes that host the desired data segments by checking the advertised messages and answer queries from the client. Furthermore, DARQ [15] and [11] utilising Hadoop and MapReduce are among the systems that process RDF data queries in a centralised way. 2.1 Single Machine Supported Triplestores Sesame [3] was developed as a generic architecture for storage and querying of large quantities of Semantic Web data and it allowed the persistent storage of RDF data and schema information to be provided by any particular underlying storage devices. The Storage And Inference Layer (SAIL) was a single archi- tectural layer of Sesame that oﬀered RDF-speciﬁc methods to its clients and translated these methods to calls to its certain DBMS. A version of RQL (RDF Query Language) [12] was implemented in Sesame that supported querying of RDF and RDFS [2] documents at the semantic level. Other single machine supported triplestores include 3store [8] that adopted a relational DBMS as its repository for storage and Kowari3, an entirely Java- based transactional, permanent triplestore. On the Support of Ad-Hoc Semantic Web Data Sharing 3 3 On the Support of Ad-Hoc Semantic Web Data Sharing 2.4 Cloud Computing-based Triplestores SemaPlorer [16], which was awarded the ﬁrst prize at the 2008 Semantic Web Challenge Billion Triples Track, was built upon the cloud infrastructure. Seman- tic data from sources including DBPedia, GeoNames, WordNet, personal FOAF ﬁles, and Flickr, was integrated and leveraged in the SemaPlorer application. SemaPlorer allowed users to enjoy, in real-time, blended browsing of an interest- ing area in diﬀerent context views. A set of 25 RDF stores in SemaPlorer was hosted on the virtual machines of Amazon’s Elastic Computing Cloud (EC2), and the EC2 virtual machine images and the semantic datasets were stored by Amazon’s Simple Storage Service (S3). SemaPlorer employed Networked Graphs [17] to integrate semantically heterogeneous data. In theory, any distributed data sources can be integrated into the data infrastructure of SemaPlorer in an ad- hoc manner on a very important premise, that is, such data sources can be located by querying SPARQL (SPARQL Protocol And RDF Query Language) endpoints [14]. However, the same premise does not hold in an ad-hoc settings since peers have no knowledge about the speciﬁc locations of these data sources in the absence of a fully distributed query forwarding and processing mechanism. 2.3 Fully Distributed Triplestores Piazza [6] was one the few unstructured P2P systems that supported data4 man- agement in Semantic Web applications. Each node provided source data with its schema, or only a schema (or ontology) to which schemas of other nodes could be mapped. Point-to-point mappings (between domain structures and document structures) were supported by Piazza whereby nodes could choose other nodes with which they would like to establish semantic connections. One of the roles that mappings in Piazza played was serving as storage descriptions that speci- ﬁed what data a node actually stored. A ﬂooding-like technique was employed to process queries and the designers claimed that they focused mostly on ob- taining semantically correct answers. The prototype system of Piazza comprised 25 nodes. 4 Jing Zhou et al. RDFPeers [4] was a distributed and scalable RDF repository. Each triple in RDFPeers was stored at three places in a multi-attribute addressable network (MAAN) by applying hash functions to the subject, predicate, and object values of the triple. Hence, data descriptions were given implicitly as identiﬁers in the one-dimensional modulo-2m circular identiﬁer space of MAAN. MAAN not only supported exact-match queries on single or multiple attributes but also could eﬃciently resolve disjunctive and range queries as well as conjunctive multi- attribute queries. RDFPeers was demonstrated to provide good scalability and fault resilience due to its roots in Chord [18]. [ ] S-RDF [20] was a fully decentralized P2P RDF repository that explored ways to eliminate resource and performance bottlenecks in large scale triplestores. Each node maintained its local RDF dataset. Triples in the dataset were further grouped into individual RDF data ﬁles according to the type of their subject. The Description Generator took an RDF data ﬁle as input to generate a description of the ﬁle, which could comprise up to several terms, with the help of ontolo- gies. Considering the semantic relationship that might exist between RDF data ﬁles hosted by diﬀerent nodes, S-RDF implemented a semantics-directed search protocol, mediated by topology reorganization, for eﬃciently locating triples of interest in a fully distributed fashion. Desired scalability was demonstrated through extensive simulations. 5 Triplestores built upon distributed hash tables are an exception to this but still not applicable to solving our problem because of the data placement issue, that is, any single RDF triple needs to be placed at some location. In our scenario, the tripletore does not have the issue, which excludes the use of distributed hash tables. 2.5 Summary In brief, triplestores that were intended to support data sharing and reusing have shifted from centralised to distributed architecture over time. The inherent reasons for this are manifold. To begin with, every single machine is unable to accommodate all existing (and coming) Semantic Web data and process them in On the Support of Ad-Hoc Semantic Web Data Sharing 5 5 memory. Then, copying large amounts of distributed data to one machine and processing them in a centralised manner will inevitably result in issues such as increased network traﬃc, deteriorated query eﬃciency, and even infrigement of copyrights sometimes. Furthermore, personal users of the future sharing Seman- tic Web data would seemingly appear the same way that Web users of today share music, video, and image ﬁles; the distinguishing features of Semantic Web data, including being structured and given well-deﬁned associations, however, urge us to come up with new distributed computing technologies. An ad-hoc setting in which Semantic Web data sharing should be supported certainly brings about more challenges. 3 Semantic Web Data Sharing – What is Still Missing? Hall pointed out in [7] that the great success of the early Web is attributed in part to the power of the network eﬀect, that is, more people will join a network to get the beneﬁts as its value increases and meanwhile people contribute more information to the network, thus further increasing its value. The network ef- fect is expected to exhibit in the Semantic Web and promote its success. Hence, research communities and organizations were encouraged to publish and share data that can be mapped onto URIs and links to other data, thus increasing the value of the data and the Semantic Web. Ever since, triplestores maintained by individual publishers for storing RDF data from particular domains came into existence. These data stores provide generic features such as data access, retrieval, and deletion. Meanhile, system designers manage to increase the scala- bility and robustness of the data stores through enabling techniques to deal with the ever-increasing volume of Semantic Web data. For Semantic Web data sharing among diﬀerent publishers, querying is an indispensable mechanism. Regardless of the paradigm in which RDF data is stored (centralised or distributed), existing query mechanisms are rather simple and most assume that the target data is within two hops away, that is, the data can be simply located by directly interrogating some central directory node5. For more complex scenarios, for example a fully distributed ad-hoc environ- ment in which the target data may be more than two hops away, Semantic Web researchers have yet to supply an eﬃcient querying solution. Approaches from the P2P computing community, in the meantime, generally do not take into ac- count the inherent semantic associations between Semantic Web data, and hence the quality of the query results they yield has room for improvement. Another important factor that makes supporting ad-hoc Semantic Web data sharing a pressing issue, relates to the paradigm in which people will share RDF data on their personal computers sometime in the future. As RDF converters are becoming available for many kinds of application data, we anticipate that Jing Zhou et al. 6 large amounts of RDF data will be generated in personal computers. These data might be references to literature (e.g. BibTex), information in spreadsheets (e.g. Excel), or even data from the GPS (Global Positioning System) receiver. 3 Semantic Web Data Sharing – What is Still Missing? They would be carried around and shared with others at will–just like what we do with document ﬁles, music ﬁles, or video ﬁles in our computers. In most cases, Semantic Web data sharing among personal computers will typically occur in an ad-hoc environment6 where querying becomes much more complicated in the absence of a central directory node. Though it has long been recognised and followed that publishing and sharing large amounts of RDF data on the Web is an important approach to making the Semantic Web a reality, we argue that providing eﬃcient techniques and technologies to support ad-hoc Semantic Web data sharing, which is currently missing from the overall approach, is a complementary and indispensable so- lution the importance of which should never be underestimated. In an ad-hoc environment, Semantic Web data will only be shared in a desired way if ef- ﬁcient mechanisms for describing, manipulating, querying, and analysing data are developed. 4 An Architecture for Ad-Hoc Semantic Web Data Sharing In this section, we will present the main components in an architecture for ad- hoc Semantic Web data sharing based on the P2P paradigm. This is followed by describing the way that all the components in the architecture collaborate to resolve a query for RDF data in an ad-hoc scenario. 7 Nodes that are attached to any node on the Chord ring are not taken into account. 6 This is very much like the way that Internet users share music and video ﬁles in a P2P fashion. 7 4.1 Building an Architecture on Top of Chord Our decentralized RDF data repository consists of many individual nodes or peers (each representing a user and her client program) and extends Chord [18] with RDF-speciﬁc retrieval techniques. We assume that some of the nodes are willing to host indices for other nodes and self-organize into a Chord ring. Nodes that are reluctant to do so will need to be attached to one of the nodes on the ring. In Fig. 1 we show a peer network of ﬁve nodes7 in a 4-bit identiﬁer space. Node identiﬁers N1, N4, N7, N12, and N15 correspond to actual nodes that are willing to host indices for other peers. In the meantime, node identiﬁers D1, D2, and D3 represent three actual nodes that are attached to N12 and share information about their RDF data with N12. Hence, N12 becomes associated with all RDF data shared by D1, D2, and D3. For instance, if N4 stores in its ﬁnger table an item regarding a pointer to D3 which stores speciﬁc RDF triples, the item will only involve node identiﬁer N12 rather than D3. On the Support of Ad-Hoc Semantic Web Data Sharing 7 7 N7 N4 N1 N12 N15 D1 D2 D3 Fig. 1. A peer network of ﬁve nodes in a 4-bit identiﬁer space Fig. 1. A peer network of ﬁve nodes in a 4-bit identiﬁer space Each node has an IP address by which it may be contacted. The overall index will be spread across the nodes on the ring. Unlike RDFPeers [4], our system applies hash functions to the subject (s), predicate (p), object (o), subject and predicate (sp), predicate and object (po), and subject and object (so) types of an RDF triple and stores the information (a pointer to the node, for instance) about the node that shares the triple at six places on a Chord ring. As shown in Fig. 2, each node on the Chord ring is comprised of seven com- ponents: the RDF Converter, SPARQL Query Engine, RDF Data Repository, Pre- and Post-processor, Query Rewriter, Query Forwarder, and Result Merger. Both the RDF Converter and the SPARQL Query Engine can be obtained by downloading from the Web and some coding is needed for enabling them to interact with other components in the architecture. The RDF Data Repository, or the triplestore, is assumed to be implemented by XML databases, and then is mainly responsible for data storage. 4.1 Building an Architecture on Top of Chord The functionality of the other components is described as follows. – The Pre- and Post-processor accepts incoming queries from either the exter- nal application or any other peer in the P2P network. It directs queries for RDF triples to the Query Rewriter for further processing. When the targe RDF triples are ﬁnally obtained, the result will be transmitted to the Pre- and Post-processor for proper presentation to the query originator. – The Query Rewriter is responsible for reformulating the queries from the Pre- and Post-processor whenever needed. For instance, on the receipt of a query that speciﬁes a set of disjunctive ranges for attribute (subject, predicate, or object) values, the Query Rewriter may create several sub-queries for each attribute value if the targe triples are hosted by remote nodes. It may also transform a query into the SPARQL format for resolving by the SPARQL Query Engine. – The Query Forwarder receives queries from the Query Rewriter and, accord- ing to the query forwarding protocol, will pass on the queries to correspond- ing target nodes. g g – The Result Merger collects and compiles query results regarding all the re- mote nodes that may contain targe RDF triples and then returns the result 8 Jing Zhou et al. External application SINGLE NODE Query Rewriter Pre- and Post-processor Result Merger Query Forwarder SPARQL Query Engine GPS receiver data Excel files BibTex files RDF Data Repository P2P network RDF Converter P2P network Fig. 2. Single node in ad-hoc Semantic Web data sharing system RDF Converter Result Merger Query Rewriter Fig. 2. Single node in ad-hoc Semantic Web data sharing system to the Query Rewriter. The latter will rewrite the original query in the SPARQL format with explicit information on the named graphs that will be interrogated by the SPARQL query. 8 Note that during the ﬁrst phase, all queries (except the original one) processed by the Query Rewriter, Query Forwarder, and Result Merger are meant to discover the 4.2 Resolving a Query for RDF Data in an Ad-Hoc Scenario Data preparation and distributed index building On any single node, application-speciﬁc data from personal ﬁles can be transformed into RDF triples by the RDF Converter in batches and then inserted into the RDF Data Repos- itory. To support eﬃcient queries on decentralized RDF triples, we exploit the overlay structure of Chord to build a distributed index for locating these triples. We store pointers to the node that maintains a speciﬁc RDF triple six times, each obtained by applying hash functions to the class type of the subject, predi- cate, object, subject and predicate, subject and object, and predicate and object of the triple. Each pointer will be stored at the successor node of the hash key of the corresponding attribute type combination. Decentralized query processing Generally, resolving a query for remote RDF triples in the proposed network is divided into two phases. In the ﬁrst phase8, a query from the external application is submitted to a single node On the Support of Ad-Hoc Semantic Web Data Sharing 9 which will instruct its Pre- and Post-processor to accept the incoming query. The query is further passed onto the Query Rewriter which will reformulate the query when needed and then send it to the Query Forwarder. The Query For- warder determines the target node to which the query should be routed. When the Result Merger obtains the query result about the potential target nodes in the network, it will provide the information to the Query Rewriter. The Query Rewriter retrieves the original query, reformulates it in the SPARQL format us- ing the information from the Result Merger, and submits it to the SPARQL Query Engine. In the second phase, the SPARQL Query Engine resolves the SPARQL query by querying against all relevant named graphs. The SPARQL query result containing the RDF data of interest is returned to the Pre- and Post-processor for presentation to the external application. 5 Conclusions and Future Work We proposed in this position paper a P2P architecture for Semantic Web data sharing in an ad-hoc environment. To fully support the implementation of the architecture, we also need to address the following issues in the future work: (1) decentralized query processing mechanisms that resolve exact-match queries, disjunctive queries, conjunctive queries, and range queries for RDF data and (2) enhancement techniques for RDF data query performance by taking advantage of the semantic relationship between Semantic Web data and by dynamically changing the network topology based on the local knowledge of peer nodes. Acknowledgments. The ﬁrst author is grateful for the discussion with Prof. Gregor v. Bochmann from Ottawa University, Canada. This work is funded by China Postdoctoral Science Foundation (No.20100470557). We would also like to acknowledge the input of the National Natural Science Foundation of China (No. 61035003, 60933004, 60970088, 60903141, 61072085, 61103198), National High-tech R&D Program of China (863 Program) (No.2012AA011003), National Science and Technology Support Program2012BA107B02). ( ) 2. Brickley, D. and Guha, R.: Rdf vocabulary description language 1.0: Rdf schem World Wide Web Consortium, http://www.w3.org/TR/rdf-schema/ (2004) potential peers that may contain the RDF data of interest. The target RDF data is obtained in the second phase by issuing a SPARQL query. 3. Broekstra, J., Kampman, A., and van Harmelen, F.: Sesame: A Generic Architec- ture for Storing and Querying RDF and RDF Schema. In: the 2nd International Semantic Web Conference, pp.54–68. Springer-Verlag London, UK (2002) 1. Abadi, D. J., Marcus, A., Madden, S. R., and Hollenbach, K.: Scalable Semantic Web Data Management Using Vertical Partitioning. 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https://openalex.org/W3134488845	https://periodicos.ufsc.br/index.php/biotemas/article/download/76552/45722	Portuguese	null	Análise comparativa de protocolos para extração de DNA genômico em Prosopis juliflora (Sw.) DC	Biotemas	2,021	cc-by	3,832	Luiz Henrique Tolentino Santos 1,4 Cibelle Santos Dias 2,4 Jardyelle Carvalho Lima 4 Messulan Rodrigues Meira 2,4 Carlos Bernard Moreno Cerqueira Silva 1,2,3,4* Universidade Estadual do Sudoeste da Bahia 1 Programa de Pós-graduação em Zootecnia 2 Programa de Pós-graduação em Ciências Ambientais 3 Departamento de Ciências Exatas e Naturais 4 Laboratório de Genética Molecular Aplicada CEP 45.700-000, Itapetinga – BA, Brasil * Autor para correspondência csilva@uesb.edu.br Universidade Estadual do Sudoeste da Bahia 1 Programa de Pós-graduação em Zootecnia 2 Programa de Pós-graduação em Ciências Ambientais 3 Departamento de Ciências Exatas e Naturais 4 Laboratório de Genética Molecular Aplicada CEP 45.700-000, Itapetinga – BA, Brasil * Autor para correspondência csilva@uesb.edu.br Submetido em 18/08/2020 Aceito para publicação em 14/12/2020 Resumo Objetivou-se comparar a eficiência entre sete protocolos adaptados para a extração de DNA genômico da espécie Prosopis juliflora (Sw). DC. Os protocolos utilizados tiveram como base em seus tampões: SDS 10%; CTAB 5%; Sorbitol, CTAB 3% e Sorbitol; SDS 0,7% e NaCl; CTAB 2%; CTAB 2% e NaCl 1,4M. Em todos os testes, eliminou-se o uso de β-mercaptoetanol, nitrogênio líquido, proteinase K e RNAses. As amostras consistiram de primórdios folhiares de cada espécime em triplicata. A quantidade e a qualidade do DNA extraído foram avaliadas através de eletroforese em gel de agarose a 1% e da leitura das absorbâncias em espectrofotômetro. Para as reações de amplificação, utilizou-se o iniciador ISSR. Entre os tampões de extração testados, os de SDS 10%, de CTAB 5% e de CTAB 3% e Sorbitol foram os mais eficientes para P. juliflora. Desses três protocolos, o CTAB 3% e Sorbitol apresentou melhor nível de pureza apesar da menor quantidade de DNA (123 ng/µL) se comparado aos tampõs de SDS 10% e de CTAB 5% (312 e 321 ng/µL, respectivamente, e com alto teor de contaminantes). Conclui-se que é possível o uso de protocolos de extração sem aditivos nocivos à saúde e com menor custo no processo de extração. Palavras-chave: Ácidos nucleicos; Algaroba; Genética molecular; PCR Biotemas, 34 (1): 1-7, março de 2021 ISSNe 2175-7925 Biotemas, 34 (1): 1-7, março de 2021 ISSNe 2175-7925 Biotemas, 34 (1): 1-7, março de 2021 ISSNe 2175-7925 1 http://dx.doi.org/10.5007/2175-7925.2021.e76552 Este periódico está licenciado conforme Creative Commons Atribuição 4.0 Internacional. Abstract Comparative analysis of protocols for genomic DNA extraction in Prosopis juliflora (Sw.) DC. The objective of the present research was to compare the efficiency among seven protocols adapted for the extraction of genomic DNA from the species Prosopis juliflora (Sw.) DC. The protocols used were based on their buffers: SDS 10%, CTAB 5%, Sorbitol, CTAB 3% and Sorbitol, SDS 0.7% and NaCl, CTAB 2%, CTAB 2% and NaCl Revista Biotemas, 34 (1), março de 2021 L. H. T. Santos et al. 2 1.4M. In all tests, the use of β-mercaptoethanol, liquid nitrogen, proteinase K and RNAses was eliminated. The samples consisted of leaf primordia in each specimen in triplicate. The quantity and quality of the extracted DNA were evaluated by electrophoresis in 1% agarose gel and by reading the absorbances in a spectrophotometer. For amplification reactions, the ISSR primer was used. Among the tested extraction buffers, SDS 10%, CTAB 5% and CTAB 3%, and Sorbitol were the most efficient for P. juliflora. Of these three protocols, CTAB 3% and Sorbitol showed a better level of purity despite the lower amount of DNA (123 ng/µL) compared to 10% SDS and 5% CTAB buffers (312 and 321 ng/µL, respectively, and high content of contaminants). We conclude that it is possible to use extraction protocols without additives that are harmful to human health and at a lower cost in the extraction process. Key words: Algaroba; Molecular genetics; Nucleic acids; PCR Introdução específicos devido à presença de metabólitos secundários e polissacarídeos comuns a cada espécie. Portanto, a adequação de protocolos é indispensável para obtenção de DNA puro e íntegro, e para melhor eficiência na amplificação do material genômico em estudos moleculares (OLIVEIRA et al., 2017; DIAS et al., 2018). Prosopis juliflora (Sw.) DC é, conforme The Legume Phylogeny Working Group (LPWG, 2017), uma espécie arbórea, da família das leguminosas (Leguminoseae, subfamília Caesalpinoideae). Originária da região tropical andina e bem adaptada às regiões semi-áridas de baixa precipitação. Popularmente conhecida como algaroba ou algarobeira, essa espécie possui diversos usos bem como importância econômica (DOS SANTOS; DIODATO, 2017), entre eles: a alimentação humana e de animais, a produção de madeira e a recuperação de áreas degradadas. Pode, ainda, ser utilizada, entre outras possibilidades, como inseticida, analgésico, anti-helmíntico, antibiótico, probióticos e como suplemento nutricional (AHIRWAL et al., 2017; HENCIYA et al., 2017; DHIVYA et al., 2018). Pelas razões apresentadas nos parágrafos anteriores, a realização de estudos associados à proposição, comparação, seleção e otimização de protocolos para extração de ácidos nucleicos em diferentes espécies tem sido reportada na literatura (OLIVEIRA et al., 2015; MATOS-OLIVEIRA et al., 2017; DIAS et al., 2018). Porém, para a algaroba, estudos de estabelecimento de protocolo e seleção de iniciadores ainda são incipientes. Diante do exposto, objetivou-se comparar a eficiência na extração de DNA genômico de sete protocolos para espécies vegetais em Prosopis juliflora (Sw.) DC. Embora a espécie possua muito potencial de uso pelo ser humano e estudos de diveridade genética sejam o ponto de partida para se efetivar ações de conservação e de melhoramento de espécies com potencial econômico (LOPES et al., 2011; BESSEGA et al., 2018; FALEIRO et al., 2018), pouco se conhece quanto à diversidade genética de P. juliflora. Para estudos dessa natureza, faz-se necessária a obtenção de ácidos nucleicos, em quantidades e qualidades que possibilitem a amplificação do material genômico e a seleção de iniciadores potenciais (MICHEL-LÓPEZ et al., 2013). Revista Biotemas, 34 (1), março de 2021 Material e Métodos A coleta das amostras foliares de três espécimes de Prosopis juliflora (Sw.) DC foi realizada no Instituto Federal de Educação, Ciência e Tecnologia Baiano (IF Baiano) em Itapetinga, Bahia, Brasil (coordenadas geográficas: 15°14’38,57”S, 40°13’38,95”W), em um ecótono de Caatinga e floresta estacional decidual e semi-decidual. As amostras encontram-se cadastradas no Sistema Nacional de Gestão do Patrimônio Genético e Conhecimento Tradicional Associado – SisGen (número: AFF0E34). O processo de extração de DNA é parte fundamental em estudos moleculares, sendo sua eficiência diretamente relacionado à pureza, à quantidade e à qualidade do material genômico obtido (DA ROCHA et al., 2017). A extração de DNA de tecidos vegetais requer protocolos O material vegetal foi condicionado em isopor com gelo e transferido para o Laboratório de Genética Molecular Aplicada (LGMA) da Universidade Estadual Revista Biotemas, 34 (1), março de 2021 Comparação de protocolos para extração de DNA em P. juliflora Comparação de protocolos para extração de DNA em P. juliflora 3 Bórico-EDTA) durante 90 min a 90 V. Os resultados foram comparados com o marcador de peso molecular (DNA Lambda; Invitrogen) nas concentrações de 25 e 75 ng/µL. O gel foi visualizado em luz UV e registrado em sistema de fotodocumentação Kodak (KODAK MI Software). De posse das leituras espectrofotométricas, mensurou-se as médias e o desvio-padrão com o auxílio do Software BioEstat® 5.0 (AYRES et al., 2007). do Sudoeste da Bahia (UESB, campus Juvino de Oliveira), no município de Itapetinga, Bahia, onde foi armazenado em freezer -18°C e, posteriormente, foram realizadas as extrações do DNA. Os testes de extração de DNA genômico foram realizados de acordo com sete protocolos previamente publicados, com modificações apresentadas na Tabela 1. Para todos os protocolos foram realizadas três extrações em duplicata. A maceração das folhas prosseguiu-se de 0,4 g de tecido foliar e 3 mL de solução tampão referente a cada protocolo, sendo conduzido com auxílio de almofariz e pistilo em temperatura ambiente até total rompimento das estruturas foliares. Portanto não houve adição de nitrogênio líquido, β-mercaptoetanol, proteinase K e RNAses em nenhum dos protocolos, respectivamente. As padronizações do material genômico foram obtidas por meio da ressuspensão em 60 µL de água ultrapura (q.s.p.). Para avaliar a qualidade e a viabilidade de uso do DNA, eles foram submetidos à amplificação por PCR (Polymerase Chain Reaction), utilizando o marcador molecular tipo ISSR (Inter Simple Sequence Repeats) DiCA3’RG (5’- CAC ACA CAC ACA CAC ARG-3’). L. H. T. Santos et al. 4 Conforme os resultados observados com a eletroforese, foi possível identificar diferença na eficiência da extração de DNA genômico de P. juliflora entre os protocolos (Figura 1). Material e Métodos As amplificações foram realizadas com a seguinte programação: 1) desnaturação: 5 minutos a 94°C; 2) 35 ciclos: desnaturação – 50 segundos a 94°C; anelamento – 50 segundos a 48°C e extensão – 1 minuto a 72°C; e 3) extensão final: 5 minutos a 72°C. Os padrões de amplificação foram avaliados por eletroforese a 120 minutos sob uma corrente elétrica de 120 V em gel de agarose a 2% (m/v) com solução de corrida TBE 0,5X (Tris-Ácido Bórico-EDTA). As amplificações foram comparadas com o marcador tamanho (1 kb plus; Invitrogen) e os resultados visualizados e registrados conforme acima descrito. Os ácidos nucleicos foram quantificados por espectrofotometria nas razões de absorbância em ng/ µL a A260/230 e A260/280, utilizando o BioDrop® µLITE (Whitehead Scientific). Para melhor atribuição dos resultados, as amostras foram avaliadas também em gel de agarose a 1% (m/v) corado com GelRed® (Biotium) e corrida em solução TBE 0,5X (Tris-Ácido TABELA 1: Descrição dos protocolos utilizados para os testes de extração de DNA genômico de Prosopis juliflora (Sw.) DC. Protocolos Referências Tampão de Extração Modificações P1 Sunnucks e Hales (1996) SDS* 10% Proteinase K P2 Doyle e Doyle (1990) CTAB** 5% Nitrogênio líquido, β-mercaptoetanol, RNAse P3 Método modificado por Štorchová et al. (2000) Sorbitol β-mercaptoetanol P4 Método modificado por Russell et al. (2010) CTAB* * 3% e Sorbitol Nitrogênio líquido, β-mercaptoetanol, RNAse P5 Mogg e Bond (2003) SDS* 0,7% e NaCl 500 mM Proteinase K e RNAse P6 Barnwell et al. (1998) CTAB 2% Nitrogênio líquido P7 Método modificado por Silva et al. (2014) CTAB* * 2% e NaCl 1,4M Nitrogênio líquido, β-mercaptoetanol, RNAse, Proteinase K * Sodium Dodecyl Sulfate. ** Cetyl Trimethylammonium Bromide. Modificações: Reagentes não utilizados no referido protocolo de extração. os protocolos utilizados para os testes de extração de DNA genômico de Prosopis juliflora (Sw.) DC. ABELA 1: Descrição dos protocolos utilizados para os testes de extração de DNA genômico de Proso * Sodium Dodecyl Sulfate. ** Cetyl Trimethylammonium Bromide. Modificações: Reagentes não utilizados no referido protocolo de extração. Revista Biotemas, 34 (1), março de 2021 Discussão lise das células para liberação dos nucleotídeos e uma adequada remoção de proteínas, restos celulares e de organelas. Na amplificação do DNA com o marcador tipo ISSR, a principal vantagem observada para os procedimentos foi a ausência de nitrogênio líquido e do β-mercaptoetanol. Os quais conferiram baixo custo e maior segurança operacional, uma vez que esse último reagente é tóxico e acarreta vários prejuízos à saúde humana. O β-mercaptoetanol se caracteriza por ser um antioxidante muito utilizado em protocolos de extração do DNA genômico em plantas (AUSUBEL et al., 2005). Entretanto, sua remoção não inviabilizou a extração de DNA em quantidade e qualidade necessárias para uso em reação de PCR quando se utilizou os protocolos com tampão CTAB5%, CTAB 3% e Sorbitol. Outros estudos corroboram esses resultados, demostrando não haver necessidade da utilização do β-mercaptoetanol na extração de DNA (SCHMITT et al., 2014; SILVA et al., 2014). Para que o DNA seja considerado puro, ele deve apresentar uma razão de absorbância A260/A280 média de 1,8 (NELSON; COX, 2004), o que de fato não ocorreu nas extrações realizadas (cujas razões de absorbância média A260/A280 variaram de 0,44 a 1,76). O mesmo foi observado para a quantificação, pois, para amostras isentas de RNA, esperavam-se valores A260/A280 entre 1,8 e 2,0, sendo o valor obtido abaixo do esperado para todos os protocolos. Esse fato pode ser explicado pela ausência de RNAse nos protocolos. Em relação à absorbância A260/A230, que é um indicador secundário de pureza do DNA, seus resultados inferem a presença de fenol e clorofórmio e de acordo as especificações do fabricante do BioDrop® µLITE, para considerar o DNA “puro”, os valores desta absorbância devem estar entre 2,0 e 2,2. Ademais, é sabido que o gênero Prosopis inclui árvores com altos níveis de polissacarídeos, compostos fenólicos e outros constituintes químicos (RATHORE, 2009), sendo provável que tais compostos secundários tenham relação direta com os baixos valores observados para as razões (A260/A230) de absorbância. A utilização de nitrogênio líquido no processo de extração de DNA possibilita uma melhor neutralização enzimática e favorece o rompimento do envoltório celular. Em contrapartida, sua utilização eleva os custos do procedimento. Resultados juliflora 5 FIGURA 2: Padrão de amplificação dos fragmentos de DNA genômico de Prosopis juliflora (Sw.) DC obtidos por meio de sete protoc de extração avaliados com o iniciador ISSR – 5’ (CA)8RG 3’. FIGURA 2: Padrão de amplificação dos fragmentos de DNA genômico de Prosopis juliflora (Sw.) DC obtidos por meio de sete proto de extração avaliados com o iniciador ISSR – 5’ (CA)8RG 3’. FIGURA 2: Padrão de amplificação dos fragmentos de DNA genômico de Prosopis juliflora (Sw.) DC obtidos por meio de sete protocolos de extração avaliados com o iniciador ISSR – 5’ (CA)8RG 3’. ação dos fragmentos de DNA genômico de Prosopis juliflora (Sw.) DC obtidos por meio de sete protocolos dos com o iniciador ISSR – 5’ (CA)8RG 3’. * 1kb DNA Ladder. Resultados O DNA genômico da espécie Prosopis juliflora (Sw.) DC apresentou pureza média de 0,56±0,17 A260/A230 e 1,16±0,15 A260/A280 (Tabela 2). A partir dos resultados observados para os protocolos P1 (SDS 10%), P2 (CTAB 5%) e P4 (CTAB 3% e Sorbitol), foi possível verificar, a despeito dos valores de baixa pureza estimada a partir de todos os protocolos, a presença de marcas (DNA) definidas, as quais não foram observadas nos demais protocolos. Na amplificação com o marcador tipo ISSR, (CA)8RG, observou-se a eficiência dos protocolos P1 (SDS 10%), P2 (CTAB5%) e P4 (CTAB 3% e Sorbitol), o que confirma o sucesso desses protocolos para a extração de DNA (Figura 2). Os protocolos P1, P2 e P4 (SDS 10%, CTAB 5% e CTAB 3% e Sorbitol) apresentaram melhores performances para a espécie, sendo que o tampão SDS 10% e o CTAB 5% apresentaram boa concentração de DNA (312 e 321 ng/µL), porém com alto teor de contaminantes. Já o CTAB 3% e Sorbitol apresentou menor quantidade de DNA (123 ng/µL), mas se destacou em nível de pureza. Dos testes, o protocolo P6 (CTAB 2%) apresentou-se menos eficiente. TABELA 2: Quantificação e pureza de DNA genômico de Prosopis juliflora (Sw.) DC a partir das absorbâncias obtidas por espectrofotômetro. Protocolos A260/A230 A260/A280 Concentração (ng/µL) Média ±dp* Média± dp* Média± dp* P1 0,421±0,154 1,191±0,353 312,25±152,69 P2 0,808±0,204 1,343±0,170 321,333±118,88 P3 0,399±0,044 1,298±0,073 415,400±252,55 P4 1,344±0,341 1,759±0,041 123,655±71,89 P5 0,295±0,080 0,965±0,176 130,482±110,84 P6 0,220±0,143 0,440±0,169 48,635±12,375 P7 0,476±0,257 1,155±0,119 263,417±168,39 dp: Desvio Padrão. TABELA 2: Quantificação e pureza de DNA genômico de Prosopis juliflora (Sw.) DC a partir das absorbâncias obtidas por espectrofotômetro. ão e pureza de DNA genômico de Prosopis juliflora (Sw.) DC a partir das absorbâncias obtidas por ômetro. FIGURA 1: DNA genômico de Prosopis juliflora (Sw.) DC obtido a partir dos protocolos de extração. FIGURA 1: DNA genômico de Prosopis juliflora (Sw.) DC obtido a partir dos protocolos de ex λ- DNA Lambda nas concentrações de 25 e 75 ng/µL. g p j fl ( ) p p ç * λ- DNA Lambda nas concentrações de 25 e 75 ng/µL. * λ- DNA Lambda nas concentrações de 25 e 75 ng/µL. Revista Biotemas, 34 (1), março de 2021 Comparação de protocolos para extração de DNA em P. Revista Biotemas, 34 (1), março de 2021 Discussão No presente estudo, a ausência de nitrogênio líquido não foi determinante para a qualidade e quantidade do DNA extraído, os quais apresentaram resultados satisfatórios para utilização em plataforma A obtenção de DNA de boa qualidade tem impacto direto sobre os resultados de técnicas como a reação em cadeia da polimerase (PCR). Sendo que os procedimentos de extração de DNA devem proporcionar uma eficiente Revista Biotemas, 34 (1), março de 2021 L. H. T. Santos et al. 6 different levels of human disturbance and altitude. Ecology and Evolution, Sheffield, v. 8, n. 22, p. 11309-11321, 2018. de PCR. Outros estudos reportaram que a ausência de nitrogênio líquido no processo de maceração torna custo operacional menos oneroso (MICHEL-LÓPEZ et al., 2013; DOS SANTOS; ARAÚJO, 2017). DA ROCHA, V. D.; ZÓRTEA, K. E. M.; CARDOSO, E. S.; BISPO, R. B.; TIAGO, A. V.; ROSSI, A. A. B. Efeito da idade da folha na qualidade do DNA extraído de Piper aduncum L. Revista de Ciências Agroambientais, Alta Floresta, v. 15, n. 2, p. 218-222, 2017. Os resultados apresentados possibilitaram a seleção de três dos protocolos de extração de DNA genômico: SDS10%, CTAB5% puro e CTAB 3% com Sorbitol. Esses métodos foram propostos por Sunnucks e Hales (1996), Doyle e Doyle (1990) e por Russell et al. (2010). Por fim, este estudo certifica que a utilização desses protocolos com adaptações metodológicas reduz os insumos nocivos à saúde humana e o custo elevado, podendo ser utilizados em estudos de diversidade genética de Prosopis juliflora (Sw.) DC. DHIVYA, K.; VENGATESWARI, G.; ARUNTHIRUMENI, M.; KARTHI, S.; SENTHIL-NATHAN, S.; SHIVAKUMAR, M. S. Bioprospecting of Prosopis juliflora (Sw.) DC seed pod extract effect on antioxidant and immune system of Spodoptera litura (Lepidoptera: Noctuidae). Physiological and Molecular Plant Pathology, East Lansing, v. 101, p. 45-53, 2018. DIAS, C. S.; SANTOS, L. H. T.; LIMA, J. C.; SOARES, A. B. L.; SANTOS, E. S. L.; CERQUEIRA-SILVA, C. B. M. Comparação de protocolos para extração de DNA genômico de Calotropis procera Ait. R. Br. (Apocynaceae: Asclepiadoideae). Multi-Science Journal, Urataí, v. 1, n. 13, p. 277-281, 2018. DOS SANTOS, E. M.; ARAÚJO, R. R. Testes de comparação de protocolos de extração de DNA e de maceração de tecido de Platonia insignis Mart. (Clusiaceae). Revista Brasileira de Biociências, Porto Alegre, v. 15, n. 4, p. 199-202, 2017. Agradecimentos Aos órgãos de fomento à pesquisa: CNPq, CAPES, pela concessão das bolsas de doutoramentos; à FAPESB, pelo apoio financeiro referente ao Projeto de Intra PIE 0014/2016, e aos Programa de Pós-Graduação em Zootecnia e Ciências Ambientais da UESB – Campus Itapetinga, pela possibilidade da pesquisa e estrutura física. DOS SANTOS, J. P. S.; DIODATO, M. A. Histórico da implementação da algaroba no Rio Grande do Norte. Pesquisa Florestal Brasileira, Colombo, v. 37, n. 90, p. 201-212, 2017. DOYLE, J. J.; DOYLE, J. L. Isolation of plant DNA from fresh tissue. Focus, Gaithesburg, v. 12, p. 13-15, 1990. FALEIRO, F. G.; AMABILE, R. F.; SILVA, C. B. M. C. Marcadores moleculares aplicados ao melhoramento genético de plantas. In: AMABILE, R. F.; VILELA, M. S.; PEIXOTO, J. R. (Ed.). Melhoramento de plantas: variabilidade genética, ferramentas e mercado. Brasília: Sociedade Brasileira de Melhoramento de Plantas, 2018. p. 53-76. Referências HENCIYA, S.; SETURAMAN, P.; JAMES, A. R.; TSAI, Y. H.; NIKAM, R., WU, Y. C.; CHANG, F. R. Biopharmaceutical potentials of Prosopis spp. (Mimosaceae, Leguminosa). Journal of Food and Drug Analysis, Taipé, v. 25, n. 1, p. 187-196, 2017. AHIRWAL, J.; MAITI, S. K.; REDDY, M. S. Development of carbon, nitrogen and phosphate stocks of reclaimed coal mine soil within 8 years after forestation with Prosopis juliflora (Sw.) Dc. Catena, Amsterdam, v. 156, p. 42-50, 2017. LOPES, M. A.; FÁVERO, A. P.; FERREIRA, M. A. J. F.; FALEIRO, F. G.; FOLLE, S. M.; GUIMARÃES, E. P. Pré-Melhoramento de plantas. Estado da Arte e experiências de sucesso. Brasilia: Embrapa Informações Tecnologicas, 2011. 614 p. AUSUBEL, F.; BRENT, R.; KINGSTON, R.; MOORE, D.; SEIDMAN, J.; SMITH, J.; STRUHL, K. Current Protocols in Molecular Biology, tomo 1-4. New York: Greene & John Wiley, 2005. 4.648 p. LPWG – THE LEGUME PHYLOGENY WORKING GROUP. Phylogeny and classification of the Leguminosae. Taxon, Vienna, v. 66, n. 1, p. 44-77, 2017. AYRES, M.; AYRES, M. 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https://openalex.org/W2191696637	https://www.notulaebotanicae.ro/index.php/nbha/article/download/9949/7854	English	null	Chromatic, Phenolic and Antioxidant Properties of <i style='mso-bidi-font-style:normal'>Sorghum bicolor</i> Genotypes	Notulae botanicae Horti Agrobotanici Cluj-Napoca	2,015	cc-by	4,698	Introduction proliferation of carcinoma cells (Awika et al., 2009). The aim of this work was to evaluate and compare chromatic characteristics, the phenolic compounds content and the antioxidant capacity levels of sorghum genotypes Grown in Nuevo León, México. Sorghum (Sorghum bicolor (L.) Moench) is native from Africa grown in tropical, subtropical and arid regions around the world, and is the fifth most produced cereals in the world, in addition is used as food in Africa and as feed in the western hemisphere (Dykes et al., 2013). According to the data of Food and Fisheries Statistics Service (SIAP) the annual production of sorghum grain in México increased from 6.1 millions of tonnes in 2009 to 8.3 millions of tonnes in 2014, being animal feed their main use (SIAP, 2015). New ways to use sorghum for food purposes are looking to promote its consume taking into account some technological and nutritional components which includes gluten free leavened breads, cakes, cookies, tortillas, snacks and noodles (Taylor et al., 2006). In addition, a great interest has focused on sorghum grain and its co-products as a source of functional and nutraceutical components with human health promoting actions including phenolics (Althwab et al., 2015), since they have a high antioxidant activity provided by phenolic acids, condensed tannins and anthocyanins (Awika and Rooney, 2004), that can induce phase II detoxifying enzymes and inhibit Chromatic, Phenolic and Antioxidant Properties of Sorghum bicolor Genotypes José Juan LÓPEZ-CONTRERAS1, Francisco ZAVALA-GARCÍA1, Vania URÍAS-ORONA2, Guillermo Cristian G. MARTÍNEZ-ÁVILA1, Romeo ROJAS1, Guillermo NIÑO-MEDINA1* Autónoma de Nuevo León, Facultad de Agronomía, Francisco Villa S/N, Col. Ex-Hacienda El Canadá, C.P. 66050, General Escobedo, Nuevo León, México; guillermo.ninomd@uanl.edu.mx; nino.medina.g@gmail.com (corresponding author) 1Universidad Autónoma de Nuevo León, Facultad de Agronomía, Francisco Villa S/N, Col. Ex-Hacienda El Canadá, C.P. 66050, General Escobedo, Nuevo León, México; guillermo.ninomd@uanl.edu.mx; nino.medina.g@gmail.com (corresponding author) 2Universidad Autónoma de Nuevo León, Facultad de Salud Pública y Nutrición, Av. Dr. Eduardo Aguirre Pequeño y Yuriria, C.P. 64460, Col. Mitras Centro, Monterrey, Nuevo León, México México; guillermo.ninomd@uanl.edu.mx; nino.medina.g@gmail.com (corresponding author) 2Universidad Autónoma de Nuevo León, Facultad de Salud Pública y Nutrición, Av. Dr. Eduardo Aguirre Pequeño y Yuriria, C.P. 64460, Col. Mitras Centro, Monterrey, Nuevo León, México Universidad Autónoma de Nuevo León, Facultad de Salud Pública y Nutrición, Av. Dr. Eduardo Aguirre Pequeño y Yuriria, C.P. 64460, Col. Mitras Monterrey, Nuevo León, México Received: 21 May 2015. Received in revised form: 10 Sept 2015. Accepted: 12 Sept 2015. Published online: 10 Dec 2015. Abstract Chromatic, phenolic and antioxidant properties were evaluated in ten sorghum genotypes grown in Nuevo León, México. Lightness, Chroma and hue angle ranged from 64 to 83, 12 to 20 and 61 to 82 respectively, indicating that colour of the samples were located in the gray orange-yellow zone of the hue circle. Based on these results, samples were classified in three colour groups being Very Soft Orange, Slightly Desaturated Orange and Grayish Orange. Results in phenolics ranged from 796 to 15,949, 175 to 12,674 and 193 to 25,780 µgCE g-1 in total phenolics by Folin-Ciocalteu, total flavonoids by Aluminum Chloride and condensed tannins by Vanillin-HCl respectively. On the other hand, antioxidant capacity ranged from 1.20 to 93.83, 30.25 to 156.08 and 2.62 to 98.50 μmolTE g-1 in 2,2-diphenyl-1-picrylhydrazyl, 3-ethyl-benzothiazoline-6-sulfonic acid and Ferric Reducing Antioxidant Power respectively. Significant differences (p≤0.05) were observed in statistical analysis for both individual and group colour samples in chromatic, phenolics and antioxidant activity evaluations, showing ‘Rox Orange’ genotype and Grayish Orange colour group the highest levels. Keywords: ABTS, condensed tannins, DPPH, FRAP, total flavonoids, total phenolics, Sorghum bicolor Keywords: ABTS, condensed tannins, DPPH, FRAP, total flavonoids, total phenolics, Sorghum bicolor Statistical analysis l f Analysis of variance was used to assess statistical differences among sorghum genotypes with a 5% confidence level. When significance difference was found, Tukey’s multiple range tests were carried out to separate means using Minitab 14.0 (Minitab Inc., 2004). p The condensed tannins content was determined as described by Sun et al. (1998) based in the reaction of vanillin-H2SO4. Briefly, 0.25 mL of phenolic extract was mixed with 0.65 mL 1% vanillin solution and 0.65 mL of 25% H2SO4 (both dissolved in methanol). Reaction was left for 15 min at 30 °C and finally the absorbance of samples was measured at 500 nm. Phenolic extracts h d d One hundred milligrams of sorghum samples were homogenized with 3 mL of 80% methanol in a screw cap culture tube and stirred for 4 h at 200 rpm. After that, samples were centrifuged at 2,600 g, supernatants were recovered and stored at -20 °C until they were used for phenolics and antioxidant capacity analysis. FRAP (Ferric Reducing Antioxidant Power) was determined using a working solution prepared by mixing 300 mM C2H3NaO2·3H2O (pH 3.6), 10 mM TPTZ (2,4,6- tripyridyl-s-triazine, in HCl 40 mM) and 20 mM FeCl3·6H2O in 10:1:1 proportion; the FRAP assay was prepared by mixing 0.2 mL of phenolic extract with 3.3 mL of FRAP working solution, reaction was left for 30 min in the dark at 37 °C and the absorbance of samples was taken at 593 nm. Antioxidant capacity assays were performed according to Thaipong et al. (2006) with modifications, Trolox was used as standard (0 to 200 µmol L-1) and results were expressed as micromoles of Trolox equivalents per gram of sample (µmolTE g-1) using a linear equation. Phenolics The total phenolics content was determined using a colorimetric method reported by Chun and Kim (2004) based on the reaction of Folin-Ciocalteu reagent. Briefly, 0.2 mL of phenolic extract was placed in 2.6 mL of distilled water, oxidized with 0.2 mL of Folin-Ciocalteu reagent and after 5 min neutralized with 2 mL of 7% Na2CO3 solution. The reaction was left for 90 min and finally absorbance of samples was measured at 750 nm. y p The total flavonoids content was evaluated according to Ivanova et al. (2011) based on the reaction of aluminium chloride. Briefly, 0.2 mL of phenolic extract was placed in 3.5 mL of distilled water, followed by 0.15 mL of 5% NaNO2, after 5 min 0.15 mL of 10% AlCl3 was added and finally 5 min later 1.0 mL of 1M NaOH was added. Reaction was left for 15 min and finally the absorbance of samples was measured at 510 nm. Plant material Seeds of ten sorghum genotypes with different geographic origins and varying pigmentations including ‘46038B’, ‘7B’, ‘FAUANL-3’ ‘WB’ from Nuevo León, México; ‘RB Norteño’, ‘RB Paloma’ from Tamaulipas, México; ‘Keller’ ‘Rox Orange’ from Kentucky, United States; ‘SPV4511-2’ from Telangana, India; and ‘Tanol-1’ (commercial genotype, origin unknown) were grown in the experimental field of the Agronomy Faculty of Universidad Autónoma de Nuevo León located in Marín, Nuevo León (25º52’13.5’’N and 100º02’22.5’’ W) during 2014 spring summer cycle. The sowing was performed on March 5th on a clay soil and experimental units involved 4 furrows of 5 m López-Contreras JJ et al. / Not Bot Horti Agrobo, 2015, 43(2):366-370 367 Table 1. Chromatic characteristics of sorghum genotypes Genotype Colour View Colour parameter L C* h ‘46038B’ 83.61±0.12a 15.18±0.14f 82.10±0.03a ‘7Bʼ 81.35±0.16b 14.82±0.23f 80.51±0.05b ‘FAUANL-3’ 70.75±0.13f 20.41±0.16a 66.79±0.08f ‘Keller’ 69.79±0.02g 15.83±0.04e 69.37±0.04d ‘RB Norteño’ 78.14±0.02d 15.97±0.01e 73.64±0.02c ‘RB Paloma’ 79.53±0.01c 17.87±0.01c 80.70±0.06b ‘Rox Orange’ 73.33±0.27e 12.01±0.09g 61.23±0.05h SPV4511-2ʼ 66.05±0.05h 18.74±0.03b 66.15±0.03f ‘Tanol-1’ 64.68±0.01i 17.16±0.01d 63.29±0.02g ‘WB’ 77.75±0.39d 15.31±0.19f 68.49±0.20e Values with different letters within a column are significantly different (p≤0.05). Data are expressed as means values of three samples ± standard deviation Table 1. Chromatic characteristics of sorghum genotypes long and 0.8 m spaced from each other and it was established a 0.1 m distance between plants in simple line. A day before sowing, fertilisation of soil was performed with poultry manure (3 tonnes ha-1). Table 1. Chromatic characteristics of sorghum genotypes Genotype Colour View Colour parameter L* C* h ‘46038B’ 83.61±0.12a 15.18±0.14f 82.10±0.03a ‘7Bʼ 81.35±0.16b 14.82±0.23f 80.51±0.05b ‘FAUANL-3’ 70.75±0.13f 20.41±0.16a 66.79±0.08f ‘Keller’ 69.79±0.02g 15.83±0.04e 69.37±0.04d ‘RB Norteño’ 78.14±0.02d 15.97±0.01e 73.64±0.02c ‘RB Paloma’ 79.53±0.01c 17.87±0.01c 80.70±0.06b ‘Rox Orange’ 73.33±0.27e 12.01±0.09g 61.23±0.05h SPV4511-2ʼ 66.05±0.05h 18.74±0.03b 66.15±0.03f ‘Tanol-1’ 64.68±0.01i 17.16±0.01d 63.29±0.02g ‘WB’ 77.75±0.39d 15.31±0.19f 68.49±0.20e Values with different letters within a column are significantly different (p≤0.05). Data are expressed as means values of three samples ± standard deviation ( ) Four irrigations were carried out during whole cycle (March 7th, March 20th, April 15th and May 5th), all sorghum genotypes were harvested at physiological maturity (July 5th) and complete grains (endosperm and bran) were milled and sieved to obtain flour with a particle size ≤ 0.5 mm (mesh 35). Antioxidant capacity p y DPPH (2,2-diphenyl-1-picrylhydrazyl) was evaluated using a k l h d b b d d DPPH (2,2-diphenyl-1-picrylhydrazyl) was evaluated using a working solution 60 µM with and absorbance adjusted to 0.7 at 517 nm; the assay was carried out by mixing 0.2 mL of phenolic extract with 3.3 mL of the DPPH working solution, the reaction was left for 30 min in the dark and the reduction of DDPH was determined. ABTS (3-ethyl-benzothiazoline-6-sulfonic acid) was carried out using a working solution obtained by mixing one mL 7.4 mM of ABTS and one mL of 2.6 mM of K2S2O8 and allowing them to react for 12 h in the dark, after that time absorbance of working solution was adjusted to 0.7 at 734 nm diluting with methanol; the ABTS assay was performed by mixing 0.2 mL of phenolic extract with 3.3 mL of ABTS working solution, reaction was left for 2 h in the dark and the reduction of ABTS was measured. Chromatic measurements f h Chromatic measurements Sixty grams of every sorghum sample were placed in a Petri dish and colour was measured on the surface flour using a CR- 300 Konica Minolta Chroma Meter (Tokyo, Japan). Chromatic parameters were obtained using CIELAB (L, a, b) colour system where L defines Lightness (0=black, 100=white), a* indicate red (positive a) or green value (negative a) and b* indicate yellow (positive b) or blue value (negative b). In addition C* (Chroma; saturation level of h) and h (hue angle; 0°=red, 90°=yellow, 180°=green, 270°=blue) were obtained using CIELAB colour values as C= (a2+b2) and h=arctan (b/a) (Commission Internationale de l’Ecleirage, 2004). Colour view was obtained by online software ColorHexa colour converter using L, C* and h values (ColorHexa, 2015) and sorghum genotypes were grouped by colour based on h values. Chromatic characteristics p For total phenolics, total flavonoids and condensed tannins assays, catechin was used as standard (0 to 200 mg L-1) and results were expressed as micrograms of catechin equivalents per gram of dry sample (µgCE g-1) using a linear equation. There were significant differences in L, C and h (p≤0.05) between samples (Table 1). Lightness values ranged from 64 to 83 such indicate that all samples were more white than black; López-Contreras JJ et al. / Not Bot Horti Agrobo, 2015, 43(2):366-370 368 Table 2. Chromatic characteristics of sorghum genotypes grouped by colour Genotype Number of genotypes Colour View Colour parameter L* C* h VSO 3 81.49±1.77a 15.96±1.44ab 81.10±0.75a SDO 5 72.50±4.88b 17.25±2.05a 68.88±2.73b GO 2 69.00±4.74b 14.58±2.82b 62.26±1.12c Values with different letters within column are significantly different (p≤0.05). VSO, SDO and GO are expressed as means value of nine, fifteen and six samples ± standard deviation respectively Table 2. Chromatic characteristics of sorghum genotypes grouped by colour (GO), including ʻRox Orangeʼ and ʻTanol-1ʼ. When samples are grouped based on colour, VSO showed significant differences (p≤0.05) in L* being 1.12 and 1.18-fold higher than SDO and GO groups respectively; SDO was 1.08 and 1.18-fold higher than VSO and GO in C* respectively, showing significant differences (p≤0.05); in addition three colour groups showed significant differences (p≤0.05) in h being VSO group 1.17 and 1.30-fold higher than SDO and GO respectively (Table 2). Phenolics Significant differences (p≤0.05) were observed in total phenolics, total flavonoids and condensed tannins between samples (Table 3). In all phenolics analysis ‘Rox Orange’ showed the highest levels followed by ‘Tanol-1’ while the lowest levels were in ‘46038B’, in addition the first one was 20, 72 and 133- fold higher than the latter one in total phenolics, total flavonoids and condensed tannins respectively. Table 3. Phenolics in sorghum genotypes Table 3. Phenolics in sorghum genotypes Genotype Phenolics (µgCE g-1) Total phenolics Total flavonoids Condensed tannins ‘46038B’ 796±65h 175±15h 193±31h ‘7B’ 1,100±22g 270±41g 1,343±166g ‘FAUANL-3’ 2,394±75d 2,131±115d 5,830±224d ‘Keller’ 5,570±526c 3,694±234c 8,064±896c ‘RB Norteño’ 1,795±51e 660±116f 4,300±536e ‘RB Paloma’ 2,329±190d 452±118fg 1,121±304g ‘Rox Orange’ 15,949±208a 12,674±615a 25,780±1513a SPV4511-2ʼ 2,026±75d 1,444±104e 2,361±173f ‘Tanol-1’ 8,749±79b 7,325±415b 22,245±1321b ‘WB’ 1,330±60f 201±43gh 2,327±176f Values with different letters within column are significantly different (p≤0.05). Chromatic characteristics Data are expressed as means values of three samples ± standard deviation Total phenolics values obtained ranged from 796 to 15,949 µgCE g-1 and were between data reported by Awika et al. (2005) that found from 1,000 to 21,000 µg g-1 analyzing whole grains of brown, black and white sorghum genotypes grown in Texas (United States). Total flavonoids content ranged from 175 to 12,674 µgCE g-1 and data obtained for most of the samples are in the range of those reported by Herald et al. (2012) who analyzed flours of tannin and non-tannin sorghums grown in Kansas (United States) and found a range from 500 to 6,810 µg g-1 except for ‘Rox Orange’ and ‘Tanol-1’ that were higher than the data reported by these authors. Table 4. Phenolics in sorghum genotypes grouped by colour Genotype Number of genotypes Phenolics (µgCE g-1) Total phenolics Total flavonoids Condensed tannins VSO 3 1,408±710c 299±137c 885±557c SDO 5 2,570±1592b 1,679±1302b 4,576±2295b GO 2 12,349±3946a 10,000±2967a 24,013±2316a Values with different letters within column are significantly different (p≤0.05). VSO, SDO and GO are expressed as means value of nine, fifteen and six samples ± standard deviation respectively Table 4. Phenolics in sorghum genotypes grouped by colour p y Condensed tannins levels ranged from 193 to 25,780 µgCE g-1 and excepting ‘Rox Orange’ and ‘Tanol-1’ which had content higher than 20,000 µgCE g-1, the rest eight samples were between values reported by Dykes et al. (2005) who analyzed sorghum grains of varying genotypes developed in Texas (United States) finding levels from 200 to 15,500 µg g-1. According to Dykes et al. (2013), sorghum genotypes with values of condensed tannins less than 2,000 µg g-1 measured by vanillin-HCl assay are considered tannin-free, taking that into account ‘46036B’, ‘7B’ and ‘RB Paloma’ can be classified as tannin-free sorghum genotypes. values of a* and b* (data not shown) were positive in all samples which indicate that they are more red than green and more yellow than blue. According to McGuire (1992), to include h and C* is a more appropriate measurement of colour since the first one is how we perceive colour and the second one is the level of saturation of the colour perceived. In general, colour saturation of all samples were low with C* values ranging from 12 to 20 located in the gray zone of the hue circle. Chromatic characteristics (2009) and they found lower average values than our GO group samples with 27 µmolTE g-1 in DPPH and 103 µmolTE g-1 in ABTS. In addition, Luthria et al. (2013) evaluated the antioxidant capacity of the breeding lines pearled kernels ‘PR6E14’ and ‘PR6E6’ grown in Texas (United States) by FRAP, reporting an average value around 80 µmolTE g-1 which were higher than all our colour groups. g g g p Although there are many reports about different sorghum genotypes from around the world in terms of phenolics and antioxidant capacity, there are not a colour system classification of sorghum genotypes based on CIELAB chromatic characteristics and so difficult comparisons among reports. In addition, data variation in phenolics and antioxidant capacity analysis among different reports using same plant material is mainly attributed to solvent and technique used for extraction of phenolic compounds and such topic is analyzed by Luthria (2006). Table 6. Antioxidant capacity of sorghum genotypes grouped by colour Genotype Number of genotypes Antioxidant capacity (µmolTE g-1) DPPH ABTS FRAP VSO 3 1.83±0.52b 44.21±8.79b 3.94±1.03b SDO 5 4.33±3.90b 38.51±6.99b 6.16±2.96b GO 2 65.73±30.79a 121.73±37.64a 63.34±38.51a Values with different letters within column are significantly different (p≤0.05). VSO, SDO and GO are expressed as means value of nine, fifteen and six samples ± standard deviation respectively Table 6. Antioxidant capacity of sorghum genotypes grouped by colour Antioxidant capacity Significant differences (p≤0.05) between samples were observed in DPPH, ABTS and FRAP antioxidant capacity assays. Results of antioxidant capacity were 1.20 to 93.83, 30.25 to 156.08 and 2.62 to 98.50 µmolTE g-1 in DPPH, ABTS and FRAP respectively (Table 5). ‘Rox Orange’ showed highest levels in three antioxidant capacity assays followed by ‘Tanol-1ʼ, in addition ‘46038B’ showed the lowest levels in DPPH and FRAP and ‘RB Norteño’ obtained lowest level in ABTS. ‘Rox Orange’ was 78 and 37-fold higher than ‘46038B’ in DPPH and FRAP respectively and 5.15-fold higher than ‘RB Norteño’ in ABTS. p y g Results of DPPH and ABTS assays are into the values reported by Awika et al. (2003) who found 6 to 202 µmolTE g-1 in DPPH and 6 to 226 µmolTE g-1 in ABTS analyzing sorghum of varied colours including white, red, brown and black genotypes grown in Texas (United States), although ‘Hi Tannin’ and ‘Sumac’ genotypes include in that study were higher than all our samples; in addition, Moraes et al. (2015) evaluated the antioxidant capacity of ‘SC21’ genotype (brown pericarp, pigmented testa) by FRAP assay reporting 90 µmolTE g-1 respectively, which is around levels found in ‘Rox Orange’ of our study. Acknowledgments Authors would like to acknowledge the funding provided by Fondo PAICYT-UANL 2010 Project CN450-10. Guillermo Niño-Medina thank to José Juan López-Contreras for be part of the project as undergraduate student, to Dr. Francisco Zavala- García for conducted field experimental work and to Dr. Alejandro Isabel Luna-Maldonado for facilities in the use of Chroma meter. y In samples grouped by colour, the behaviour in all antioxidant capacity assays was GO > SDO > VSO, significant difference (p≤0.05) was observed between GO in relation to VSO and SDO, but VSO and SDO did not present significant difference (p≥0.05) between them (Table 6). GO group was 15.1 and 35.9, 3.1 and 2.7, 10.4 and 16.3-fold higher than SDO and VSO in DPPH, ABTS and FRAP respectively. Conclusions ‘Rox Orange’ showed the lowest values in h colour parameter and highest levels of phenolic compounds and antioxidant capacity evaluated. Although there are not a colour system classification of sorghum genotypes, we proposed to group our samples as Very Soft Orange (VSO), Slightly Desaturated Orange (SDO), Grayish Orange (GO) based on their L, C and h values, and also genotypes were classified as non tannin- containing, moderate tannin-containing and high tannin- containing respectively, based on their condensed tannins content. In addition, behaviour in phenolics of samples group by colour were Grayish Orange ≥ Slightly Desaturated Orange ≥ Very Soft Orange while in antioxidant capacity was Grayish Orange ≥ Slightly Desaturated Orange = Very Soft Orange. Finally, the ten evaluated sorghum genotypes grown in Nuevo León, México, were in agreement with most of the data available in literature for content of phenolics and levels of antioxidant capacity of sorghum genotypes from different regions around the world, although data variation among different reports is mainly attributed to solvent and technique used for extraction. their results higher than ours. Taking into account levels of condensed tannins, sorghum genotypes of the present study can be classified as tannin-free, moderate containing-tannin and high containing-tannin for samples within VSO, SDO and GO groups respectively. Chromatic characteristics Antioxidant capacity of sorghum genotypes Genotype Antioxidant capacity (µmolTE g-1) DPPH ABTS FRAP ‘46038B’ 1.20±0.02g 48.12±0.17d 2.62±0.06h ‘7B’ 1.93±0.08f 51.82±0.43c 4.88±0.02e ‘FAUANL-3’ 1.82±0.25f 35.30±0.59f 4.01±0.33g ‘Keller’ 11.77±0.08c 46.57±0.88de 11.77±0.27c ‘RB Norteño’ 2.17±0.48ef 30.25±0.36h 4.88±0.05e ‘RB Paloma’ 2.37±0.16e 32.69±0.45g 4.32±0.26fg ‘Rox Orange’ 93.83±0.92a 156.08±0.69a 98.50±0.25a SPV4511-2ʼ 3.48±0.15d 33.98±0.66fg 5.60±0.27d ‘Tanol-1’ 37.64±0.61b 87.37±0.47b 28.19±0.50b ‘WB’ 2.40±0.12e 46.43±0.46e 4.55±0.02f Values with different letters within column are significantly different (p≤0.05). Data are expressed as means values of three samples ± standard deviation Table 6. Antioxidant capacity of sorghum genotypes grouped by colour Genotype Number of genotypes Antioxidant capacity (µmolTE g-1) DPPH ABTS FRAP VSO 3 1.83±0.52b 44.21±8.79b 3.94±1.03b SDO 5 4.33±3.90b 38.51±6.99b 6.16±2.96b GO 2 65.73±30.79a 121.73±37.64a 63.34±38.51a Values with different letters within column are significantly different (p≤0.05). VSO, SDO and GO are expressed as means value of nine, fifteen and six samples ± standard deviation respectively Table 5. Antioxidant capacity of sorghum genotypes Genotype Antioxidant capacity (µmolTE g-1) DPPH ABTS FRAP ‘46038B’ 1.20±0.02g 48.12±0.17d 2.62±0.06h ‘7B’ 1.93±0.08f 51.82±0.43c 4.88±0.02e ‘FAUANL-3’ 1.82±0.25f 35.30±0.59f 4.01±0.33g ‘Keller’ 11.77±0.08c 46.57±0.88de 11.77±0.27c ‘RB Norteño’ 2.17±0.48ef 30.25±0.36h 4.88±0.05e ‘RB Paloma’ 2.37±0.16e 32.69±0.45g 4.32±0.26fg ‘Rox Orange’ 93.83±0.92a 156.08±0.69a 98.50±0.25a SPV4511-2ʼ 3.48±0.15d 33.98±0.66fg 5.60±0.27d ‘Tanol-1’ 37.64±0.61b 87.37±0.47b 28.19±0.50b ‘WB’ 2.40±0.12e 46.43±0.46e 4.55±0.02f Values with different letters within column are significantly different (p≤0.05). Data are expressed as means values of three samples ± standard deviation Table 5. Antioxidant capacity of sorghum genotypes DPPH and 15.54 µmolTE g-1 in ABTS, these results were higher than SDO group of our study which had similar characteristics of genotypes analyzed by these authors. Four tannin-containing sorghum grains from Texas (United States) and Kari (Kenya) were analyzed by Awika et al. (2009) and they found lower average values than our GO group samples with 27 µmolTE g-1 in DPPH and 103 µmolTE g-1 in ABTS. In addition, Luthria et al. (2013) evaluated the antioxidant capacity of the breeding lines pearled kernels ‘PR6E14’ and ‘PR6E6’ grown in Texas (United States) by FRAP, reporting an average value around 80 µmolTE g-1 which were higher than all our colour groups. DPPH and 15.54 µmolTE g-1 in ABTS, these results were higher than SDO group of our study which had similar characteristics of genotypes analyzed by these authors. Four tannin-containing sorghum grains from Texas (United States) and Kari (Kenya) were analyzed by Awika et al. Chromatic characteristics g g yp When sorghum genotypes were grouped by colour (Table 4), the behaviour in all phenolics analysis was GO > SDO > VSO and significant difference (p≤0.05) between the colour groups was observed. GO group was 4.8 and 8.7, 5.9 and 33.4, 5.24 and 27.1-fold higher than SDO and VSO in total phenolics, total flavonoids and condensed tannins respectively. p y There are several studies of phenolics in different sorghum genotypes but most of them did not report colour data and only described visual characteristics. Afify et al. (2012) analyzed phenolics of three white sorghum genotypes grown in Giza (Egypt) and found an average content of 1,121, 532 and 84 µg g-1 in total phenolics, total flavonoids and condensed tannins respectively; the description of genotypes evaluated by these authors are similar to VSO colour group of the present study and the values in total phenolics and condensed tannins were lower than our results but data obtained in total flavonoids were higher. Kobue-Lekalake et al. (2007) evaluated containing-tannin (red) and tannin-free (white) genotypes developed in Botswana and South Africa, which had visual characteristics similar than ours VSO and GO colour groups respectively and they found a total phenolics average of 15,066 µg g-1 for containing-tannin and 2,333 µg g-1 for tannin-free sorghums. In addition, they found 52,300 µg g-1 of condensed tannins in containing-tannin genotypes, being g y The hue angle ranged from 61 to 82 indicating that all values were in the orange zone (red-yellow) colour on the hue circle. The values of C* and h of all genotypes analyzed in our study are in agreement with most of the data reported by Dykes et al. (2011) for lemon-yellow (obtained from a* and b) sorghum genotypes grown in two locations of Texas (United States), but L values of these authors are different since they found a range from 44 to 63 being their genotypes darker than ours. g g p There are not a defined classification nomenclature colour of sorghum genotypes according to their chromatic values, but based on L, C and h values obtained we classified our sorghum genotypes in three colour groups: 1) Very Soft Orange (VSO), including ʻ46038Bʼ, ʻ7Bʼ and ʽRB Palomaʼ; 2) Slightly Desaturated Orange (SDO), including ʻFAUANL-ʼ3, ʻKellerʼ, ʻRB Norteñoʼ, ʻSPV4511-2ʼ and ʻWBʼ; 3) Grayish Orange López-Contreras JJ et al. / Not Bot Horti Agrobo, 2015, 43(2):366-370 369 Table 5. References Afify AE-MMR, El-Beltagi HS, Abd El-Salam SM, Omran AA (2012). Biochemical changes in phenols, flavonoids, tannins, vitamin E, β- carotene and antioxidant activity during soaking of three white sorghum varieties. 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https://openalex.org/W2745734064	https://www.research-collection.ethz.ch/bitstream/20.500.11850/192547/2/Fulltext%28publishedversion%29.pdf	English	null	Folate Receptor-Positive Gynecological Cancer Cells: In Vitro and In Vivo Characterization	Pharmaceuticals	2,017	cc-by	11,785	ETH Library Folate Receptor-Positive Gynecological Cancer Cells: In Vitro and In Vivo Characterization Klaudia Siwowska 1, Raffaella M. Schmid 1, Susan Cohrs 1, Roger Schibli 1,2 and Cristina Müller 1,* 1 Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, Villigen-PSI 5232, Switzerland; klaudia.siwowska@psi.ch (K.S.); raffaella.schmid@psi.ch (R.M.S.); susan.cohrs@psi.ch (S.C.); roger.schibli@psi.ch (R.S.) 1 Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, Villigen-PSI 5232, Switzerland; klaudia.siwowska@psi.ch (K.S.); raffaella.schmid@psi.ch (R.M.S.); susan.cohrs@psi.ch (S.C.); roger.schibli@psi.ch (R.S.) g p ( ) 2 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich 8093, Switzerland * Correspondence: cristina.mueller@psi.ch; Tel.: +41-56-310-44-54 g p 2 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich 8093, Switzerland * C d i ti ll @ i h T l 41 56 310 44 54 g p 2 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich 8093, Switzerland * Correspondence: cristina.mueller@psi.ch; Tel.: +41-56-310-44-54 2 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich 8093, Switzerland * Correspondence: cristina.mueller@psi.ch; Tel.: +41-56-310-44-54 * Correspondence: cristina.mueller@psi.ch; Tel.: +41-56-310-44-54 Received: 6 May 2017; Accepted: 9 August 2017; Published: 15 August 2017 Received: 6 May 2017; Accepted: 9 August 2017; Published: 15 August 2017 Abstract: The folate receptor alpha (FR) is expressed in a variety of gynecological cancer types. It has been widely used for tumor targeting with folic acid conjugates of diagnostic and therapeutic probes. The cervical KB tumor cells have evolved as the standard model for preclinical investigations of folate-based (radio) conjugates. In this study, a panel of FR-expressing human cancer cell lines— including cervical (HeLa, KB, KB-V1), ovarian (IGROV-1, SKOV-3, SKOV-3.ip), choriocarcinoma (JAR, BeWo) and endometrial (EFE-184) tumor cells—was investigated in vitro and for their ability to grow as xenografts in mice. FR-expression levels were compared in vitro and in vivo and the cell lines were characterized by determination of the sensitivity towards commonly-used chemotherapeutics and the expression of two additional, relevant tumor markers, HER2 and L1-CAM. It was found that, besides KB cells, its multiresistant KB-V1 subclone as well as the ovarian cancer cell lines, IGROV-1 and SKOV-3.ip, could be used as potentially more relevant preclinical models. They would allow addressing speciﬁc questions such as the therapeutic efﬁcacy of FR-targeting agents in tumor (mouse) models of multi-resistance and in mouse models of metastases formation. Keywords: folate receptor; folic acid; ovarian cancer; cervical cancer; endometrial cancer; choriocarcinoma, KB; KB-V1; IGROV-1; SKOV-3; SKOV-3.ip Originally published in: 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. pharmaceuticals Pharmaceuticals 2017, 10, 72; doi:10.3390/ph10030072 1. Introduction An overview in this regard would not only facilitate the design of future research in the ﬁeld, but allow for the selection of an appropriate tumor cell type when testing combination therapies with chemotherapeutics or other treatment modalities. Moreover, it may allow a better understanding of the differences between in vitro and in vivo models as well as the challenges which may occur when translating in vitro results to the in vivo situation. There is a number of human gynecological cancer cell lines which are known, or mentioned in the literature, to express the FR [27]. These include cell lines of cervical and ovarian cancer, as well as choriocarcinoma and endometrial tumor types. The use of the cervical adenocarcinoma cells is most common in the ﬁeld, due to the very high FR-expression level in KB cells [31–34]. KB cells are a subclone of HeLa cells, which is the ﬁrst human epithelial cancer cell line established in culture and probably the best-known cell line in past and current research [35]. The multidrug-resistant KB-V1 cell line has been derived from KB cells by culturing them with increasing amounts of vinblastine [36]. The multi-drug resistance (MDR)-1 gene of KB-V1 cells encodes P-glycoprotein responsible for decreased intracellular accumulation of anticancer agents, such as vinca alkaloids, doxorubicin, daunorubicin, paclitaxel, actinomycin D or etoposide [37–39]. Strong attention should be drawn towards ovarian cancer, since this cancer type is known to express the FR with highest frequency (~90% of the cases) in patients [3]. The human ovarian adenocarcinoma cell line, IGROV-1, was proposed as a model for human ovarian cancer in 1985 [40], and later employed for FR-targeting studies [41]. SKOV-3 is another ovarian cancer cell line found to express the FR [42–44]. Yu et al. reported on the development of SKOV-3.ip cells, generated by isolating tumor cells from the ascites of a mouse injected with SKOV-3 intraperitoneally [43]. The SKOV-3.ip subclone was characterized with a higher level of c-erbB-2/neu expression, as well as more aggressive peritoneal carcinomatosis. It was proposed as a more relevant model of ovarian cancer since multiple metastasis-like tumors grow in the abdomen when the cells are injected into the peritoneum of mice [43]. Two choriocarcinoma cell lines, JAR and BeWo, were mentioned in the literature to be FR-positive [45–47]. These cell lines were used as models for malignant tumors of the trophoblast [48–50]. 1. Introduction The folate receptor alpha (FR) has emerged as an interesting tumor target due to its overexpression in a variety of tumor types, including several gynecological cancers of epithelial origin [1–3]. The occurrence of FRs in normal tissue is limited, with kidneys being the most important site of physiological FR-expression [4,5]. Due to favorable FR-targeting properties, the vitamin folic acid has been investigated extensively as a ligand to deliver attached diagnostic and therapeutic payloads for imaging and therapy of FR-expressing cancer [6]. This targeting concept is based on the accessibility of folic acid for chemical derivatization allowing the conjugation of even bulky entities without losing FR-binding afﬁnity [7]. Tumor targeting using radionuclides conjugated to folic acid was shown to be effective for nuclear imaging using single photon emission computed tomography (SPECT) and positron emission tomography (PET) in numerous pre-clinical experiments as well as in the clinics [8–10]. Moreover, folate conjugates of ﬂuorescent probes have been developed for intraoperative imaging of ovarian tumors allowing more radical cytoreductive surgery in patients [11]. With regard to FR-targeted tumor therapy, many approaches have been reported in the literature, among those the most promising being the coupling of folic acid with anticancer drugs [12–18]. A number of otherwise highly toxic agents Pharmaceuticals 2017, 10, 72; doi:10.3390/ph10030072 www.mdpi.com/journal/pharmaceuticals 2 of 17 Pharmaceuticals 2017, 10, 72 have been used in conjunction with folic acid to allow speciﬁc accumulation in FR-expressing tumor cells for cancer therapy in clinical trials [19–24]. The choice of an appropriate tumor model to investigate the concept of FR-targeting in preclinical settings remains challenging. In this regard, KB tumor cells are most often used and, hence, they are considered as the “gold standard” for this purpose [17]. In the past, this cell line was believed to be a human nasopharyngeal epidermal carcinoma cell line [25–28], however, later it became obvious that the KB cell line was established via contamination by HeLa cells, a cervical cancer cell line [29,30]. KB cells are readily used for any investigation with regard to FR-targeting due to their high FR-expression level as well as fast growth and general ease of culturing. The question arises, however, whether this model is the most appropriate for preclinical research. As no comprehensive study exists, in which different FR-positive tumor cell lines are investigated and compared, the in vitro and in vivo characterization of such cell lines appeared important. 2.1. In Vitro Culturing of FR-Expressing Cancer Cell Lines 2. Results and Discussion 2.1. In Vitro Culturing of FR-Expressing Cancer Cell Lines 2. Results and Discussion All cell lines were grown in folate-deﬁcient RPMI medium (FFRPMI) with fetal calf serum (FCS) as the only source of folate. The expression of the FR allowed these cell lines to grow at very low folate concentrations when most FR-negative cell lines would not survive. Different morphology and conﬂuency levels were observed for each cancer cell line even among the same tumor type as shown in the microscopic images (Figure 1). There was a tendency of faster growth of KB-V1 and SKOV-3.ip cells as compared to the parental cells, KB and SKOV-3, respectively. Most of the cell lines showed tight adherence to the culture ﬂasks, however, KB-V1 and BeWo cells were more challenging to culture and for being used in experiments as they showed weak adherence, requiring surface-coated cell culture ﬂasks and well-plates. 2.1. In Vitro Culturing of FR-Expressing Cancer Cell Lines All cell lines were grown in folate-deficient RPMI medium (FFRPMI) with fetal calf serum (FCS) as the only source of folate. The expression of the FR allowed these cell lines to grow at very low folate concentrations when most FR-negative cell lines would not survive. Different morphology and confluency levels were observed for each cancer cell line even among the same tumor type as shown in the microscopic images (Figure 1). There was a tendency of faster growth of KB-V1 and SKOV-3.ip cells as compared to the parental cells, KB and SKOV-3, respectively. Most of the cell lines showed tight adherence to the culture flasks, however, KB-V1 and BeWo cells were more challenging to culture and for being used in experiments as they showed weak adherence, requiring surface-coated cell culture flasks and well-plates. Figure 1. Microscopic images of (A) HeLa cells; (B) KB cells and (C) KB-V1 cells; (D) IGROV-1 cells; (E) SKOV-3 cells and (F) SKOV-3.ip cells; (G) JAR cells; (H) BeWo cells and (I) EFE-184 cells. Magnification 20×. Figure 1. Microscopic images of (A) HeLa cells; (B) KB cells and (C) KB-V1 cells; (D) IGROV-1 cells; (E) SKOV-3 cells and (F) SKOV-3.ip cells; (G) JAR cells; (H) BeWo cells and (I) EFE-184 cells. Magniﬁcation 20×. Figure 1. Microscopic images of (A) HeLa cells; (B) KB cells and (C) KB-V1 cells; (D) IGROV-1 cells; (E) SKOV-3 cells and (F) SKOV-3.ip cells; (G) JAR cells; (H) BeWo cells and (I) EFE-184 cells. Magnification 20×. 1. Introduction Finally, there was an indication that endometrial EFE-184 tumor cells also express the FR (oral communication). This cell line may be of interest to be used as a model of endometrial cancer, since clinically, this cancer type is also reported to express the FR with high frequency (~90%) [3]. In the present study, the aim was to characterize these FR-positive cell lines in vitro and in vivo. The relative FR-expression level and the ability to bind folate radioconjugates were investigated in cultured cells. The most promising cancer cell lines were tested with regard to their ability to grow in mice. FR-expression was evaluated on tissue sections of xenografts using in vitro autoradiography and immunohistochemistry. Finally, the in vivo targeting was demonstrated using a recently-developed folate radioconjugate (177Lu-cm10 [51]) in the four tumor mouse models that revealed the greatest potential to be used for FR-targeting research. 3 of 17 Pharmaceuticals 2017, 10, 72 2.1. In Vitro Culturing of FR-Expressing Cancer Cell Lines 2. Results and Discussion Figure 1. Microscopic images of (A) HeLa cells; (B) KB cells and (C) KB-V1 cells; (D) IGROV-1 cells; (E) SKOV-3 cells and (F) SKOV-3.ip cells; (G) JAR cells; (H) BeWo cells and (I) EFE-184 cells. Magniﬁcation 20×. f p f Western Blot technique was used to determine relative FR- 2.2. Determination of FR-Expression Levels of Cells Cultured In Vitro The value obtained for KB cells was set as 100% and the percentage of the signals of the other cell lines was calculated for each single western blot (n = 5–6) and expressed as the average ± standard deviation. Figure 2. Quantiﬁcation of signal intensity obtained from western blot for FR-expression in cervical, ovarian, choriocarcinoma and endometrial cancer cell lines. The value obtained for KB cells was set as 100% and the percentage of the signals of the other cell lines was calculated for each single western blot (n = 5–6) and expressed as the average ± standard deviation. Pharmaceuticals 2017, 10, 72 4 of 16 Figure 2. Quantification of signal intensity obtained from western blot for FR-expression in cervical, ovarian, choriocarcinoma and endometrial cancer cell lines. The value obtained for KB cells was set as 100% and the percentage of the signals of the other cell lines was calculated for each single western Figure 2. Quantification of signal intensity obtained from western blot for FR-expression in cervical, ovarian, choriocarcinoma and endometrial cancer cell lines. The value obtained for KB cells was set as 100% and the percentage of the signals of the other cell lines was calculated for each single western blot (n = 5–6) and expressed as the average ± standard deviation. Figure 2. Quantiﬁcation of signal intensity obtained from western blot for FR-expression in cervical, ovarian, choriocarcinoma and endometrial cancer cell lines. The value obtained for KB cells was set as 100% and the percentage of the signals of the other cell lines was calculated for each single western blot (n = 5–6) and expressed as the average ± standard deviation. Figure 2. Quantification of signal intensity obtained from western blot for FR-expression in cervical, ovarian, choriocarcinoma and endometrial cancer cell lines. The value obtained for KB cells was set 100% d th t f th i l f th th ll li l l t d f h i l t As next step, the ability of these cell lines to actively accumulate folate conjugates via FR-mediated uptake was investigated in vitro using a radiolabeled folate conjugate (177Lu-cm10, [51]) previously developed in our group (Figure 3). In cervical cancer cells, the total uptake of the radiofolate was in the range of 21–42% of added activity whereof about 12% and 15% were internalized after 2 h and 4 h incubation, respectively (Figure 3A). f p f Western Blot technique was used to determine relative FR- 2.2. Determination of FR-Expression Levels of Cells Cultured In Vitro IGROV-1 and SKOV-3.ip cells showed high radiofolate uptake reaching 60–70% of added activity. Interestingly, these ovarian cancer cells showed higher radiofolate uptake than KB cells, despite lower expression of FRs. These findings are in agreement with literature reports where it is stated that the FR-expression level is not proportional to the uptake of folates [7]. The uptake in SKOV-3 cells was more comparable to the uptake in cervical cancer cell lines. JAR and BeWo cells showed equally high uptake and internalization comparable to HeLa, KB, KB-V1 and SKOV-3 cells. Slightly reduced values were found in the case of EFE-184 cells in comparison to JAR and BeWo. Generally, the internalized fraction was about one third up to half of the total uptake (referring to the sum of surface-bound and internalized fraction) of radiofolate. In addition, experiments with excess folic acid to block FRs prior to the addition of the radiofolate resulted in reduced uptake and internalization to less than 1% which unambiguously indicated FR-specific binding of the radiofolate (Figure 3). As next step, the ability of these cell lines to actively accumulate folate conjugates via FR-mediated uptake was investigated in vitro using a radiolabeled folate conjugate (177Lu-cm10, [51]) previously developed in our group (Figure 3). In cervical cancer cells, the total uptake of the radiofolate was in the range of 21–42% of added activity whereas about 12% and 15% were internalized after 2 h and 4 h incubation, respectively (Figure 3A). IGROV-1 and SKOV-3.ip cells showed high radiofolate uptake reaching 60–70% of added activity. Interestingly, these ovarian cancer cells showed higher radiofolate uptake than KB cells, despite lower expression of FRs. These ﬁndings are in agreement with literature reports where it is stated that the FR-expression level is not proportional to the uptake of folates [7]. The uptake in SKOV-3 cells was more comparable to the uptake in cervical cancer cell lines. JAR and BeWo cells showed equally high uptake and internalization comparable to HeLa, KB, KB-V1 and SKOV-3 cells. Slightly reduced values were found in the case of EFE-184 cells in comparison to JAR and BeWo. Generally, the internalized fraction was about one third up to half of the total uptake (referring to the sum of surface-bound and internalized fraction) of radiofolate. f p f Western Blot technique was used to determine relative FR- 2.2. Determination of FR-Expression Levels of Cells Cultured In Vitro q p lines (Figure 2, Supplementary Materials Figure S1). Among the tested cervical cancer cell lines, KB cells revealed the most prominent FR-expression, followed by KB-V1 and HeLa cells, the latter showing clearly reduced levels despite being frequently used in FR-targeting research [52,53]. In ovarian cancer cells FR-expression levels were almost identical in IGROV-1 and SKOV-3.ip tumor cells. In SKOV-3 cells, however, FR-expression was lower and comparable to the expression level in HeLa cells. In the choriocarcinoma cells, JAR and BeWo, the FR was detected as well, but at lower levels. The western blot signal obtained with EFE-184 cells was very weak, indicating low FR-expression levels. Comparison of FR-expression in all investigated cancer cell lines, independent of the tumor type, revealed the following sequence: KB > KB-V1 > SKOV-3.ip > IGROV-1 > HeLa ≈ SKOV-3 ≈ JAR > BeWo > EFE-184. Based on these results, IGROV-1 or SKOV-3.ip ovarian cancer cell lines appeared most promising after KB and KB-V1 cells to be used for FR-targeting. Western Blot technique was used to determine relative FR-expression levels in all cancer cell lines (Figure 2, Supplementary Materials Figure S1). Among the tested cervical cancer cell lines, KB cells revealed the most prominent FR-expression, followed by KB-V1 and HeLa cells, the latter showing clearly reduced levels despite being frequently used in FR-targeting research [52,53]. In ovarian cancer cells FR-expression levels were almost identical in IGROV-1 and SKOV-3.ip tumor cells. In SKOV-3 cells, however, FR-expression was lower and comparable to the expression level in HeLa cells. In the choriocarcinoma cells, JAR and BeWo, the FR was detected as well, but at lower levels. The western blot signal obtained with EFE-184 cells was very weak, indicating low FR-expression levels. Comparison of FR-expression in all investigated cancer cell lines, independent of the tumor type, revealed the following sequence: KB > KB-V1 > SKOV-3.ip > IGROV-1 > HeLa ≈SKOV-3 ≈JAR > BeWo > EFE-184. Based on these results, IGROV-1 or SKOV-3.ip ovarian cancer cell lines appeared most promising after KB and KB-V1 cells to be used for FR-targeting. 4 of 17 16 Pharmaceuticals 2017, 10, 72 Pharmaceuticals 2017, 10, 7 Figure 2. Quantification of signal intensity obtained from western blot for FR-expression in cervical, ovarian, choriocarcinoma and endometrial cancer cell lines. f p f Western Blot technique was used to determine relative FR- 2.2. Determination of FR-Expression Levels of Cells Cultured In Vitro In addition, experiments with excess folic acid to block FRs prior to the addition of the radiofolate resulted in reduced uptake and internalization to less than 1% which unambiguously indicated FR-speciﬁc binding of the radiofolate (Figure 3). blot (n = 5–6) and expressed as the average ± standard deviation. As next step, the ability of these cell lines to actively accumulate folate conjugates via FR-mediated uptake was investigated in vitro using a radiolabeled folate conjugate (177Lu-cm10, [51]) previously developed in our group (Figure 3). In cervical cancer cells, the total uptake of the radiofolate was in the range of 21–42% of added activity whereof about 12% and 15% were internalized after 2 h and 4 h incubation, respectively (Figure 3A). IGROV-1 and SKOV-3.ip cells showed high radiofolate uptake reaching 60–70% of added activity. Interestingly, these ovarian cancer cells showed higher radiofolate uptake than KB cells, despite lower expression of FRs. These findings are in agreement with literature reports where it is stated that the FR-expression level is not proportional to the uptake of folates [7]. The uptake in SKOV-3 cells was more comparable to the uptake in cervical cancer cell lines. JAR and BeWo cells showed equally high uptake and internalization comparable to HeLa, KB, KB-V1 and SKOV-3 cells. Slightly reduced values were found in the case of EFE-184 cells in comparison to JAR and BeWo. Generally, the internalized fraction was about one third up to half of the total uptake (referring to the sum of surface-bound and internalized fraction) of radiofolate. In addition, experiments with excess folic acid to block FRs prior to the addition of the radiofolate resulted in reduced uptake and internalization to less than 1% which unambiguously indicated FR-specific binding of the radiofolate (Figure 3). Figure 3. Cont. Figure 3. Cont. Figure 3. Cont. 5 of 17 16 Pharmaceuticals 2017, 10, 72 Pharmaceuticals 2017, 10, Figure 3. Total uptake (up) and internalization (int) of 177Lu-folate in (A) cervical cancer cells; (B) ovarian cancer cells; (C) choriocarcinoma cells and endometrial cancer cell. Figure 3. Total uptake (up) and internalization (int) of 177Lu-folate in (A) cervical cancer cells; (B) ovarian cancer cells; (C) choriocarcinoma cells and endometrial cancer cell. Figure 3. Total uptake (up) and internalization (int) of 177Lu-folate in (A) cervical cancer cells; (B) ovarian cancer cells; (C) choriocarcinoma cells and endometrial cancer cell. Figure 3. f p f Western Blot technique was used to determine relative FR- 2.2. Determination of FR-Expression Levels of Cells Cultured In Vitro Total uptake (up) and internalization (int) of 177Lu-folate in (A) cervical cancer cells; (B) ovarian cancer cells; (C) choriocarcinoma cells and endometrial cancer cell. 2.3. Tumor Cell Characterization beyond FR-Expression 2.3. Tumor Cell Characterization beyond FR-Expression 2.3.1. Expression of L1-Cell Adhesion Molecule 2.3.1. Expression of L1-Cell Adhesion Molecule As a further characterization of these cancer cell lines we determined the expression levels of L1-cell adhesion molecule (L1-CAM), a frequently expressed antigen in ovarian cancer known to correlate with the aggressiveness of cancer (Supplementary Materials Figure S2A) [54–56]. L1-CAM was detected in all three cervical cancer cell lines. In ovarian cancer cells, SKOV-3 and SKOV-3.ip cells, showed significant expression of L1-CAM whereas in IGROV-1 cells the expression level appeared to be lower. L1-CAM-expression may be of relevance, as it was shown that downregulation of L1-CAM in IGROV-1 cells led to decreased cell proliferation [57]. In line with this observation, the treatment of SKOV-3.ip cells with an antibody against L1-CAM showed significantly decreased proliferation [58]. Interestingly, choriocarcinoma cells did not show any expression of L1-CAM, however, high expression levels were found in EFE-184 cells. Since L1-CAM was previously associated with a poor prognosis in endometrial cancer [59,60], it is likely that EFE-184 cells are representative for an aggressive cancer cell type. 2 3 2 Expression of Human Epidermal Growth Factor Receptor 2 As a further characterization of these cancer cell lines we determined the expression levels of L1-cell adhesion molecule (L1-CAM), a frequently expressed antigen in ovarian cancer known to correlate with the aggressiveness of cancer (Supplementary Materials Figure S2A) [54–56]. L1-CAM was detected in all three cervical cancer cell lines. In ovarian cancer cells, SKOV-3 and SKOV-3.ip cells, showed signiﬁcant expression of L1-CAM whereas in IGROV-1 cells the expression level appeared to be lower. L1-CAM-expression may be of relevance, as it was shown that downregulation of L1-CAM in IGROV-1 cells led to decreased cell proliferation [57]. In line with this observation, the treatment of SKOV-3.ip cells with an antibody against L1-CAM showed signiﬁcantly decreased proliferation [58]. Interestingly, choriocarcinoma cells did not show any expression of L1-CAM, however, high expression levels were found in EFE-184 cells. Since L1-CAM was previously associated with a poor prognosis in endometrial cancer [59,60], it is likely that EFE-184 cells are representative for an aggressive cancer cell type. 2.3.3. Sensitivity towards Chemotherapeutics The characterization of the investigated cancer cell lines was additionally addressed by determination of their sensitivity towards the treatment with commonly used chemotherapeutics (Table 1). 5-Fluorouracil (5-FU), gemcitabine (GEM) and pemetrexed (PMX) are antimetabolites which are employed or tested as radiosensitizing agents for application in radio-oncology [69–71]. 5-FU reduced cell viability when applied in the micromolar range. KB cells showed reduced sensitivity to 5-FU as compared to KB-V1 (Table 1), despite the latter being characterized as multi-drug resistant (MDR) [36]. These ﬁndings are in agreement with previous studies suggesting that the MDR-1 gene expression does not cause resistance against 5-FU [72]. IGROV-1 and SKOV-3.ip demonstrated values in the same range, whereas the SKOV-3 cells were less sensitive towards 5-FU. BeWo cells were 2-fold more sensitive than JAR and EFE-184 cells. The IC50 values for all investigated cell lines treated with GEM were in the nanomolar range. From the most sensitive to the most resistant cancer cell line IC50, values varied over two orders of magnitude. KB cells were less sensitive than HeLa cells and KB-V1 cells were the most sensitive among cervical cancer cells. These ﬁndings are in line with the literature, where it was reported that multidrug resistant cells are more sensitive toward gemcitabine treatment than their parental cell lines [73]. Among ovarian cancer cell lines, SKOV-3.ip was the least sensitive. JAR cells were less sensitive than BeWo cells, which revealed to be most sensitive among all tested cell lines. Finally, EFE-184 cells showed an IC50 value which was in the same range as for ovarian cancer cells. Table 1. IC50 values of cells treated with 5-ﬂuorouracil (5-FU), gemcitabine (GEM), pemetrexed (PMX), cisplatin (CIS), doxorubicin (DOX) and paclitaxel (PCX). Cancer Type Cell Line 5-FU IC50 (µM) GEM IC50 (nM) PMX IC50 (nM) CIS IC50 (µM) DOX IC50 (nM) PCX IC50 (nM) Cervical HeLa 16.2 56.4 8.9 ** 2.8 36.2 9.5 KB 28.6 * 120 4.5 1.9 15.4 2.7 KB-V1 9.1 12.8 5.3 0.3 211 597 Ovarian IGROV-1 2.0 11.8 41.8 0.8 23.9 5.0 SKOV-3 8.0 20.8 14.9 * 5.6 96.7 ** 4.0 SKOV-3.ip 3.1 45.0 * 6.4 * 1.9 449 ** 9.6 Choriocarcinoma JAR 8.2 30.6 33.8 0.9 9.8 3.1 BeWo 4.2 1.2 6.4 0.4 2.2 1.6 Endometrial EFE-184 9.7 * 15.8 n.d. Human epidermal growth factor receptor 2 (HER2) is an epid 2.3.2. Expression of Human Epidermal Growth Factor Receptor-2 overexpressed in 10–15% of breast cancers and associated with a poor prognosis [61]. It is a common marker of breast cancer, however, also found in ovarian cancer, with the incidence indicated between 8% and 66% depending on the literature [62]. Although the significance of HER2 is clearly established in breast cancer, its role is not as clear in ovarian cancer. Treatment of ovarian cancer with trastuzumab, an anti-HER2 antibody resulted in an overall response rate of only ~7% in patients with HER2-positive ovarian cancer [63], whereas in breast cancer patients the overall Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor receptor 2, overexpressed in 10–15% of breast cancers and associated with a poor prognosis [61]. It is a common marker of breast cancer, however, also found in ovarian cancer, with the incidence indicated between 8% and 66% depending on the literature [62]. Although the signiﬁcance of HER2 is clearly established in breast cancer, its role is not as clear in ovarian cancer. Treatment of ovarian cancer with trastuzumab, an anti-HER2 antibody resulted in an overall response rate of only ~7% in patients with HER2-positive ovarian cancer [63], whereas in breast cancer patients the overall response rate was 15–18% [64]. The detailed investigation of the role of HER2 in ovarian cancer and other non-breast cancers is currently 6 of 17 Pharmaceuticals 2017, 10, 72 an important topic of research. Therefore, we set out to investigate the cell lines with regard to HER2-expression (Supplementary Materials Figure S2B). Data on HER2-expression in cervical cancer is not consistently reported in the literature [65], however, in our study expression of HER2 was not detected in cervical cancer cell lines. Among the ovarian cell lines tested in this study, HER2-expression was found in SKOV-3 and SKOV-3.ip tumor cells, in line with the literature [66]. However, other than in previous studies, we did not ﬁnd much difference in HER2-expression levels among these cell lines. HER2 was also reported to be expressed at a moderate level in IGROV-1 cells [67], however, in the present study it was not detected in this cell line. In choriocarcinoma, the HER2-expression was reported to be associated with an invasive phenotype [68]. While JAR cells did not show expression of HER2, a signal was detected for BeWo cells, potentially indicating a more invasive phenotype of this choriocarcinoma cell line. 2.3.3. Sensitivity towards Chemotherapeutics 1.6 390 90.9 * At the highest applied concentration ~20% cells were still viable; ** at the highest applied concentration ~30% cells were still viable. In one case cells could not be killed entirely, even with very high amounts of the drug. In this case, the IC50 could not be determined (n.d.). * At the highest applied concentration ~20% cells were still viable; ** at the highest applied concentration ~30% cells were still viable. In one case cells could not be killed entirely, even with very high amounts of the drug. In this case, the IC50 could not be determined (n.d.). Among all three antimetabolites, PMX was most effective in reducing tumor cell viability resulting in IC50 values in the low nanomolar range with only slight variability among different cell lines. Cervical cancer cell lines were more sensitive than ovarian and choriocarcinoma cell lines and EFE-184 7 of 17 Pharmaceuticals 2017, 10, 72 cells emerged as the most resistant, as ~50% viable cells were found even with very high concentrations of PMX. Cisplatin (CIS), doxorubicin (DOX) and paclitaxel (PCX) are important chemotherapeutics since they are used as a standard therapy of ovarian cancer [74,75]. All investigated cell lines showed similar sensitivity to CIS in the low micromolar range and even the more aggressive versions, KB-V1 and SKOV-3.ip did not show any resistance against this chemotherapeutic agent. In the case of DOX and PCX, the multidrug resistant cell line, KB-V1 revealed to be more resistant than KB or HeLa cell lines as expected and previously shown [76]. Among ovarian cancer cells, SKOV-3.ip cells were much more resistant to DOX as compared to SKOV-3 and IGROV-1 cells. This can be considered as another indication that SKOV-3.ip cells are an aggressive subtype of ovarian cancer cells. On the other hand, no difference in sensitivity was determined towards PCX among ovarian cancer cells as previously reported [77]. BeWo cells reacted again more sensitive to the treatment with DOX and PCX as compared to JAR cells. EFE-184 cells proved again to be resistant, demonstrated by much higher IC50 values after treatment with DOX and PCX as compared to choriocarcinoma cells. Sensitivity of FR-positive cell lines to the commonly used chemotherapeutics is of crucial interest for the investigation of FR-targeted therapeutics, as these novel therapy concepts might be a solution in chemoresistant tumors. 2.4. Gynecologic Tumor Xenograft Mouse Models Based on the in vitro results, cervical and ovarian cancer cell lines appeared more promising to be used in vivo than JAR, BeWo and EFE-184 cells. These FR-expressing cancer cell lines were, therefore, investigated with regard to their potential to grow as xenografts in nude mice. Since it was reported that HeLa, KB and KB-V1 cells can be grown in CD-1 nude mice [27], this strain was used for in vivo experiments. The KB tumor mouse model is the best established and has been used for a large number of in vivo investigations of radiofolates in the past [18,51,78–81]. KB tumors are characterized with a fast growth and a solid, ﬁrm structure. In this study, it was conﬁrmed that KB-V1 tumor cells also grow fast in nude mice when inoculated subcutaneously. It appeared that KB-V1 tumors were better vascularized compared to KB tumors as was visible by a more reddish color of KB-V1 xenografts. The growth of HeLa cells in CD-1 nude mice was very slow and in some cases, the xenografts started to shrink after about 2–3 weeks and disappeared completely. The ovarian cancer cell lines were also grown as subcutaneous xenografts in CD-1 nude mice. IGROV-1 and SKOV-3.ip reached a tumor size suitable for in vivo experiments within about 2 weeks as reported previously [82]. On the other hand, SKOV-3 cells grew very slowly and the resulting tumor xenografts remained small even several weeks after tumor cell inoculation. PC-3 cells, used as FR-negative control, were also grown in CD-1 nude mice. 2.3.3. Sensitivity towards Chemotherapeutics Cell lines generally considered as invasive or aggressive, such as KB-V1 and SKOV-3.ip, overexpress the FR at very high levels and may be more susceptible to the FR-targeted therapies. 2.5.2. Determination of FR-Expression Using Immunohistochemistry Figure 4. Quantification of in vitro autoradiography results in tumor tissu cell line were set as 100% and compared with the other tissues. FR-expression levels in tumor xenografts were additionally investigated by immunohistochemistry and a semi-quantitative analysis was performed (Figure 5, Supplementary Materials, Figures S4 and S5). Similar to the result of the autoradiography, HeLa tumor tissue showed a 10% reduced staining, indicating lower FR-expression levels as compared to KB and KB-V1 tumor tissue which showed an intense staining signal. In comparison to the FR-staining of KB tumor tissue the signal was reduced by 6% in the case of IGROV-1 tumor tissue which was in agreement with the in vitro autoradiography results (Figure 4). The signal obtained for the SKOV-3 tumor tissue was slightly higher than the signal obtained for SKOV-3.ip tumor tissue (12% and 20% lower signal than for KB tumor tissue, respectively). This data was not in line with the autoradiography results possibly due to the fact that the tissue texture of SKOV-3.ip tumors was different than the tissue of the other tumors. The analysis of the results revealed signiﬁcantly higher values of all tumor tissue sections as compared to PC-3 tumor tissue, which served as a negative control. Absence of tissue staining was obtained in negative control experiments performed on tissue sections treated without the primary antibody (Supplementary Materials Figure S4). 2.5.2. Determination of FR-Expression Using Immunohistochemistry FR-expression levels in tumor xenografts were additionally investigated by immunohistochemistry and a semi-quantitative analysis was performed (Figure 5, Supplementary Materials, Figures S4 and S5). Similar to the result of the autoradiography, HeLa tumor tissue showed a 10% reduced staining, indicating lower FR-expression levels as compared to KB and KB-V1 tumor tissue which showed an intense staining signal. In comparison to the FR-staining of KB tumor tissue the signal was reduced by 6% in the case of IGROV-1 tumor tissue which was in agreement with the in vitro autoradiography results (Figure 4). The signal obtained for the SKOV-3 tumor tissue was slightly higher than the signal obtained for SKOV-3.ip tumor tissue (12% and 20% lower signal than for KB tumor tissue, respectively). This data was not in line with the autoradiography results possibly due to the fact that the tissue texture of SKOV-3.ip tumors was different than the tissue of the other tumors. The analysis of the results revealed significantly higher values of all tumor tissue sections as compared to PC-3 tumor tissue, which served as a negative control. 2.5.1. Determination of FR-Expression Using Autoradiography FR-expression levels were compared in tumor xenografts of cervical and ovarian carcinoma cells as well as in PC-3 xenograft using the technique of in vivo autoradiography. Based on the obtained signal, it was revealed that FR-expression in KB and KB-V1 tumors was comparable, but much lower in the case of HeLa tumor tissue (Figure 4, Supplementary Materials Figure S3). Among the ovarian tumor tissue sections, the most intense signal was obtained for the IGROV-1 tumors, whereas the signal intensity of SKOV-3.ip tumor tissue was in the range of HeLa tumor sections, indicating similar FR-expression levels. Only the signal of SKOV-3 tumor tissue was much lower. Incubation of the tumor tissue sections with excess folic acid blocked the receptors and reduced the signal to background levels which conﬁrmed FR-speciﬁc binding of the radiofolate. The FR-negative PC-3 tumor sections served 8 of 17 Pharmaceuticals 2017, 10, 72 as negative control revealing a signal of ~1% (Figure 4, Supplementary Materials Figure S3). In general, these ﬁndings were in line with those of western blot analysis with the exception being SKOV-3 and SKOV-3.ip cells, which showed high FR-expression in vitro, but when grown as xenografts in mice, FR-expression appeared to be signiﬁcantly reduced. Pharmaceuticals 2017, 10, 72 8 of 16 signal to background levels which confirmed FR-specific binding of the radiofolate. The FR-negative PC-3 tumor sections served as negative control revealing a signal of ~1% (Figure 4, Supplementary as negative control revealing a signal of ~1% (Figure 4, Supplementary Materials Figure S3). In general, these ﬁndings were in line with those of western blot analysis with the exception being SKOV-3 and SKOV-3.ip cells, which showed high FR-expression in vitro, but when grown as xenografts in mice, FR-expression appeared to be signiﬁcantly reduced. Pharmaceuticals 2017, 10, 72 8 of 16 signal to background levels which confirmed FR-specific binding of the radiofolate. The FR-negative PC-3 tumor sections served as negative control revealing a signal of ~1% (Figure 4, Supplementary l I l h f d l h h f bl l h Figure 4. Quantiﬁcation of in vitro autoradiography results in tumor tissues. Values obtained for KB cell line were set as 100% and compared with the other tissues. the exception being SKOV-3 and SKOV-3.ip cells, which showed high FR-expression in vitro, but when grown as xenografts in mice, FR-expression appeared to be significantly reduced. Figure 4. Quantiﬁcation of in vitro autoradiography results in tumor tissues. 2.5.1. Determination of FR-Expression Using Autoradiography Values obtained for KB cell line were set as 100% and compared with the other tissues. 2.6. Biodistribution Experiments The tumor growth was investigated starting from day 4 after inoculation of tumor cells by measuring tumor xenografts every second day (Supplementary Material, Figure S6). Comparison of accumulated radiofolate in different tumor types was performed based on tumor-to-kidney ratios in order to standardize the results to kidney uptake which should be equal for each mouse independent of the xenograft type (Figure 6). The analysis revealed the highest accumulation of activity in IGROV-1 tumors at both investigated time points after injection. A possible explanation for these ﬁndings may be the fact, that IGROV-1 tumors were smaller (116 ± 70 mm3 at day 14 after inoculation) in comparison to other FR-positive tumors and possibly better vascularized (Supplementary Materials Figure S6). KB tumor xenografts grew very fast (189 ± 73 mm3 at day 12 after inoculation) and appeared to be less vascularized. This was conﬁrmed by SPECT/CT images where it was seen that the activity was mainly accumulated in the outer rim of the tumor but not homogenously distributed within the whole tumor xenografts (Supplementary Material Figure S7). KB-V1 tumor cells were also found to grow fast even though the tumors were smaller (123 ± 86 mm3 at day 12 after cell inoculation). Nevertheless, both KB and KB-V1 tumors accumulated high amounts of activity which was in line with high levels of FR-expression in these tumor types as demonstrated by autoradiography and immunohistochemistry experiments (Figures 4 and 5, Supplementary Material Figures S3–S5). SKOV-3.ip tumors reached a tumor volume (134 ± 48 mm3 at day 12 after cell inoculation) and were in the same range as KB-V1 tumors. Tumor-to-kidney ratios of accumulated activity in SKOV-3.ip tumor-bearing mice were higher as compared to PC-3 tumor-bearing mice which served as a negative control, however, the differences were minimal and not signiﬁcant at the 24 h time point. Thus, it may be that in the case of SKOV-3.ip tumors, the accumulation of the radiofolate was mostly due to the blood ﬂow rather than as a consequence of FR-speciﬁc uptake. Figure 6. Tumor-to-kidney ratios of accumulated radioactivity in tumor-bearing mice 4 h and 24 h after injection of the radiofolate. The tumor-to-kidney ratios of all groups of mice bearing FR-positive tumor types (KB, KB-V1 or IGROV-1, respectively) were signiﬁcantly different (p < 0.05) than the tumor-to-kidney ratio in PC-3 tumor-bearing mice. 2.5.2. Determination of FR-Expression Using Immunohistochemistry Figure 4. Quantification of in vitro autoradiography results in tumor tissu cell line were set as 100% and compared with the other tissues. Absence of tissue staining was obtained in negative control experiments performed on tissue sections treated without the primary antibody (Supplementary Materials Figure S4). Figure 5. Immunohistochemistry results showing FR-expression in (A) HeLa; (B) KB; (C) KB-V1; (D) IGROV-1; (E) SKOV-3 and (F) SKOV-3.ip tumors. Tissue images are shown in magnification 40×. Figure 5. Immunohistochemistry results showing FR-expression in (A) HeLa; (B) KB; (C) KB-V1; (D) IGROV-1; (E) SKOV-3 and (F) SKOV-3.ip tumors. Tissue images are shown in magniﬁcation 40×. Figure 5. Immunohistochemistry results showing FR-expression in (A) HeLa; (B) KB; (C) KB-V1; (D) IGROV-1; (E) SKOV-3 and (F) SKOV-3.ip tumors. Tissue images are shown in magnification 40×. Figure 5. Immunohistochemistry results showing FR-expression in (A) HeLa; (B) KB; (C) KB-V1; (D) IGROV-1; (E) SKOV-3 and (F) SKOV-3.ip tumors. Tissue images are shown in magniﬁcation 40×. 9 of 17 Pharmaceuticals 2017, 10, 72 2.6. Biodistribution Experiments An exception was the tumor-to-kidney ratio of SKOV-3.ip tumor-bearing mice which was signiﬁcantly different (p < 0.05) from the ratios in PC-3 tumor-bearing mice only at 4 h p.i. but not (p > 0.05) at 24 h p.i. of the radiofolate. Figure 6. Tumor-to-kidney ratios of accumulated radioactivity in tumor-bearing mice 4 h and 24 h after injection of the radiofolate. The tumor-to-kidney ratios of all groups of mice bearing FR-positive tumor types (KB, KB-V1 or IGROV-1, respectively) were signiﬁcantly different (p < 0.05) than the tumor-to-kidney ratio in PC-3 tumor-bearing mice. An exception was the tumor-to-kidney ratio of SKOV-3.ip tumor-bearing mice which was signiﬁcantly different (p < 0.05) from the ratios in PC-3 tumor-bearing mice only at 4 h p.i. but not (p > 0.05) at 24 h p.i. of the radiofolate. In agreement with this analysis, it was found that the absolute tumor uptake 24 h after injection of the radiofolate was highest for IGROV-1 tumor xenografts (~34% IA/g) followed by KB (~22% IA/g), KB-V1 (~17% IA/g) and SKOV-3.ip tumors (~13% IA/g). A clearly reduced accumulation of activity was found in PC-3 tumors (~6% IA/g) at the same time point (Supplementary Materials Tables S1 and S2). Pharmaceuticals 2017, 10, 72 10 of 17 10 of 17 3. Conclusions A crucial aspect for the development of FR-targeted imaging and therapeutic agents is to use a suitable model for preclinical investigations. Until now, KB tumor cells have been the “gold standard” for in vitro and in vivo FR-targeting research, however, other tumor models may be of interest in order to take the diversity of naturally occurring cancers into account. In this study, we investigated tumor cells of cervical, ovarian and endometrial origin as well as choriocarcinoma cells. KB, KB-V1, IGROV-1 and SKOV-3.ip cells revealed to be appropriate for in vitro experiments and could be efﬁciently grown in mice allowing tumor targeting in vivo. KB cells were conﬁrmed to be a very useful model for FR-targeting research. KB-V1 tumor cells are a valid alternative, which would be of particular interest when multiresistance should be investigated. IGROV-1 tumor cells are favorable when the research refers to ovarian cancer, however, these cells appeared to be more challenging than KB tumor cells in terms of reproducible in vivo growth. Finally, the SKOV-3.ip tumor cell line would be attractive for the performance of research on mouse models with metastases-like tumors. It has to be kept in mind, however, that the SKOV-3.ip cell line expresses the FR at lower levels than it is the case for IGROV-1 tumor cells. Using these additional tumor cell lines can enable investigation of folate-based therapeutics in more detail as they would allow addressing speciﬁc questions such as their therapeutic efﬁcacy in tumor (mouse) models of multi-resistance and in models of metastases formation. 4.2. Preparation of 177Lu-Folate The 177Lu-folate was prepared under standard labeling conditions as previously reported [18,51]. Quality control of the prepared 177Lu-folate was performed via reversed-phase high performance liquid chromatography as previously reported [51]. The radiochemical purity of 177Lu-folate was always >97%. 4.1. General Pemetrexed (PMX, AlimtaTM, Eli Lilly, Indianapolis, IN, USA), doxorubicin (DOX, doxorubicin hydrochloride, Sigma-Aldrich, St. Louis, MO, USA) and gemcitabine (GEM, GemzarTM, Eli Lilly) were obtained as lyophilized powders and dissolved in sterile NaCl 0.9% according to the instructions of the manufacturer. 5-Fluorouracil (5-FU, Fluorouracil-TevaTM, Teva Pharma AG, Basel, Switzerland), cisplatin (CIS, Actavis, Actavis Switzerland AG, Regensdorf, Switzerland) and paclitaxel (PCX, Paclitaxel SandozTM, Sandoz Pharmaceuticals AG, Rotkreuz, Switzerland) were obtained as solutions for injection. The solutions were diluted in sterile NaCl 0.9% to obtain the required concentration. The folate conjugate cm10 (referred to as folate herein) [51], was kindly provided by Merck & Cie, Schaffhausen, Switzerland. No carrier added lutetium-177 (177Lu) was obtained from Isotope Technologies Garching (ITG) GmbH, Munich, Germany. 4.4. Cell Internalization Experiments Materials and methods of cell internalization experiments are reported in Supplementary Materials. Graphs were prepared using GraphPad Prism software (version 7.0, La Jolla, CA, USA). Data represents the average of two to four different experiments. 4.3. Cell Lines and Cell Culture HeLa cells (cervical carcinoma cell line, ACC-57), KB cells (cervical carcinoma cell line, subclone of HeLa cells, ACC-136), KB-V1 cells (cervical carcinoma cell line, multi-drug resistant (MDR) subclone of KB cells, ACC-14), BeWo cells (choriocarcinoma cell line, ACC-458), JAR cells (choriocarcinoma cell line, ACC-462) EFE-184 cells (endometrial carcinoma, ACC-230) and PC-3 cells (human prostate cancer cell line, FR-negative, ACC-465) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). SKOV-3 cells (ovarian adenocarcinoma cell line, ECACC Cat-N◦91091004) were purchased from Culture Collections, Public Health England, Salisbury, United Kingdom. SKOV-3.ip, an ovarian carcinoma cell line established from ascites of a nude mouse developed after intraperitoneal injection of SKOV-3 cells [43], were kindly provided by Dr. 11 of 17 Pharmaceuticals 2017, 10, 72 Ilse Novak (Paul Scherrer Institut, Villigen, Switzerland). IGROV-1 cells (human ovarian carcinoma cell line) were a kind gift from Dr. Gerrit Jansen (Department of Rheumatology, Free University Medical Center, Amsterdam, The Netherlands). Cervical (HeLa, KB, KB-V1), ovarian (SKOV-3, SKOV-3.ip, IGROV-1), choriocarcinoma (JAR, BeWo) and endometrial carcinoma cells (EFE-184) were cultured in folate-deﬁcient RPMI medium (FFRPMI, Cell Culture Technologies GmbH, Gravesano, Switzerland) supplemented with 10% FCS, L-glutamine and antibiotics. PC-3 cells were cultured in standard RPMI 1640 medium supplemented with 10% FCS, L-glutamine and antibiotics. Routine cell culture was performed twice a week using trypsin-EDTA (0.25%, Gibco) for detachment of the cells. Standard cell culture ﬂasks were used for all cells except BeWo and KB-V1 which were cultured in cell culture ﬂasks with a hydrophilic surface, obtained after microwaving process (Corning). All experiments with these two cell lines were performed in poly-L-lysine coated well-plates. 4.6. In Vitro Autoradiography Tumor xenografts collected from mice were ﬁxed in embedding material (Cryo-M-Bed, Bright) and frozen at −80 ◦C. Tumor tissue sections of 5–10 µm thickness were prepared using a cryostat (Bright OTF Cryostat, OTF/AS-001/MR/V/304/X, Huntingdon, UK). Data represents the average of three different experiments. The detailed procedure of the autoradiography experiments is described in Supplementary Materials. 4.5. Western Blot Western blot was performed with cell lysates (~40 µg protein) using anti-FR antibody (Abcam, Cambridge, UK, mouse Ab, ab3361, 1:500), anti-L1CAM antibody (chCE-7, IgG1-subtype chimeric monoclonal human antibody [83], 5 µg/mL) and anti-HER2/erbB-2 antibody (Cell Signaling Technology, Danvers, MA, USA, rabbit Ab, #2165, 1:1000). The detailed procedure is described in Supplementary Materials. Western Blot signal was quantiﬁed using ImageJ software (version 1.51k, NIH, Rockville, MD, USA). Region of interest (ROI) was chosen manually, based on the largest band in the blot. The same ROI was applied in all remaining rows, with the protein band in the middle of the ROI frame. The mean signal of each ROI was standardized to the signal of KB cells, which was set as 100%. The result is an average of percentage from ﬁve to six different experiments. 4.7. Immunohistochemistry Tumor xenografts were embedded in parafﬁn and cut into 5 µm-thick sections using a manual rotary microtome (Leica RM2235, Leica Biosystems, Wetzlar, Germany). Removal of parafﬁn was performed with xylene, followed by rehydration with decreasing ethanol concentrations. Citrate buffer (10 mM trisodium citrate/0.05% Tween buffer, pH 6) was used for antigen retrieval in 95 ◦C for 30 min. Endogenous peroxidase was blocked by incubation of the slides in a solution of 3% H2O2. Unspeciﬁc binding was prevented by incubation of the slides in 10% FCS for 60 min. Avidin/biotin blocking kit (SP-2001, Vector Laboratories, Burlingame, CA, USA) was used according to the manufacturer’s protocol. The primary anti-FR antibody (Abcam, ab67422) was added in a concentration of 0.5 µg/100 µL and slides were incubated overnight at 4 ◦C. Slides were incubated with biotinylated secondary antibody (Abcam, ab97049, 1:200) for 30 min followed by addition of the Avidin-Biotin Complex kit (ABC Reagent kit, Elite, Vectastain, Vector Laboratories) and incubation for 30 min. DAB peroxidase substrate kit (Vector Laboratories, SK-4100) was used for the development of the signal and hematoxylin (Novolink™, Leica Biosystems) for counterstaining. The sections were 12 of 17 Pharmaceuticals 2017, 10, 72 treated with increasing concentrations of ethanol before treatment with xylene for ﬁxation. Pictures were obtained using a light microscope (Axio Lab.A1, Zeiss, Oberkochen, Germany). treated with increasing concentrations of ethanol before treatment with xylene for ﬁxation. Pictures were obtained using a light microscope (Axio Lab.A1, Zeiss, Oberkochen, Germany). 4.8. Animal Experiments In vivo experiments were conducted in accordance with the Swiss law of animal protection. Athymic nude mice (Crl:CD-1-Foxn1 nu, referred herein as CD-1 nude) were purchased from Charles River Laboratories (Sulzfeld, Germany). Animals were inoculated with a suspension of the tumor cells (5–7 × 106 tumor cells in 100 µL PBS) subcutaneously on the right shoulder or both shoulders and 5 × 106 tumor cells in the case of a biodistribution study. All animals were fed with a folate-deﬁcient rodent diet (ssniff Spezialdiäten GmbH, Soest, Germany). 4.9. Biodistribution Experiments Biodistribution studies were performed 12–14 days after inoculation of the tumor cells when the tumor xenografts reached a volume between 63 mm3 and 189 mm3. 177Lu-folate conjugate (3 MBq, 0.5 nmol per mouse) was injected in a volume of 100 µL PBS into a lateral tail vein. The animals were sacriﬁced at 4 h (n = 4) and 24 h (n = 4) after administration of the radioconjugate. Blood and selected tissues and organs were collected, weighed, and radioactivity was measured using a γ-counter (Perkin Elmer, Wallac Wizard 1480, Waltham, MA, USA). The results were listed as a percentage of the injected radioactivity per gram of tissue mass (% IA/g), using counts of a deﬁned volume of the original injection solution measured at the same time resulting in decay-corrected values. The signiﬁcance of the data was determined using a one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test (GraphPad Prism Software, version 7.00). A p value of <0.05 was considered statistically signiﬁcant. Supplementary Materials: The following ﬁgures are available online at www.mdpi.com/1424-8247/10/3/ 72/s1, Figure S1: Western blot analysis of the folate receptor (FR) in different gynecologic cancer cell lines, Figure S2: Western blot analysis of L1-CAM (A) and HER2 (B) in different gynecologic cancer cell lines, Figure S3: Autoradiography results, Figure S4: Immunohistochemistry results of FR-expression in FR-expressing tumors, Figure S5: Semi-quantitative analysis of FR expression levels determined by immunohistochemistry, Figure S6: Tumor growth in mice inoculated with different cell lines, Figure S7: SPECT/CT images of tumor-bearing mice injected with 177Lu-folate, Tables S1 and S2: Results of biodistribution experiment. Acknowledgments: We thank Konstantin Zhernosekov (Isotope Technologies Garching GmbH, Munich, Germany) for providing 177Lu for this study. The project was ﬁnancially supported by the Swiss National Science Foundation (Grant 310030_156803). Klaudia Siwowska was funded by a Swiss Government Excellence Scholarship. Author Contributions: K.S. performed the in vitro and in vivo experiments, analyzed the data and wrote the manuscript. R.M.S. and S.C. assisted the in vitro and in vivo experiments and reviewed the manuscript. R.S. reviewed the manuscript and gave inputs and advice. C.M. designed and supervised the experiments, coordinated the studies and data analysis and reviewed and revised the manuscript. Conﬂicts of Interest: There is no conﬂict of interest. 4. Holm, J.; Hansen, S.I.; Hoiermadsen, M.; Bostad, L. A high-afﬁnity folate binding-protein in proximal tubule cells of human kidney. Kidney Int. 1992, 41, 50–55. [CrossRef] [PubMed] References 1. Garin-Chesa, P.; Campbell, I.; Saigo, P.E.; Lewis, J.L.; Old, L.J.; Rettig, W.J. Trophoblast and ovarian cancer antigen LK26—Sensitivity and speciﬁcity in immunopathology and molecular identiﬁcation as a folate-binding protein. Am. J. Pathol. 1993, 142, 557–567. [PubMed] 1. 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https://openalex.org/W2473628047	http://deepblue.lib.umich.edu/bitstream/2027.42/134597/1/13048_2016_Article_250.pdf	English	null	Developmental programming: rescuing disruptions in preovulatory follicle growth and steroidogenesis from prenatal testosterone disruption	Journal of ovarian research	2,016	cc-by	9,247	* Correspondence: vasantha@umich.edu 1Department of Pediatrics, University of Michigan, 7641A Med Sci II, Ann Arbor, MI 48109-5622, USA Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Developmental programming: rescuing disruptions in preovulatory follicle growth and steroidogenesis from prenatal testosterone disruption A Veiga-Lopez1,2, J Moeller1, D. H. Abbott3 and V Padmanabhan1* Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 DOI 10.1186/s13048-016-0250-y Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 DOI 10.1186/s13048-016-0250-y RESEARCH Open Access Developmental programming: rescuing disruptions in preovulatory follicle growth and steroidogenesis from prenatal testosterone disruption Background g With well over 5 million U.S. women affected, women with polycystic ovary syndrome (PCOS) are frequent patients in infertility clinics, seeking assistance in becoming pregnant [1]. A PCOS diagnosis is reached when two of the follow- ing three criteria are met: hyperandrogenism, oligo- or anovulation, and/or polycystic ovaries [2–4]. The reduced pregnancy rate in PCOS patients has been attributed to the oligo-anovulatory condition of the syndrome; this, in part, stems from a disrupted intrafollicular milieu, which in- cludes reductions in cortisone [5], insulin growth like fac- tor (IGF) I and II [6], and progesterone (P4) [7], increases in anti-Mullerian hormone (AMH) [8], testosterone, andro- stenedione, and proteomic dysregulation [9]. The compro- mised intrafollicular steroidal milieu in PCOS women likely accounts for the poor quality of oocytes [10, 11]. A recent meta-analysis study found removal of oocytes from the dis- rupted endogenous steroidal environment of PCOS women and maturing them in vitro helps achieve better conception rates [12]. Understanding the dysregulation of the intrafolli- cular milieu is essential for developing strategies to over- come infertility in PCOS. Controlled ovarian stimulation During their third breeding season (~2.5 years of age), the follicular response to a controlled ovarian stimula- tion protocol modified from that previously described [25] was tested in all females (see Fig. 1a). All females received 2 ml of prostaglandin F2α (PGF2α, 5 mg/ml; Lutalyse, Pfizer Animal Health, MI) and an intravaginal P4 control internal drug release device (CIDR; Eazi- Breed CIDR sheep inserts, Pfizer Animal Health, NY) on day 0 that was replaced on day 7. Beginning on day 1, 10 μg/kg body weight of acyline, a GnRH antagonist (GnRHa) procured from the National Hormone and Peptide Program, was administered every 12 h for 10 days. This was followed by administration of 8 de- creasing doses (two doses at each concentration) of FSH (0.6, 0.4, 0.3, and 0.1 mg/kg; Folltropin-V, Bioniche Ani- mal Health, GA) starting on day 11. The P4 CIDR was removed after the sixth FSH dose. Using the milder T60-90 phenotype [19] and a con- trolled ovarian stimulation protocol that effectively stimulates follicular development [20], we tested the hypothesis that prenatal T excess compromises mat- uration of the preovulatory follicle and disrupts the intrafollicular milieu in sheep. Since i) prenatal T- treated sheep manifest functional hyperandrogenism and insulin resistance [13]; ii) treatment with an an- drogen antagonist or insulin sensitizer improves ovu- latory function in women with PCOS [21], the reproductive phenotype of whom prenatal T-treated sheep recapitulate; and iii) postnatal insulin sensitizer-treatment prevents a progressive loss in cy- clicity of prenatal T-treated sheep [22], this study aimed to parse out the relative postnatal contribution of androgen and insulin towards dysfunctional follicle Abstract Background: Prenatal testosterone (T) excess from days 30-90 of gestation disrupts gonadotropin surge and ovarian follicular dynamics and induces insulin resistance and functional hyperandrogenism in sheep. T treatment from days 60-90 of gestation produces a milder phenotype, albeit with reduced fecundity. Using this milder phenotype, the aim of this study was to understand the relative postnatal contributions of androgen and insulin in mediating the prenatal T induced disruptions in ovarian follicular dynamics. Methods: Four experimental groups were generated: 1) control (vehicle treatment), 2) prenatal T-treated (100 mg i. m. administration of T propionate twice weekly from days 60-90 of gestation), 3) prenatal T plus postnatal anti-androgen treated (daily oral dose of 15 mg/kg/day of flutamide beginning at 8 weeks of age) and 4) prenatal T and postnatal insulin sensitizer-treated (daily oral dose of 8 mg/day rosiglitazone beginning at 8 weeks of age). Follicular response to a controlled ovarian stimulation protocol was tested during their third breeding season. Main outcome measures included the determination of number and size of ovarian follicles and intrafollicular concentrations of steroids. Results: At the end of the controlled ovarian stimulation, the number of follicles approaching ovulatory size (≥6 mm) were ~35 % lower in prenatal T-treated (6.5 ± 1.8) compared to controls (9.8 ± 2.0). Postnatal anti-androgen (10.3 ± 1.9), but not insulin sensitizer (5.0 ± 0.9), treatment prevented this decrease. Preovulatory sized follicles in the T group had lower intrafollicular T, androstenedione, and progesterone compared to that of the control group. Intrafollicular steroid disruption was partially reversed solely by postnatal insulin sensitizer treatment. Conclusions: These results demonstrate that the final preovulatory follicular growth and intrafollicular steroid milieu is impaired in prenatal T-treated females. The findings are consistent with the lower fertility rate reported earlier in these females. The finding that final follicle growth was fully rescued by postnatal anti-androgen treatment and intrafollicular steroid milieu partially by insulin sensitizer treatment suggest that both androgenic and insulin pathway disruptions contribute to the compromised follicular phenotype of prenatal T-treated females. Keywords: Steroids, Testosterone, Androgen antagonist, Insulin sensitizer Page 2 of 10 Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 responses of T60-90 prenatal T-treated sheep to controlled ovarian stimulation in adulthood. Prenatal and postnatal treatments p All procedures used were approved by the Institutional Animal Care and Use Committee of the University of Michigan and conducted at the University of Michigan Sheep Research Facility. Animal husbandry details have been published previously [23]. Mature Suffolk ewes (2 to 3 years in age) maintained under a natural photo- period were mated and date of mating confirmed based on rump paint marks left by a raddled ram. Pregnant ewes were blocked by weight and body score and ran- domly assigned to one of two treatment groups: 36 ani- mals in the prenatal T treatment group received 100 mg T propionate (Sigma-Aldrich Corp., St. Louis, MO) twice weekly in 2 ml of corn oil, i.m. from days 60 to 90 of gestation, while 12 controls received an equal volume of vehicle. Before puberty beginning at 8 weeks of age, prenatal T-treated females received either andro- gen antagonist, flutamide (Sigma-Aldrich, Corp.) (n = 11), the insulin sensitizer, rosiglitazone (Avandia; GlaxoS- mithKline, Durham, NC) (n = 12), or no treatment (n = 13). Flutamide was administered orally at a dose of 15 mg/kg/ ewe/day and rosiglitazone orally at a dose of 0.11 mg/kg/ ewe/day as previously described [24]. Increasing evidence from several species (rhesus mon- keys, sheep, rats, and mice) has demonstrated a link be- tween prenatal exposure to testosterone (T) and development of a PCOS-like phenotype [13, 14]. Specif- ically, prenatal T-treatment disrupts the intrafollicular steroidal balance in preovulatory follicles (5-7 mm) and reduces embryonic potential in rhesus monkeys [11]. In sheep, prenatal T excess from days 30 to 90 of gestation (T30-90) enhances follicular recruitment and persistence [13] and causes disruptions in several key mediators of folliculogenesis [15–18]. A milder PCOS-like phenotype with reduced fecundity was found in sheep treated pre- natally from days 60-90 of gestation (T60-90), where only 40 % of such prenatal T-treated females became pregnant [19]. These T60-90 females also developed in- sulin resistance [13]. Follicular dynamics CIDR: intravaginal P4 control internal drug release device, PGF2α: prostaglandin F2α, FSH: follicle stimulating hormone, GnRH: gonadotropin releasing hormone, OVX: ovariectomy, replac.: replacement, US: transrectal ultrasonography. Grey and black arrows indicate time of GnRH antagonist and FSH administration, respectively. b Mean (± SEM) number of 2-3 mm (top panel) and 4-6 mm (bottom panel) before GnRH antagonist (PreGnRHa), after GnRH antagonist (PostGnRHa), before the 3rd and 6th FSH doses (FSH3 and FSH6, respectively), and ovariectomy (OVX) in control (white bars), T (filled bars), T + F (dotted bars), and T + R (stripped dars) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for details of prenatal/postnatal treatment details. F: flutamide; R: rosiglitazone; n.s.: not significant; * P < 0.01, ** P < 0.001 Fig. 1 a Scheme depicting synchronization and controlled ovarian stimulation protocol used in the study. See text for details. CIDR: intravaginal P4 control internal drug release device, PGF2α: prostaglandin F2α, FSH: follicle stimulating hormone, GnRH: gonadotropin releasing hormone, OVX: ovariectomy, replac.: replacement, US: transrectal ultrasonography. Grey and black arrows indicate time of GnRH antagonist and FSH administration, respectively. b Mean (± SEM) number of 2-3 mm (top panel) and 4-6 mm (bottom panel) before GnRH antagonist (PreGnRHa), after GnRH antagonist (PostGnRHa), before the 3rd and 6th FSH doses (FSH3 and FSH6, respectively), and ovariectomy (OVX) in control (white bars), T (filled bars), T + F (dotted bars), and T + R (stripped dars) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for details of prenatal/postnatal treatment details. F: flutamide; R: rosiglitazone; n.s.: not significant; * P < 0.01, ** P < 0.001 collapsed follicle was not useful to undertake histological studies. after the last GnRHa dose, and after the 3rd and 6th FSH dose (Fig. 1a). Two hours after the 8th FSH dose, a sub- set (n = 6/group) of females were ovariectomized follow- ing procedures previously described [27], and all follicles ≥3 mm were dissected [20]. After recording their diameter, follicular fluid was aspirated and frozen at -20 °C. Prior to measurements, all follicular fluids were diluted 1:100 in 1x PBS supplemented with 1 % BSA. After follicular dissection and aspiration, the Follicular dynamics To monitor changes in follicular dynamics, transrectal ultrasonography was performed as previously described [26] using a scanner (Aloka SSD-900 V, Aloka Co. Ltd., Wallington, CT) fitted to a 7.5 MHz linear array trans- ducer. Number of follicles ≥2 mm and corpora lutea were determined prior to the start of GnRHa treatment, Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Page 3 of 10 Fig. 1 a Scheme depicting synchronization and controlled ovarian stimulation protocol used in the study. See text for details. CIDR: intravaginal P4 control internal drug release device, PGF2α: prostaglandin F2α, FSH: follicle stimulating hormone, GnRH: gonadotropin releasing hormone, OVX: ovariectomy, replac.: replacement, US: transrectal ultrasonography. Grey and black arrows indicate time of GnRH antagonist and FSH administration, respectively. b Mean (± SEM) number of 2-3 mm (top panel) and 4-6 mm (bottom panel) before GnRH antagonist (PreGnRHa), after GnRH antagonist (PostGnRHa), before the 3rd and 6th FSH doses (FSH3 and FSH6, respectively), and ovariectomy (OVX) in control (white bars), T (filled bars), T + F (dotted bars), and T + R (stripped dars) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for details of prenatal/postnatal treatment details. F: flutamide; R: rosiglitazone; n.s.: not significant; * P < 0.01, ** P < 0.001 Fig. 1 a Scheme depicting synchronization and controlled ovarian stimulation protocol used in the study. See text for details. CIDR: intravaginal P4 control internal drug release device, PGF2α: prostaglandin F2α, FSH: follicle stimulating hormone, GnRH: gonadotropin releasing hormone, OVX: ovariectomy, replac.: replacement, US: transrectal ultrasonography. Grey and black arrows indicate time of GnRH antagonist and FSH administration, respectively. b Mean (± SEM) number of 2-3 mm (top panel) and 4-6 mm (bottom panel) before GnRH antagonist (PreGnRHa), after GnRH antagonist (PostGnRHa), before the 3rd and 6th FSH doses (FSH3 and FSH6, respectively), and ovariectomy (OVX) in control (white bars), T (filled bars), T + F (dotted bars), and T + R (stripped dars) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for details of prenatal/postnatal treatment details. F: flutamide; R: rosiglitazone; n.s.: not significant; * P < 0.01, ** P < 0.001 Fig. 1 a Scheme depicting synchronization and controlled ovarian stimulation protocol used in the study. See text for details. Follicular size dynamics GnRHa treatment decreased the number of 4-6 mm, but not 2-3 mm, follicles (P < 0.001; Fig. 1b). FSH adminis- tration increased number of 2-3 mm follicles (P < 0.001) by the third dose followed by a decline by the 6th FSH dose (P < 0.001). This decline in 2-3 mm was accom- panied by a marked increase (P < 0.001) in the num- ber of 4-6 mm follicles (P < 0.001). At ovariectomy, a further decline in 2-3 mm follicles and an increase in 4-6 mm follicles (P < 0.001) were found. GnRHa- and FSH-induced changes in 2-3 and 4-6 mm follicles in all treatment groups did not differ from the control group. A4, T, E2, estrone, and P4 were assayed in the Assay Services Laboratories at the Wisconsin National Primate Research Center using a QTRAP 5500 LC-MS/MS (AB Sciex, USA) equipped with an atmospheric pressure chemical ionization source. The system included two Shimadzu LC20ADXR pumps and a Shimadzu SIL20ACXR autosampler. Thirty μl samples were injected onto a Phenomenex Kinetex 2.6u C18 100A, 100 × 2.1 mm column (Phenomenex) for separation. LC- MS/MS results were generated in positive-ion mode with optimized voltages. Calibration curve concentra- tions for estrogens were 1.56-0.003 ng/ml and 3.91- 0.0076 ng/ml ng/ml for remaining steroids. Linearity was r > 0.9990 and curve fit was linear with 1/x weight- ing. Interassay coefficients of variation were determined by a pool of human serum and ranged from 6.09- 19.47 % for all steroids. Assay sensitivities for A4, E2, es- trone, P4 and T were 0.015, 0.005, 0.0015, 0.015, and 0.0325 ng/ml, respectively. When all follicles ≥2 mm were considered, FSH in- creased the total number of follicles by the third FSH dose (P < 0.001; Fig. 2), increasing further until the sixth FSH dose (P < 0.001) but not beyond. In contrast, GnRHa administration significantly reduced ≥4 mm fol- licles (P < 0.001; Fig. 2). An increase in ≥4 mm follicles was observed following the third FSH dose and beyond (P < 0.001). There were no differences in follicular classes ≥2 mm and ≥4 mm between control and all treatment groups. An increase was evident by the sixth FSH dose in ≥6 mm follicles (P < 0.001), culminating in a 3-fold in- crease at ovariectomy (P < 0.001; Fig. 2). Intrafollicular steroids Follicular fluid concentrations of androstenedione (A4), estradiol (E2), estrone, P4, and T were measured by quadruple linear ion trap mass spectrometer (LC-MS/ MS) from one 3 mm, one 4 mm, and two 5-6 mm folli- cles that were randomly selected from each Page 4 of 10 Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Page 4 of 10 Page 4 of 10 ovariectomized female. Samples (400 μl) were extracted after diluting with ultrapure water (500 μl). An internal deuterated standard and 1 ml of 2-methoxy-2-methyl- propane was added to each sample, vortexed vigorously, and incubated for 5 min at room temperature. The steroid-containing organic phase was air-dried and re- suspended in 100 μl of ethanol and 500 μl of water. A second liquid-liquid extraction was performed with di- chloromethane. The steroid-containing dichloromethane phase was air-dried and samples re-suspended in NaHCO3 buffer (25 μl), and estrone and E2 were derivi- tized with 50 μl of dansyl chloride (200 mg/ml in aceto- nitrile), heated to 40 °C for 4 min, and transferred into minivials. Follicular size dynamics Prenatal T treatment reduced ≥6 mm follicle number, with an initial decline evident by sixth FSH dose and achieving signifi- cance at ovariectomy (Fig. 2). Postnatal treatment with flutamide, but not rosiglitazone, prevented the prenatal T-induced reduction in ≥6 mm follicles. Intrafollicular steroids Figure 3a shows changes in intrafollicular concentrations of steroids. In control females, intrafollicular T concentrations were higher in 3 mm vs. larger follicles (P < 0.05), while the reverse was found for intrafollicular E2 and P4, with higher concentrations found in 5-6 mm follicles (P < 0.05). Control females had an increase in E2 with follicle size (P < 0.05) that was not seen in T and T + R females. T + F females had high E2 concentrations regardless of follicle size. There was no follicle size effect on intrafollicular Tand E2 concen- trations in T and T + F females, while an increase in P4 was evident in 5-6 mm follicles of T + R females. Statistical analysis l f y For analyses of follicular dynamics, follicles were grouped as 2-3 mm, 4-6 mm, ≥2 mm, ≥4 mm, and ≥6 mm follicles. For intrafollicular steroid measure- ments, follicle classes included 3 mm, 4 mm, and 5- 6 mm in diameter. Follicle size distribution among treat- ment groups and intrafollicular steroid concentrations among follicular classes within each treatment group and within a follicular class across treatment groups were analyzed by ANOVA and linear mixed effect model with Tukey posthoc tests. Percent change in intrafollicu- lar steroid concentrations between 3 mm and 5-6 mm follicles was derived by subtracting concentration in 3 mm from that in larger follicles. Appropriate transfor- mations were applied, as needed, to account for normal- ity of data allowing analyses by parametric tests. All analyses were carried out using PASW Statistics for Windows release 18.0.1 and data presented as mean ± SEM. P < 0.05 was considered significant. No differences were found among treatment groups with intrafollicular T, A4, estrone, E2, and P4 concentra- tions in the different follicular classes. When analysis was restricted only to control and T-treated females, T females had lower T and P4 (P < 0.05) and tended to have lower A4 (P = 0.07) in 5-6 mm follicles. Overall evaluation of change in steroids between the 3 and 5- 6 mm sized follicles (Fig. 3b) revealed increases in A4, estrone, E2, and P4 and a reduction in T in control Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Page 5 of 10 Fig. 2 Mean (± SEM) number of ≥2 mm (top panel), ≥4 mm (middle panel), and ≥6 mm (bottom panel) follicles before GnRH antagonist f b f h rd d th d d l d l Fig. 2 Mean (± SEM) number of ≥2 mm (top panel), ≥4 mm (middle panel), and ≥6 mm (bottom panel) follicles before GnRH antagonist (PreGnRHa), after GnRH antagonist (PostGnRHa), before the 3rd and 6th FSH dose (FSH3 and FSH6, respectively), and ovariectomy (OVX) in control (C; white bars), T (filled bars), T + F (dotted bars), and T + R (stripped bars) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for prenatal/postnatal treatment details. F: flutamide; R: rosiglitazone; n.s.: not significant; * P < 0.01, ** P < 0.001. # represents significant different (P < 0.05) compared to the control group Fig. Statistical analysis l f 2 Mean (± SEM) number of ≥2 mm (top panel), ≥4 mm (middle panel), and ≥6 mm (bottom panel) follicles before GnRH antagonist (PreGnRHa), after GnRH antagonist (PostGnRHa), before the 3rd and 6th FSH dose (FSH3 and FSH6, respectively), and ovariectomy (OVX) in control (C; white bars), T (filled bars), T + F (dotted bars), and T + R (stripped bars) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for prenatal/postnatal treatment details. F: flutamide; R: rosiglitazone; n.s.: not significant; * P < 0.01, P < 0.001. # represents significant different (P < 0.05) compared to the control group T:E2, T + A4:E2, T + A4:estrone + E2, and T:estrone + E2 ratios were all lower (P < 0.05) in 5-6 mm vs. 3 mm folli- cles within the T + R group. No treatment effect was found in steroid ratios. Data skewness prevented detection of differences in T:E2 and T + A4:E2 ratios in T + F females. females, while reductions in A4, estrone, and P4 were observed in T females. The increase in E2 from 3 to 5- 6 mm was of a higher magnitude in control compared to T females. The changes in A4, estrone, and E2 were more pronounced in T + F than T females. The direc- tionality of changes in steroids between 3 and 5-6 mm in T + R females mirrored that of control females. Discussion Comparison of steroid ratios across follicular stages found the follicular androgen to estrogen ratios (T:E2, T + A4:E2, T + A4:estrone + E2, and T:estrone + E2) in controls were lower in 5-6 mm compared 3 mm follicles (P < 0.05) (Fig. 4). The T:E2 and T + A4:E2 ratios were also lower in 5-6 mm vs. 3 mm follicles in T females. Intrafollicular This study is the first to demonstrate that prenatal T ex- cess from days 60-90 of gestation impairs final preovula- tory follicular growth (ovulatory size: ≥6 mm; [28]) and reduces intrafollicular concentrations of T, A4, and P4 in preovulatory sized follicles of sheep undergoing Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Page 6 of 10 Fig. 3 a Mean (± SEM) intrafollicular concentrations (ng/ml) of testosterone (T), androstenedione (A4), estrone, estradiol (E2), and progesterone (P4) at the time of ovariectomy and coincidentally with the 8th FSH dose in control (C; yellow), prenatal testosterone-treated (T; red), prenatal T plus postnatal flutamide (T + F; blue), and prenatal T plus postnatal rosiglitazone (T + R; gray) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for prenatal/postnatal treatment details. Within group comparisons: asterisks represent differences within group within hormone and between follicular sizes. Posthoc analyses performed only when overall ANOVA among all three sizes was significant. Comparisons between C and T group are represented by a ≠b if P < 0.05 and by a’ ≠b’if P = 0.07. F: flutamide; R: rosiglitazone. Data obtained from one 3 mm, one 4 mm, and two 5-6 mm follicles randomly selected from each female. b Percent change in intrafollicular steroids of T, A4, estrone, E2, and P4 between 3 and 5-6 mm follicles in C (yellow), T (red), T + F (blue), T + R (black) groups. Percent change was calculated Fig. 3 a Mean (± SEM) intrafollicular concentrations (ng/ml) of testosterone (T), androstenedione (A4), estrone, estradiol (E2), and progesterone (P4) at the time of ovariectomy and coincidentally with the 8th FSH dose in control (C; yellow), prenatal testosterone-treated (T; red), prenatal T plus postnatal flutamide (T + F; blue), and prenatal T plus postnatal rosiglitazone (T + R; gray) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for prenatal/postnatal treatment details. Within group comparisons: asterisks represent differences within group within hormone and between follicular sizes. Discussion Posthoc analyses performed only when overall ANOVA among all three sizes was significant. Comparisons between C and T group are represented by a ≠b if P < 0.05 and by a’ ≠b’if P = 0.07. F: flutamide; R: rosiglitazone. Data obtained from one 3 mm, one 4 mm, and two 5-6 mm follicles randomly selected from each female. b Percent change in intrafollicular steroids of T, A4, estrone, E2, and P4 between 3 and 5-6 mm follicles in C (yellow), T (red), T + F (blue), T + R (black) groups. Percent change was calculated by subtracting the overall mean values between the 3 and 5-6 mm size within each group Prenatal T programming of follicular dynamics 4 Mean (± SEM) intrafollicular steroid ratios of testosterone to estradiol (T: E2), T plus androstenedione (A4) to E2 (T + A4:E2), T + A4:estrone + E2, T:estrone + E2, and P4:E2 in 3 mm, 4 mm, and 5-6 mm follicles at the time of ovariectomy and coincidentally with the 8th FSH dose in control (C; yellow), prenatal testosterone-treated (T; red), prenatal T plus postnatal flutamide (T + F; blue), and prenatal T plus postnatal rosiglitazone (T + R; gray) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for prenatal/postnatal treatment details. Within group comparisons, asterisks represent differences within group within hormone and between follicular sizes. Posthoc analyses performed only when overall ANOVA among all three sizes was significant. Data obtained from one 3 mm, one 4 mm, and two 5-6 mm follicles randomly selected from each female Intrafollicular steroid ratios 0 1 2 3 3mm 4mm 5-6mm T T+R 0 1 2 3 3mm 4mm 5-6mm C 0 1 2 3 3mm 4mm 5-6mm T:E2 T+A4:E2 T+A4: Eone+E2 T:Eone+E2 P4:E2 ** * * * * * * 0 2 4 6 3mm 4mm 5-6mm T+F Intrafollicular steroid ratios 0 1 2 3 3mm 4mm 5-6mm T T+R 0 1 2 3 3mm 4mm 5-6mm C 0 1 2 3 3mm 4mm 5-6mm T:E2 T+A4:E2 T+A4: Eone+E2 T:Eone+E2 P4:E2 * * * * * * 0 2 4 6 3mm 4mm 5-6mm T+F number of follicles ≥6 mm, but not in the number up to 4 mm, and ii) in sheep, follicles up to 4 mm size are considered FSH dependent and those beyond 4 mm as LH dependent [28], the shift from FSH to LH depend- ency appears to be impaired. One possibility is that this reduction in preovulatory sized follicles might be a func- tion of advancement in LH dependency and hence a re- quirement for LH. Because LH was not co-administered with FSH, the final transition to preovulatory size might be compromised. This premise is supported by the lack of reduction in preovulatory sized follicles in the T30-90 females [32], when follicles were stimulated concomi- tantly with LH and FSH. Findings from both studies (this study and Steckler et al. [32]) suggest the compro- mised preovulatory follicular development might be res- cued with exogenous LH supplementation. Prenatal T programming of follicular dynamics Prenatal T programming of follicular dynamics The GnRHa treatment regimen used was effective in blocking follicular growth beyond the 3 mm stage in both the control and prenatal T-treated sheep, as was the case with T30-90 females exposed to a shorter GnRHa treatment [32]. The efficacy of pFSH to recruit follicular growth in controls was comparable to that achieved with oFSH or pFSH stimulation in other sheep breeds [25, 33]. pFSH used in this study was also as ef- fective in stimulating follicular growth in the 3 treatment groups (T, T + F, and T + R) as the combined oFSH- LH controlled ovarian stimulation, a finding consistent with the lower fertility rates reported in these sheep [19]. The rescue of final growth by postnatal andro- gen antagonist, but not insulin sensitizer, administra- tion suggests the impairment of prevoulatory follicle growth is mediated via androgenic action. This, in concert with the partial rescue of the intrafollicular steroid milieu with insulin sensitizer, suggests that both androgens and insulin contribute to reproductive disruption [29–31] and reduced fecundity [17] in adult prenatal T-treated sheep. p g g y The GnRHa treatment regimen used was effective in blocking follicular growth beyond the 3 mm stage in both the control and prenatal T-treated sheep, as was the case with T30-90 females exposed to a shorter GnRHa treatment [32]. The efficacy of pFSH to recruit follicular growth in controls was comparable to that achieved with oFSH or pFSH stimulation in other sheep breeds [25, 33]. pFSH used in this study was also as ef- fective in stimulating follicular growth in the 3 treatment groups (T, T + F, and T + R) as the combined oFSH- LH Page 7 of 10 Page 7 of 10 Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Intrafollicular steroid ratios 0 1 2 3 3mm 4mm 5-6mm T T+R 0 1 2 3 3mm 4mm 5-6mm C 0 1 2 3 3mm 4mm 5-6mm T:E2 T+A4:E2 T+A4: Eone+E2 T:Eone+E2 P4:E2 * * * * * * 0 2 4 6 3mm 4mm 5-6mm T+F Fig. Prenatal T programming of follicular dynamics It is unclear if this impairment is a function of reduced number of LH receptors or altered LH signaling, both are aspects yet to be studied in this model but implicated in women with PCOS [36–38]. Intrafollicular steroid ratios Importantly, the fact that postnatal androgen antagonist, not insulin sensitizer, treatment was able to rescue the number of preovulatory-sized follicles supports the notion that i) compromised androgen receptor expression/func- tion in growing follicles or surrounding ovarian stroma is detrimental to progression beyond a critical size (4 mm in this case) and ii) that blockade of androgen action with an androgen antagonist would help overcome follicular growth arrest. In previous studies, we found prenatal T treatment from days 30-90 of gestation increases granu- losa cell androgen receptor expression in antral follicles and is supportive of functional ovarian hyperandrogenism [15]. Although insulin plays a role in follicular develop- ment [39–41], failure of insulin sensitizer treatment to rescue this follicular growth defect suggests this dysfunc- tion is driven primarily by the androgen signaling imbal- ance within the growing follicle. T+A4: Eone+E2 Fig. 4 Mean (± SEM) intrafollicular steroid ratios of testosterone to estradiol (T: E2), T plus androstenedione (A4) to E2 (T + A4:E2), T + A4:estrone + E2, T:estrone + E2, and P4:E2 in 3 mm, 4 mm, and 5-6 mm follicles at the time of ovariectomy and coincidentally with the 8th FSH dose in control (C; yellow), prenatal testosterone-treated (T; red), prenatal T plus postnatal flutamide (T + F; blue), and prenatal T plus postnatal rosiglitazone (T + R; gray) females. See Fig. 1 for synchronization and controlled ovarian stimulation protocol and text for prenatal/postnatal treatment details. Within group comparisons, asterisks represent differences within group within hormone and between follicular sizes. Posthoc analyses performed only when overall ANOVA among all three sizes was significant. Data obtained from one 3 mm, one 4 mm, and two 5-6 mm follicles randomly selected from each female Fig. 4 Mean (± SEM) intrafollicular steroid ratios of testosterone to estradiol (T: E2), T plus androstenedione (A4) to E2 (T + A4:E2), T + A4:estrone + E2, T:estrone + E2, and P4:E2 in 3 mm, 4 mm, and 5-6 mm follicles at the time of ovariectomy and coincidentally with the 8th FSH dose in control (C; yellow), prenatal Prenatal T programming of follicular steroid milieu Prenatal T programming of follicular steroid milieu The opposing follicular size-related changes in intra- follicular T and estrogens in control females were similar to previous findings [20, 42]. A higher andro- genic environment prevails in small follicles, with a shift towards a highly estrogenic milieu in preovula- tory follicles; this is consistent with increased aroma- tase activity as follicles mature [42]. The transition from low E2 in smaller follicles to high E2 in larger follicles was the most striking change (4-fold increase) in the control group. Conversely, the E2 increase was of much lower magnitude (< 1-fold) in the T group. Because androgens are the main substrate for estrogen production, the reduced magnitude of E2 increase in T females may be driven by a reduced androgenic environ- ment (T and A4) at earlier follicular stages. Androgen and regimen used with T30-90 females [32]. These findings indicate responsiveness to exogenous FSH, and hence recruitment, was not impaired by prenatal T excess or postnatal treatment with androgen antagonist or insulin sensitizer. The finding that recruitment and growth of follicles up to 4 mm size was similar across groups is not surprising, because androgens increase FSH activity [34] and are not detrimental to follicular survival and growth of preantral and early antral follicles [35]. The reduced number of follicles ≥6 mm in T females suggests the final maturation of the preovulatory follicle is impaired. Given that i) the suppression was evident in Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Page 8 of 10 Page 8 of 10 estrogen receptors [17] and steroidogenic enzymes [43] are dysregulated in granulosa and theca cells of T30-90 sheep, and these disruptions remain to be determined in T60-90 females. It is also unclear whether the lower mag- nitude of E2 increase in T females contributes to the delayed onset of LH surge that was reported earlier in these females [29]. ultimately fertility remains unclear. Paradoxically, while flutamide treatment failed to ameliorate intrafollicular steroidal defects, the same treatment prevented pubertal advancement and enhanced preovulatory LH surge ampli- tude in the T30-90 females [24]. Considering T is an aro- matizable androgen, the differing effects of flutamide in rescuing the various physiologic functions may be a func- tion of whether androgen or estrogen (via aromatization) is the programming agent [31]. Prenatal T-induced disruptions in intrafollicular steroid milieu were also reported in non-human primates [11]. Prenatal T programming of follicular steroid milieu Controlled ovarian stimulation studies in rhesus monkeys found 15-35 days of T treatment starting on gestational days 40-44, but not days 100-115, reduced intrafollicular A4 and E2 concentrations in preovulatory sized follicles [11]. Although disruptions in intrafollicular A4 and E2 parallel findings from the current study, timing and dur- ation of T exposure differ between the sheep and monkey study. Our earlier findings of reduced granulosa cell CYP19A1 expression in antral follicles in the T30-90 model [43] agrees with the reduced intrafollicular E2 in large antral follicles evidenced in the present study [43]. Considering the disrupted intrafollicular steroidal milieu of prenatal T-treated monkeys was accompanied by a re- duction in oocyte competence [11], a similar intrafollicular disruption was evidenced in prenatal T-treated sheep is likely to be associated with compromised oocyte health. This, in fact, may explain the reduced fecundity in T60-90 females [19]. The strength of the present study is that the intrafollicular steroid milieu was identified in different size follicular classes as opposed to only the preovulatory fol- licular size in monkeys. Another strength is the parallel as- sessment of follicular growth at different time points during the ovarian stimulation protocol, which was helpful in dissecting out regulation of follicular growth from steroidogenesis. g g g Despite the fact that T60-90 females are insulin re- sistant [13], the lack of rescue in the number of folli- cles that achieved a preovulatory size by rosiglitazone suggests the insulin pathway is not involved in growth of preovulatory follicles. In contrast, the intrafollicular steroid milieu of T + R follicles was more similar to that of the controls and is supportive of a role for in- sulin coupled with FSH as follicles mature in main- taining intrafollicular steroid balance [46]. The beneficial effects of insulin sensitizer therapies in enhancing insulin sensitivity and improving ovulatory function in women with PCOS [47, 48] may relate to normalization of intrafollicular steroidal milieu, as evidenced in the T + R animals. In interpreting the impact of the interventions, it is im- portant to recognize that GnRH antagonist treatment given prior to FSH stimulation to achieve a homogeneous follicu- lar pool before FSH stimulation (as achieved in this study, Fig. 1) might have played a role in determining the impact of androgen antagonist and insulin sensitizer on follicular dynamics and the intra-follicular hormone milieu. Prenatal T programming of follicular steroid milieu However, considering that GnRH antagonist treatment is the same across treatments, any variability in the starting pool of fol- licles across treatment would suggest intrinsic ovarian differences originating from the T treatment and interven- tions respectively. It needs to be recognized that GnRH agonist and antagonist treatments are routinely used in standard IVF practices [49], and hence the approach taken with this study is consistent with this practice. g Relative to interventions, postnatal flutamide treatment rescued follicular growth to where preovulatory follicle size was achieved, but treatment failed to ameliorate the disruptions in intrafollicular steroidal milieu. The steroidal transition from low E2 to a high E2 milieu between 3 to 6 mm size antral follicles seen in controls was not evident in T + F females. In addition, the directionality of change in intrafollicular concentrations of estrone and A4 in T + F animals, namely a reduction in both steroids in the 6 mm compared to 3 mm follicles as opposed to the increase in both steroids in the controls, point to an intra- follicular steroidal disruption that persists through antral follicle growth. The increase of T in the smaller 3 mm follicles of the T + F females may be a compensatory response to the blockade of androgen receptor signaling by flutamide treatment, which was present throughout the course of the study. Such a response would be analogous to the masculinizing effects of flutamide seen relative to other variables [44, 45]. To what extent the intrafollicular steroidal disruptions play a role in oocyte health and The outcomes achieved with the two interventions, namely the androgen antagonist helping rescue follicular growth and the insulin sensitizer partially rescuing intra- follicular steroidal milieu, suggest that both androgens and insulin may synergize in establishing optimal follicu- lar growth and steroidogenesis. A combined intervention involving both may help compensate for any deficiency that one intervention has in order to achieve better suc- cess. While the finding in PCOS women is that com- bined treatment is more efficacious than monotherapies in treating anovulation [50] is supportive of this possibil- ity, this remains to be tested. Availability of data and material Data will be shared. 11. Dumesic DA, Schramm RD, Peterson E, Paprocki AM, Zhou R, Abbott DH. Impaired developmental competence of oocytes in adult prenatally androgenized female rhesus monkeys undergoing gonadotropin stimulation for in vitro fertilization. J Clin Endocrinol Metab. 2002;87:1111–9 11. Dumesic DA, Schramm RD, Peterson E, Paprocki AM, Zhou R, Abbott DH. Impaired developmental competence of oocytes in adult prenatally androgenized female rhesus monkeys undergoing gonadotropin stimulation for in vitro fertilization. J Clin Endocrinol Metab. 2002;87:1111–9. Funding Thi k This work was supported by NIH P01 HD44232 (VP). Effort spent by A.V-L. during the preparation of the manuscript was supported by AgBioResearch and the United States Department of Agriculture (USDA) National Institute of Food and Agriculture, Hatch project MICL02383. 9. Ambekar AS, Kelkar DS, Pinto SM, Sharma R, Hinduja I, Zaveri K, et al. Proteomics of follicular fluid from women with polycystic ovary syndrome suggests molecular defects in follicular development. J Clin Endocrinol Metab. 2015;100:744–53. 9. Ambekar AS, Kelkar DS, Pinto SM, Sharma R, Hinduja I, Zaveri K, et al. Proteomics of follicular fluid from women with polycystic ovary syndrome suggests molecular defects in follicular development. J Clin Endocrinol Metab. 2015;100:744–53. 9. Ambekar AS, Kelkar DS, Pinto SM, Sharma R, Hinduja I, Zaveri K, et al. Proteomics of follicular fluid from women with polycystic ovary syndrome suggests molecular defects in follicular development. J Clin Endocrinol Metab. 2015;100:744–53. 10. Homburg R, Berkowitz D, Levy T, Feldberg D, Ashkenazi J, Ben-Rafael Z. In vitro fertilization and embryo transfer for the treatment of infertility associated with polycystic ovary syndrome. Fertil Steril. 1993;60:858–63. 10. Homburg R, Berkowitz D, Levy T, Feldberg D, Ashkenazi J, Ben-Rafael Z. In vitro fertilization and embryo transfer for the treatment of infertility associated with polycystic ovary syndrome. Fertil Steril. 1993;60:858–63. Authors’ contributions AV-L participated in designing the experiment and generation of the animals, provided supervision in the performance of animal experiments, performed the ultrasonography and statistical analyses, and wrote the manuscript. JM participated in generating animals and in animal experiments, DHA provide oversight for the steroid measurements, VP paticipated in designing the experiment, provided oversight for integrating the study components, participated in data interpretation and writing of the manuscript. All authors read through the manuscript and provided input in finalizing the manuscript. All authors read and approved the final manuscript. 12. Siristatidis C, Sergentanis TN, Vogiatzi P, Kanavidis P, Chrelias C, Papantoniou N, et al. In Vitro Maturation in Women with vs. without Polycystic Ovarian Syndrome: A Systematic Review and Meta-Analysis. PLoS One. 2015;10:e0134696. 12. Siristatidis C, Sergentanis TN, Vogiatzi P, Kanavidis P, Chrelias C, Papantoniou N, et al. In Vitro Maturation in Women with vs. without Polycystic Ovarian Syndrome: A Systematic Review and Meta-Analysis. PLoS One. 2015;10:e0134696. 13. Padmanabhan V, Veiga-Lopez A. Animal models of the polycystic ovary syndrome phenotype. Steroids. 2013;78:734–40. 13. Padmanabhan V, Veiga-Lopez A. Animal models of the polycystic ovary syndrome phenotype. Steroids. 2013;78:734–40. 14. Abbott DH, Dumesic DA, Levine JE, Dunaif A, Padmanabhan V. Animal models and fetal programming of PCOS. In: Azziz JE, Nestler JE, Dewailly D, editors. Contemporary endocrinology: androgen excess disorders in women: polycystic ovary syndrome and other disorders. Totowa, NJ: Humana Press Inc; 2006. p. 259–72. Ethics approval and consent to participate l ll d d d b 16. Salvetti NR, Ortega HH, Veiga-Lopez A, Padmanabhan V. Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep. Biol Reprod. 2012;87(22):1–10. 16. Salvetti NR, Ortega HH, Veiga-Lopez A, Padmanabhan V. Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep. Biol Reprod. 2012;87(22):1–10. Animals: All procedures used were approved by the Institutional Animal Care and Use Committee of the University of Michigan and conducted at the University of Michigan Sheep Research Facility (number: PRO00005623). Human subjects: Not applicable. Animals: All procedures used were approved by the Institutional Animal Care and Use Committee of the University of Michigan and conducted at the University of Michigan Sheep Research Facility (number: PRO00005623). Human subjects: Not applicable. 17. Ortega HH, Rey F, Velazquez MM, Padmanabhan V. Developmental programming: effect of prenatal steroid excess on intraovarian components of insulin signaling pathway and related proteins in sheep. Biol Reprod. 2010;82:1065–75. Conclusions Prenatal T excess from days 60-90 of gestation impairs final follicular growth and intrafollicular milieu under Veiga-Lopez et al. Journal of Ovarian Research (2016) 9:39 Page 9 of 10 Page 9 of 10 controlled ovarian stimulation protocols. This indicates an inherent ovarian defect that may contribute to the lower fertility seen in these females [19]. The differential benefit of postnatal androgen antagonist and insulin sensitizer treatment in rescuing follicular growth and steroidogenesis, respectively, raises the possibility that combined therapies during adolescence and early adult- hood may be beneficial in enhancing fertility, a premise that remains to be tested. 3. Zawadki J, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: A D, JR G, FP H, GR M, eds. Polycystic ovary syndrome. Boston: Blackwell Scientific Publications; 1992. p. 377-84. 3. Zawadki J, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: A D, JR G, FP H, GR M, eds. Polycystic ovary syndrome. Boston: Blackwell Scientific Publications; 1992. p. 377-84. 4. Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, Escobar-Morreale HF, Futterweit W, et al. 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Barreca A, Del Monte P, Ponzani P, Artini PG, Genazzani AR, Minuto F. Intrafollicular insulin-like growth factor-II levels in normally ovulating women and in patients with polycystic ovary syndrome. Fertil Steril. 1996;65:739–45. Acknowledgements W h k D l D We thank Douglas Doop for help with breeding, lambing, excellent animal care, and facility management, Gary McCalla for help with daily administration of androgen antagonist and insulin sensitizer treatments, Carol Herkimer for assistance with prenatal treatment and ovariectomies, and Kaitlyn Bates, Joe Majors, Shannon Lohman, Dr. Chunxia Lu, Sam Olson, Meg Ryan, and Rohit Shreedharan for help in various aspects of the animal experimentation. We are grateful to Dr. Fred Karsch for surgical help during ovariectomies. 7. Lambert-Messerlian G, Taylor A, Leykin L, Isaacson K, Toth T, Chang Y, et al. Characterization of intrafollicular steroid hormones, inhibin, and follistatin in women with and without polycystic ovarian syndrome following gonadotropin hyperstimulation. Biol Reprod. 1997;57:1211–6. 7. Lambert-Messerlian G, Taylor A, Leykin L, Isaacson K, Toth T, Chang Y, et al. Characterization of intrafollicular steroid hormones, inhibin, and follistatin in women with and without polycystic ovarian syndrome following gonadotropin hyperstimulation. Biol Reprod. 1997;57:1211–6. 8. Hossein G, Arabzadeh S, Hossein-Rashidi B, Hosseini MA. Relations between steroids and AMH: impact of basal and intrafollicular steroids to AMH ratios on oocyte yield and maturation rate in women with or without polycystic ovary undergoing in vitro fertilization. Gynecol Endocrinol. 2012;28:413–7. 8. Hossein G, Arabzadeh S, Hossein-Rashidi B, Hosseini MA. Relations between steroids and AMH: impact of basal and intrafollicular steroids to AMH ratios on oocyte yield and maturation rate in women with or without polycystic ovary undergoing in vitro fertilization. Gynecol Endocrinol. 2012;28:413–7. Consent for publication Not applicable. 15. Ortega HH, Salvetti NR, Padmanabhan V. Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor balance. Reproduction. 2009;137:865–77. 15. Ortega HH, Salvetti NR, Padmanabhan V. Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor balance. Reproduction. 2009;137:865–77. Author details 1D f 1Department of Pediatrics, University of Michigan, 7641A Med Sci II, Ann Arbor, MI 48109-5622, USA. 2Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA. 3Department of Obstetrics and Gynecology and Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA. 18. 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https://openalex.org/W4312240724	https://journal.unnes.ac.id/nju/index.php/kemas/article/download/33715/13418	English	null	Related Factors of Anxiety Level in Covid-19 Patient during Self Quarantine	Kemas	2,022	cc-by	5,547	Abstract Article Info Article History: Submitted December 2021 Accepted March 2022 Published July 2022 Keywords: DOI https://doi.org/10.15294/ kemas.v18i1.33715 Coronavirus disease-19 was a pandemic in the world. One of the efforts to reduce the spread of Covid-19 cases was to self-quarantine. Self-quarantine impacted emotional mental disorders in the form of anxiety. This study aimed to identify factors related to the anxiety of Covid-19 patients who were undergoing self-quarantine. METHODS: This study used a descriptive research design with a cross-sectional study. The population was all confirmed COVID-19 patients who underwent self-quarantine in Wonogiri Re gency as many as 70 respondents. Samples used clustered randomized sampling with 40 respondents. The instrument used a sociodemographic questionnaire, and anxiety was measured using Hamilton Rating Scale-Anxiety. Data were analyzed using multiple linear regression. RESULTS: The results showed that the mean age was 33.58 ± 11.08 years. Most participants were women 52.5%, 87.5% of the respondents had high educa tion background, 37.5% were medical workers, 37.5% were private workers, and 95% of the participants had received information about covid-19. The mean of the time they were self-quarantined was 8.1 ± 4.77 days. 55% of the participants had no anxiety, 15% had mild anxiety, 10% had moderate anxiety, and 20% had severe anxiety. Factors that affect the level of anxiety are age (p-value = 0.047), occupation (p-value = 0.031), educa tion (p-value = 0.035) and length of self-quarantine (0.023). CONCLUSION: The con clusion said that age, occupation, education, and length of self-quarantine have a strong relationship and have a significant effect on anxiety. Social support is needed to reduce the anxiety of COVID-19 patients during self-quarantine. were urgently needed because COVID-19 has an impact on several aspects, namely political, economic, social, cultural, defense, and security aspects, as well as the welfare of the people in Indonesia (Kemenkes RI, 2020). Fear of the Covid-19 pandemic caused cognitive distress, negative emotions, aggressiveness, and reduced sleep quality or numbness (Cao et al., 2020). One of the management efforts for patients with confirmed Covid-19 was self-quarantined with monitoring, especially for patients without symptoms and mild symptoms. Self-quarantine at home/quarantine facilities for a maximum of ten days from the onset of symptoms plus three days free of symptoms of fever and respiratory problems. If symptoms were more than 10 days, then quarantine was continued  Correspondence Address: Badan Kependudukan dan Keluarga Berencana Nasional, Indonesia. Email : ns.haha354@gmail.com KEMAS 18 (1) (2022) 83-91 Related Factors of Anxiety Level in Covid-19 Patient during Self Quarantine Putri Halimu Husna1, Nita Yunianti Ratnasari1, Marni2 1Academic of Nursing Giri Satria Husada Wonogiri 2Duta Bangsa University Surakarta Introduction Coronavirus disease-19 (Covid-19) is a disease caused by the betacorona type coro- navirus. It is named by the World Health Organization (WHO) SARS-CoV-2 and the name of the disease Coronavirus Disease 2019 (COVID-19). This coronavirus was a pathogen in respiratory diseases. The virus transmission is between humans, so it is fast (PDPI et al., 2020). Symptoms of COVID-19 were symptoms of acute respiratory distress such as fever, cough, and shortness of breath. The average incubation period was 5-6 days, with the incubation period up to 14 days. In severe cases of COVID-19, it could cause pneumonia, acute respiratory syndrome, kidney failure, and even death. Countermeasures and prevention pISSN 1858-1196 eISSN 2355-3596 pISSN 1858-1196 eISSN 2355-3596 Putri Halimu Husna, et all. / Related Factors of Anxiety Level in Covid-19 Patient during Self Quarantine feelings of uncertainty, insecurity, helplessness, and isolation. The Covid-19 pandemic caused emotional and mental disorders that have a physical and psychological impact on every individual, especially individuals who have to isolate themselves due to Covid-19. Individuals who have to live in particular quarantine homes partially experienced mental and emotional disorders. Signs of emotional and mental disorders symptoms are experienced in the form of somatic complaints, namely anxiety, tension, and neglected daily activities(Ozamiz- Etxebarria et al., 2020). Health concerns and anxiety associated with an epidemic or pande- mic could have a significant psychological impact (e.g., stress, intrusive negative thoughts, avoidance) and might be associated with ineffective or unfavorable preventive behavior. A person’s anxiety response to an epidemic/ pandemic could vary from one person to another (Ahmad & Murad, 2020). Anxiety during the COVID-19 pandemic could be caused by several factors. Namely, predisposing factors include the COVID 19 pandemic, spending >9 hours at home, excessive online information seeking, more common in women, economic status, having a baby, married status, student status, learning environment, and internet network. Factors that could prevent or reduce anxiety in this literature are reinforcing factors were emotion regulation, resilience, supportive intervention, religious coping, family support, limiting exposure to information media and physical activity or sports (Brooks et al., 2020).f until symptoms disappear plus 3 symptom-free days. Quarantine could be done independently 93.8% at home or in public facilities prepared by the government (PDPI et al., 2020). The management of self-quarantine for COVID-19 patients who were asymptomatic and had mild symptoms made some people feel restless and anxious. Introduction It would raise the risk of other mental health disorders during the Covid-19 pandemic in the community. Anxiety, lack of social contact, and fewer opportunities to deal with stress were major concerns (Fegert et al., 2020). The related factors of anxiety needed to be explored further to overcome anxiety so that it did not have an impact on other health problems if anxiety was not overcome. Based on data from the World Health Organization (WHO), as of September 10, 2021, the number of confirmed COVID-19 patients was 223,022,538 people, and the number of patients who died from Covid-19 was 4,602,882 people (World Health Organization, 2021). In Indonesia, as of September 10, 2021, the number of confirmed COVID-19 patients was 4,158,731 (+5,367) cases, the number of patients who died from Covid-19 was 128,431 (3.3%), the number of patients recovered was 3,901,766 (93.8%), and the number of active cases was 118,534 (2.9%)(Kemenkes RI, 2020). In Central Java Province, as of September 11, 2021, the number of confirmed patients was 6,102 cases, the number of patients recovered was 439,042 cases, and the number of patients who died was 31,596 (Tengah, 2021). The number of Covid-19 cases in Wonogiri Regency as of September 10, 2021 namely 142 confirmed cases (48 hospitalized and 94 self- quarantine), 10,199 confirmed recovered cases, and 1,353 confirmed deaths (Wonogiri, 2021). Based on Cao et al. study, during the Covid-19 pandemic in Hubei Province, China, there were 62 students (0.9%) experiencing severe anxiety, 196 (2.7%) students had moderate anxiety, and 1,518 (21.3%) students experiencing mild anxiety (Cao et al., 2020). Self-quarantine was one of the efforts to reduce the spread of Covid-19, but self- quarantine caused mental and emotional problems in the form of anxiety. The factors that caused anxiety during self-quarantine in COVID-19 patients need to be explored more deeply to develop possible preventive measures and therapeutic interventions. It was what underlies the authors to examine the factors related to the anxiety of Covid-19 patients who were self-quarantining. From some literature mentioned, the number of anxiety diagnoses increased during and after the Covid-19 pandemic. Anxiety diagnosis often presented with symptoms of poor sleep and depression (Nicolini, 2020). Anxiety was a disguised feeling of fear accompanied by Methodh The study took place in the Wonogiri District Health Office Work Area. It is in the southern part of Central Java in Indonesia. During this study piloted, the number of 84 KEMAS 18 (1) (2022) 83-91 Covid-19 cases in Wonogiri Regency as of December 2020, namely 108 confirmed cases (38 hospitalized and 70 self-quarantine). A cross-sectional study took place in Wonogiri from December 2020 – May 2021 to assess the anxiety level of the covid-19 patient during self- quarantine. The population was all confirmed COVID-19 patients who underwent self- quarantine as of December 2020, as many as 70 people (Wonogiri, 2021). The primary criterion for the inclusion of participants in the study was people with covid-19 who self quarantined without comorbid. The sampling technique used was Cluster Random Sampling. Samples were taken from 2 sub-districts with the highest number of cases, as many as Wonogiri and Selogiri sub-districts with 40 respondents. swallowing, wind abdominal pain, burning sensations, abdominal fullness, nausea, vomiting, borborygmi, looseness of bowels, loss of weight, constipation; [12] Genitourinary symptoms: Frequency of micturition, urgency of micturition, amenorrhea, menorrhagia, development of frigidity, premature ejacula- tion, loss of libido, impotence; [13]Autonomic symptoms: Dry mouth, flushing, pallor, tendency to sweat, giddiness, tension headache, raising of hair; [14] Behavior: Fidgeting, restless- ness or pacing, tremor of hands, furrowed brow, strained face, sighing or rapid respiration, facial pallor, swallowing, etc (Ramdan, 2019). Each symptom was assigned a score of 0-4 (0: no symptoms; 1: mild symptoms; 2: moderate symptoms; 3: severe symptoms; 4: very severe symptoms). The scores of the 14 symptoms are added up, and the total score obtained was used to determine a person’s anxiety degree. The anxiety degree was classified as follows: <6: no anxiety; 7-14: mild anxiety; 15-27: moderate; 28-41: severe anxiety; >41: severe. The questionnaire was in the form of a Google Form distributed via WhatsApp. Therefore, the researcher was with the respondents when they filled out the questionnaire. Sociodemographic data measurement use a questionnaire containing age, gender, education, occupation, exposure to information, and length of self- quarantine. The researcher also provided clarifications where necessary.h Research data were collected using the Hamilton Rating Scale-Anxiety, Indonesian Version of HRS-A. The validation is through an international field trial (Maier et al, 1988) and an Indonesian field trial (Ramdan, 2019). Methodh Various instruments for measuring Anxiety have been developed and described, but very few studies used the HRS-A instrument to assess anxiety levels. HRS-A questionnaire included 14 symptoms containing: [1] Anxious mood: Worries, anticipation of the worst, fearful anticipation, irritability; [2] Tension: Feelings of tension, fatigability, startle response, moved to tears easily, trembling, feelings of restlessness, inability to relax; [3] Fears: Of dark, of strangers, of being left alone, of animals, of traffic, of crowds; [4] Insomnia: Difficulty in falling asleep, broken sleep, unsatisfying sleep and fatigue on waking, dreams, nightmares, night terrors; [5] Intellectual: Difficulty in concentration, poor memory; [6] Depressed mood: Loss of interest, lack of pleasure in hobbies, depression, early waking, diurnal swing; [7] Somatic (muscular): Pains and aches, twitching, stiffness, myoclo- nic jerks, grinding of teeth, unsteady voice, increased muscular tone; [8] Somatic (sensory): Tinnitus, blurring of vision, hot and cold flu- shes, feelings of weakness, pricking sensation; [9] Cardiovascular symptoms: Tachycardia, palpitations, pain in chest, throbbing of vessels, fainting feelings, missing beat; [10] Respiratory symptoms: Pressure or constriction in chest, choking feelings, sighing, dyspnea; [11] Gastrointestinal symptoms: Difficulty in i The subjects were invited to participate in the study while being self-quarantined. All the subjects were informed about the purpose of the study. After obtaining the informed consent, they filled out the questionnaire. The data collection took time from January to March 2021, when they were self-quarantined. Definition of operational Anxiety Level: an emotional response without a specific object that is subjectively experienced and communicated interpersonally is defined as normal, if the score is less than 6, mild anxiety if it is from 7 to 14, moderate anxiety if it is from 15 to 27 and severe anxiety if the score more than 27. After explaining the research objectives, we obtained informed consent from each participant. Ethical permission to carry out this study was granted by the Department of Research and Community Engagement, 85 Putri Halimu Husna, et all. / Related Factors of Anxiety Level in Covid-19 Patient during Self Quarantine the School of Nursing, Giri Satria Husada Wonogiri. The confidentiality of the data was ensured for all participants.h were used. Quantitative data are presented as means ± standard deviations (SD), while qualitative data are presented in frequencies and proportions (Guillén-Astete et al., 2020). Linear regression was used to evaluate factors that correlate with anxiety levels of covid-19 patients during self-quarantined. Result And Discussion And Discussion Socio-demographic variables of respondents and level of anxiety during self-quarantine Result And Discussion Table 1. Socio-demographic variables of respondents and level of anxiety during self-quarantine (n = 40) Variable n % Age M±SD 33.58± 11.08 Gender Male 19 47.5 Female 21 52.5 Education Low 5 12.5 High 35 87.5 Occupation Unemployment 6 15.0 Medical Worker 15 37.5 Government Employee 1 2.5 Private Employee 15 37.5 House wife 3 7.5 Information exposed have not received information 2 5.0 received information 38 95.0 Length of Self-Quarantine (Days) M±SD 8,1 ± 4.77 Anxiety Level Normal 22 55.0 Mild 6 15.0 Moderate 4 10.0 Severe 8 20.0 Note: M = mean; SD= Stanard Deviation Table 1. Socio-demographic variables of respondents and level of anxiety during self-quarantine Methodh The statistical significance threshold was set at a p-value less than 0.05. The data were organized and coded in Excel spreadsheets and exported to the Statistical Package for the Social Sciences (SPSS) version 20.0 program. The descriptive statistics, the absolute and relative frequency, mean, standard deviation, coefficient of variation, and minimum and maximum values Result And Discussion Note: M = mean; SD= Stanard Deviation Note: M = mean; SD= Stanard Deviation The mean age of the respondents was 33.58± 11.08 years. In their study, Kapasia et al. said that anxiety occurred in respondents aged 18-35 years. 70% of respondents faced various problems related to anxiety, and depression, due to poor internet connectivity and an unfavorable learning environment at home (Kapasia et al., 2020). The risk factors for the development of anxiety included initial or peak phase of the outbreak, female sex, younger age, marriage, social isolation, unemployment, and student status, financial hardship, low educational level, insufficient knowledge of COVID-19, epidemiological or clinical risk of disease and some lifestyle and personality variables (Santab´arbara et al., 2021). Table 1. describes the sociodemographic data of the participants. The mean age was 33.58 ± 11.08 years of the 40 participants: 52.5% were females, and 47.5% were males. Regarding educational background, 12.5% of the respondents had low education, and 87.5% had high education. In occupation status, 37.5% were medical worker, 37.5% were private worker, 15% were unemployed, 7.5% were housewife, and 2.5% was a government employee. 95% of the participants had received information about covid-19, and 5% had not received information about covid-19. The mean of the time they were self-quarantined was 8.1 ± 4.77 days. 55% of the participants had no anxiety, 15% had mild anxiety, 10% moderate, and 20% had severe anxiety. 86 KEMAS 18 (1) (2022) 83-91 In terms of gender, the most respondents were female, as many as 21 (52.5%) respondents. It was in line with Galindo-Vazques et al. research which explains that single women, who have no children, have co-morbidities, and have a history of mental health care have a higher risk of symptoms of anxiety and depression (Galindo-Vázquez et al., 2020). Older women and professionals experience higher symptoms of stress, anxiety, depression, and insomnia among healthcare workers during the Covid-19 pandemic (Dosil Santamaría et al., 2021). In infected people, females have higher anxiety symptoms than males (Pashazadeh Kan et al., 2021). In line, our study revealed that females are more likely to be affected by anxiety than males (OR = 0.81, CI = 0.37-1.74) (Kan et al., 2021). Female was the risk factor for anxiety because female are more openly and firmly in their emotions (Fu et al., 2020). Female anxiety level is higher than male due to differences in brain chemistry and hormone levels. Note: M = mean; SD= Stanard Deviation Females with anxiety had higher testosterone and estradiol composition (Fu et al., 2020). the direct diagnosis, treatment, and care of patients with COVID-19 were associated with a higher risk of symptoms of anxiety (OR, 1.57; 95% CI, 1.22-2.02; P < .001) (Lai et al., 2020). Some study has identified a high prevalence of moderate depression, anxiety, and PTSD among healthcare workers during the COVID-19 pandemic (Li et al., 2021). p Most of the respondents have received information related to Covid-19, as many as 38 (95%). Predisposing factors that caused anxiety during the COVID-19 pandemic were spending >9 hours at home, excessive online information search, more common in women, economy, having babies, marital status, student status, learning environment, and internet network. Watching/reading COVID-19 news for ≥ 2 h/ daywere associated with a high prevalence of severe to very severe depression, anxiety, and stress (Lasheras et al., 2020). Knowledge about COVID-19 transmission, treatment, prognosis, and prevention can stabilize the anxiety level of medical students during the pandemic. Ensuring that the general population receives enough timely and transparent information during health emergencies is critical for healthy psychological self-adaptation (Lasheras et al., 2020). h In terms of education, most respondents had high education as many as 35 (87.5%). It was in line with Wang et al. study, which explains that respondents with high education level has anxiety level higher than those who had low education (Wang et al., 2020). Most of the respondents’ occupations were medical personnel, as many as 15 (37.5%), and private employees as many as 15 (37.5%) respondents. Most health workers experience anxiety due to the lack of personal protective equipment and family safe during the Covid-19 pandemic. Health workers had experienced mild anxiety in as many as 52 respondents (65.0%), 11 respondents (13.8%) had moderate anxiety, two respondents (2.5%) had experienced severe anxiety and 15 respondents (18.8%) ) who do not experience anxiety (Fadli et al., 2020). Frontline health care workers engaged The mean of the time they were self- quarantined was 8.1 ± 4.77 days. Most of the studies reviewed reported negative psycholo- gical effects of quarantine during confirmed COVID-19, namely symptoms of post- traumatic stress, confusion, and anger. Stressors include longer quarantine duration, fear of infection, frustration, boredom, inadequate supplies, inadequate information, financial loss, and stigma (Brooks et al., 2020). Note: M = mean; SD= Stanard Deviation feelings of loneliness and lack of social support were among the strongest correlators with anxiety during the pandemic (Arafa et al., 2021; Horesh et al., 2020). 87 87 Putri Halimu Husna, et all. / Related Factors of Anxiety Level in Covid-19 Patient during Self Quarantine Table 2. Distribution of the scores among anxiety level of covid-19 patient during self-quarantine, according to HRS-A* Domain No. Items Mean SD Variance Min Max Anxious mood 1 1.58 1.279 1.635 0 4 Tension 1 1.50 1.320 1.744 0 4 Fears 1 1.28 0.933 0.871 0 3 Insomnia 1 1.33 1.095 1.199 0 4 Intellectual 1 0.85 0.864 0.746 0 3 Depressed mood 1 1.53 1.339 1.794 0 4 Somatic (muscular) 1 1.00 1.109 1.231 0 4 Somatic (sensory) 1 0.80 0.723 0.523 0 3 Cardiovascular symptoms 1 0.58 0.747 0.558 0 2 Respiratory symptoms 1 1.20 1.436 2.062 0 4 Gastrointestinal symptoms 1 0.75 1.056 1.115 0 3 Genitourinary symptoms 1 0.48 0.640 0.410 0 2 Autonomic symptoms 1 1.15 1.167 1.362 0 3 Behavior 1 0.88 1.090 1.189 0 4 HRS-A : Hamilton Rating Scale-Anxiety 88 Cardiovascular symptoms 1 0.58 Respiratory symptoms 1 1.20 Gastrointestinal symptoms 1 0.75 Genitourinary symptoms 1 0.48 Autonomic symptoms 1 1.15 Behavior 1 0.88 HRS-A : Hamilton Rating Scale-Anxiety The distribution of the parameters related to the HRS-A domains is presented in table 2. The symptoms of anxiety level were revealed in all 14 symptoms investigated. Regarding the gross mean scores observed, the highest ones were assigned to the anxious mood symptoms (1.58). It evaluates the perception of the respon- dents regarding worries, the anticipation of the worst, fearful anticipation, irritability, and depressed mood symptoms (1.53) evaluating the perception of the respondents regarding loss of interest, lack of pleasure in hobbies, depression, early waking, diurnal swing. Tension symptom (1.50) was related to feelings of tension, fatigability, startle response, moved to tears easily, trembling, restlessness feelings, and inability to relax. Insomnia symptoms (1.33), evaluate the perception regarding the difficulty in falling asleep, broken sleep, unsatisfying sleep and fatigue on waking, dreams, nightmares, and night terrors. The l f i p v t w i i i Table 3. Linear regression of factors Correlating A quarantine (n = 40). Note: M = mean; SD= Stanard Deviation Variables B Be Age 0.296 0.280 Gender -0.601 -0.026 Occupation 2.054 0.334 Education -4.849 -0.310 Information Exposed 3.655 0.069 Length of self quarantine -0.771 -0.314 Note:p<0.05; CI = Confidence Interval The distribution of the parameters related to the HRS-A domains is presented in table 2. The symptoms of anxiety level were revealed in all 14 symptoms investigated. Regarding the gross mean scores observed, the highest ones were assigned to the anxious mood symptoms (1.58). It evaluates the perception of the respon- dents regarding worries, the anticipation of the worst, fearful anticipation, irritability, and depressed mood symptoms (1.53) evaluating the perception of the respondents regarding loss of interest, lack of pleasure in hobbies, depression, early waking, diurnal swing. Tension symptom (1.50) was related to feelings of tension, fatigability, startle response, moved to tears easily, trembling, restlessness feelings, and inability to relax. Insomnia symptoms (1.33), evaluate the perception regarding the difficulty in falling asleep, broken sleep, unsatisfying sleep and fatigue on waking, dreams, nightmares, and night terrors. The lowest means were assigned to the genitourinary symptoms (0.48), evaluating the frequency of micturition, the urgency of micturition, amenorrhea, menorrhagia, development of frigidity, premature ejaculation, loss of libido, impotence, and the scores of cardiovascular symptoms (0.58). It assesses the perception of the respondents regarding tachycardia, palpitations, pain in the chest, throbbing of vessels, fainting feelings, and missing beat.h The highest ones were assigned to the anxious mood symptoms (1.58), which evaluates the respondents’ perception regarding worries, the anticipation of the worst, fearful anticipation, and irritability. Worrying about infection of oneself or loved ones was common in the respondents and correlated strongly with anxiety (Lei et al., 2020). The degree of worry about epidemiological infection significantly influenced psychological status, specifically anxiety (Liu X et al., 2020). Table 3. Linear regression of factors Correlating Anxiety level of Covid-19 patient during self- quarantine (n = 40). Variables Anxiety Level B Beta t p-value 95 % CI Age 0.296 0.280 2.071 0.047 0.005 – 0.587 Gender -0.601 -0.026 -0.193 0.848 -6.954 – 5.752 Occupation 2.054 0.334 2.256 0,031* 0.199 – 3.908 Education -4.849 -0.310 -2.198 0.035* -9.343 - -0.356 Information Exposed 3.655 0.069 0.472 0.640 -12.124 – 19.433 Length of self quarantine -0.771 -0.314 -2.384 0.023* -1.429 - -0.112 Note:*p<0.05; CI = Confidence Interval ble 3. Linear regression of factors Correlating Anxiety level of Covid-19 patient during self- uarantine (n = 40). Note: M = mean; SD= Stanard Deviation 88 KEMAS 18 (1) (2022) 83-91 A regression model was used to examine factors related to anxiety level during self quarantine. The results are presented in table 3 - the factors that associated with anxiety level were age (B = 0.296, β = 0.280, t = 2.071, p value = 0.047), occupation (B = 2.054, β = 0.334, t = 2.256, p value = 0,031), education (B = -4.849, β = -0.310, t = -2.198, p value = 0.035), and length of self-quarantine (B = -0.771, β = -0.314, t = -2.384, p value = 0.023).h source of information to provide psychological interventions for nurses in improving mental health during the COVID-19 pandemic. One effort to reduce anxiety levels was with peer support. Peer support could reduce stress levels in health workers (Saleha et al., 2021). A regression model was used to examine factors related to anxiety level during self quarantine. The results are presented in table 3 - the factors that associated with anxiety level were age (B = 0.296, β = 0.280, t = 2.071, p value = 0.047), occupation (B = 2.054, β = 0.334, t = 2.256, p value = 0,031), education (B = -4.849, β = -0.310, t = -2.198, p value = 0.035), and length of self-quarantine (B = -0.771, β = -0.314, t = -2.384, p value = 0.023).h Conclusion Self-quarantine in patients who are confirmed positive for Covid-19 can cause another mental health problem, namely anxiety. The factors that directly affect the anxiety level of patients undergoing independent quarantine are work, education, and the length of self- isolation. Factors that indirectly affect are age, gender, and exposure to information. The above factors together can significantly affect the anxiety level of Covid-19 patients undergoing self-quarantine. Furthermore, during self-quarantine, monitoring and provi- ding information about what to do during self-quarantine and support from families and communities around COVID-19 patients needs to be increased to reduce the anxiety of Covid-19 patients who are undergoing independent isolation. The factors that associated with anxiety level were age (B = 0.296, β = 0.280, t = 2.071, p-value = 0.047), occupation (B = 2.054, β = 0.334, t = 2.256, p-value = 0,031), education (B = -4.849, β = -0.310, t = -2.198, p-value = 0.035), and length of self-quarantine (B = -0.771, β = -0.314, t = -2.384, p-value = 0.023). It was in line with research from Lai et al. which stated that nurses, women, and frontline health workers have higher anxiety scores than other health workers (Lai et al., 2020). It was also in line with research from Perez-Cano . which stated that the majority of respondents who experience anxiety, depression, and stress due to the Covid-19 pandemic are women and have undergraduate education (Pérez- Cano et al., 2020). Respondents aged 20-60 years experienced mental-emotional disorders while in quarantine. The most complaints were psychological complaints, namely feeling anxious, tense/worried (40%), followed by complaints of neglected daily activities/tasks (37%). The next complaint was somatic com- plaints such as loss of appetite (30%) and poor sleep (30%). Older adults reported lower levels of anxiety and sadness than middle-aged adults, and middle-aged adults reported lower levels than younger participants (Losada-baltar et al., 2020). Most of the jobs in this study were health workers and private employees. 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https://openalex.org/W2973405239	https://europepmc.org/articles/pmc6804080?pdf=render	English	null	Benzoxaboroles—Novel Autotaxin Inhibitors	Molecules/Molecules online/Molecules annual	2,019	cc-by	17,308	Received: 18 August 2019; Accepted: 17 September 2019; Published: 20 September 2019 Received: 18 August 2019; Accepted: 17 September 2019; Published: 20 September 2019 Abstract: Autotaxin (ATX) is an extracellular enzyme that hydrolyses lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which has a role in the mediation of inﬂammation, ﬁbrosis and cancer. ATX is a drug target that has been the focus of many research groups during the last ten years. To date, only one molecule, Ziritaxestat (GLPG1690) has entered the clinic; it is currently in Phase 3 clinical trials for idiopathic pulmonary ﬁbrosis. Other small molecules, with diﬀerent binding modes, have been investigated as ATX inhibitors for cancer including compounds possessing a boronic acid motif such as HA155. In this work, we targeted new, improved inhibitors of ATX that mimic the important interactions of boronic acid using a benzoxaborole motif as the acidic warhead. Furthermore, we aimed to improve the plasma stability of the new compounds by using a more stable core spacer than that embedded in HA155. Compounds were synthesized, evaluated for their ATX inhibitory activity and ADME properties in vitro, culminating in a new benzoxaborole compound, 37, which retains the ATX inhibition activity of HA155 but has improved ADME properties (plasma protein binding, good kinetic solubility and rat/human plasma stability). Keywords: Autotaxin (ATX); lysophosphaditic acid; benzoxaboroles; cancer Kristina Kralji´c, Dubravko Jeli´c, Dinko Žiher, Adam Cvrtila, Snježana Dragojevi´c, Verona Sinkovi´c and Milan Mesi´c * Kristina Kralji´c, Dubravko Jeli´c, Dinko Žiher, Adam Cvrtila, Snježana Dragojevi´c, Verona Sinkovi´c and Milan Mesi´c * Fidelta Ltd., Prilaz baruna Filipovi´ca 29, 10000 Zagreb, Croatia; kristina.kraljic@glpg.com (K.K.); dubravko.jelic@glpg.com (D.J.); dinko.ziher@glpg.com (D.Ž.); adam.cvrtila@glpg.com (A.C.); snjezana.dragojevic@glpg.com (S.D.); verona.sinkovic@glpg.com (V.S.) * Correspondence: milan.mesic@glpg.com; Tel.: +385-1-8886-372 molecules molecules Article Article Molecules 2019, 24, 3419; doi:10.3390/molecules24193419 1. Introduction Autotaxin (ATX) is a lysophospholipase D enzyme that hydrolyses the bioactive lipid molecule lysophosphatidylcholine (LPC) to form lysophosphatidic acid (LPA) and choline (Figure 1) [1]. ATX is present in cerebrospinal ﬂuid, blood, peritoneal ﬂuid and synovial ﬂuid [2]. LPA is a mediator of several pathophysiological processes, such as inﬂammation, ﬁbrosis and cancer [3]. LPA modulates six diﬀerent G protein-coupled receptors (GPCRs) in the cell membrane. It has been reported that LPA has pro-tumorigenic eﬀects on ovarian, breast and prostate cancer cells [4]. High levels of LPA have also been found in the peritoneal liquids (ascites) of women suﬀering from ovarian cancer [5]. High levels of LPA (up to 10 times higher than normal) have generally been observed in cancer patients compared to healthy subjects [6]. In mice, Nagano reported that a 4 mg/kg i.v. injection of an ATX inhibitor (such as 3BoA) signiﬁcantly decreases LPA plasma levels (almost to zero), indicating that ATX is the major enzyme responsible for LPA production [7]. Molecules 2019, 24, 3419; doi:10.3390/molecules24193419 www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules 2 of 22 2 of Molecules 2019, 24, 3419 Molecules 2019 24 x Figure 1. Lysophosphatidylcholine (LPC) is hydrolyzed by autotaxin (ATX) to lysophosphatidic ac (LPA); role of LPA on downstream signaling in cells + Migration Proliferation Survival G Extracellular Intracellular LPC LPA Choline ATX Figure 1. Lysophosphatidylcholine (LPC) is hydrolyzed by autotaxin (ATX) to lysophosphatidic acid (LPA); role of LPA on downstream signaling in cells. Figure 1. Lysophosphatidylcholine (LPC) is hydrolyzed by autotaxin (ATX) to lysophosphatidic ac (LPA); role of LPA on downstream signaling in cells Figure 1. Lysophosphatidylcholine (LPC) is hydrolyzed by autotaxin (ATX) to lysophosphatidic acid (LPA); role of LPA on downstream signaling in cells. High levels of LPA have also been found in the peritoneal liquids (ascites) of women sufferi from ovarian cancer [5]. High levels of LPA (up to 10 times higher than normal) have generally be observed in cancer patients compared to healthy subjects [6]. In mice, Nagano reported that a 4 mg/ i.v. injection of an ATX inhibitor (such as 3BoA) significantly decreases LPA plasma levels (almost zero), indicating that ATX is the major enzyme responsible for LPA production [7]. In the search for non-lipid like ATX inhibitors, a thiazolidinedione carboxylic acid analog HA (Figure 2) has been reported [8]. 1. Introduction Inspired by the proteasome inhibitor bortezomib, in which t In the search for non-lipid like ATX inhibitors, a thiazolidinedione carboxylic acid analog HA51 (Figure 2) has been reported [8]. Inspired by the proteasome inhibitor bortezomib, in which the boronic acid group interacts with a N-terminal threonine oxygen nucleophile in the proteasome, the carboxylic group in HA51 (Figure 2) was similarly replaced by the boronic acid isostere. The derived compound HA130 has reported activity of 28 nM in an in vitro LPC choline release assay. Crystallography studies published by Hausman [9] further supported the hypothesis that HA130 is engaged in interactions with Thr209 residue and that the 4-ﬂuorobenzyl moiety reaches deep in a hydrophobic pocket. Molecules 2019, 24, x FOR PEER REVIEW 3 of 22 boronic acid group interacts with a N-terminal threonine oxygen nucleophile in the proteasom carboxylic group in HA51 (Figure 2) was similarly replaced by the boronic acid isostere. The d compound HA130 has reported activity of 28 nM in an in vitro LPC choline release Crystallography studies published by Hausman [9] further supported the hypothesis that HA engaged in interactions with Thr209 residue and that the 4-fluorobenzyl moiety reaches de hydrophobic pocket. R= H, Cl, Me, OMe (1-2) (1-2) Boretezomib HA51 HA155 3BoA HA130 PF-8380 S-35 Figure 2. Design and development of small molecule ATX inhibitors. Figure 2. Design and development of small molecule ATX inhibitors. residue Boretezomib HA51 Figure 2. Design and development of small molecule ATX inhibitors. Figure 2. Design and development of small molecule ATX inhibitors. The molecule HA130 is a potent inhibitor of ATX. Furthermore, in vivo proof of principle studies have demonstrated a rapid decrease of LPA levels in the plasma of mice after an intravenous administration (1 nmol/g), with no overt toxicity issues observed [8]. HA130, in a dose-dependent The molecule HA130 is a potent inhibitor of ATX. Furthermore, in vivo proof of principle studies have demonstrated a rapid decrease of LPA levels in the plasma of mice after an intravenous 3 of 22 Molecules 2019, 24, 3419 administration (1 nmol/g), with no overt toxicity issues observed [8]. HA130, in a dose-dependent manner, inhibited the ATX-mediated migration of A2058 human melanoma cells. However, the stability of HA130 in vivo was very poor (half-life less than ﬁve minutes) [10]. 1. Introduction A later publication from the same authors [11] reported on HA155, a boronic acid regioisomer of HA130, which has a better inhibition of ATX. The same was observed for HA130, as the presence of a double bond attached to the thiazolidinedione heterocycle may cause HA155 to act as a Michael acceptor and may cause in vivo instability. Therefore, in an eﬀort to improve stability, a reduction of the double bond in HA155, and the introduction of the rigid bicyclic tetrahydroisoquinoline as a central ring core have been performed [11]. More recent developments around boronic acid-based ATX inhibitors were published in 2013 by Nagano’s group [7], in which thiazolinone analogues bearing benzylboronic acid groups were investigated. The boronic acid warhead, as in HA155, was designed to bind to Zn2+ ions and to Thr209. Changes designed to optimize lipophilic interactions with the active site of ATX, aﬀorded derivative 3BoA (Figure 2) with good inhibitory properties (IC50 = 13 nM). Compound 3BoA was further tested in vitro and in vivo, showing several very promising pharmacologically related features. In brief 3BoA is more potent than HA155 as an LPA production inhibitor in incubated plasma and cell-motility assays, and it exerts a more favorable metabolic stability and decreases rapidly plasma LPA levels in mice after a 4 mg/kg intravenous administration [10]. A recent (2017) publication by Lanier et al. [12] reported a fragment based approach used to explore phenylboronic acids as warheads towards ATX inhibitors. They tested more than 650 boronic acid fragments. combining in silico computational chemistry ﬁlters and crystallography, allowing them to identify fragments that were consistent to known SAR against ATX [12]. The non-boronic acid compound, PF-8380, is one of the most potent in vitro inhibitors of ATX (Figure 2) with an IC50 = of 2.8 nM in a human enzyme assay. PF-8380 was developed by Pﬁzer in the context of identifying new anti-inﬂammatory drugs indicating promising in vivo eﬃcacy. Notably, in a separate study, PF-8380 produced a signiﬁcant anti-invasive eﬀect in glioblastoma cell lines, delaying simultaneously glioma tumor growth progression in vivo [8]. The Ovaa group has speculated that replacing carboxylic acid warheads with less acidic boronic acid warheads may be beneﬁcial for ATX inhibition. Benzoxaboroles, (cyclic boronic acid esters that have lower pKa values that are 1–2 units lower than boronic acids [13]) may have improved binding characteristics towards the ATX enzyme. 1. Introduction ( ) ( ) Figure 3. Formation of an adduct of benzoxaborole in the ATX active site according to literat [12]. (a) Nucleophilic attack on benzoxaboroles by catalytic threonine oxygen side chain; reversible covalent tetrahedral adduct Our group was interested in new ATX inhibitors for cancer treatment. Based on the previous work [9] where boronic acids were used as warheads, we designed and synthesized novel ATX inhibitors with a benzoxaborole as an isostere aiming for the same binding pattern (like HA155) to the active pocket but with better overall drug properties. Working around the potential stability issue due to presence of the double bond, we designed different linkers, introducing more rigidity. We also used the 3,5-substituted benzylic group as a hydrophobic lipid binding motif. Our group was interested in new ATX inhibitors for cancer treatment. Based on the previous work [9] where boronic acids were used as warheads, we designed and synthesized novel ATX inhibitors with a benzoxaborole as an isostere aiming for the same binding pattern (like HA155) to the active pocket but with better overall drug properties. Working around the potential stability issue due to presence of the double bond, we designed diﬀerent linkers, introducing more rigidity. We also used the 3,5-substituted benzylic group as a hydrophobic lipid binding motif. Our group was interested in new ATX inhibitors for cancer treatment. Based on the previous work [9] where boronic acids were used as warheads, we designed and synthesized novel ATX inhibitors with a benzoxaborole as an isostere aiming for the same binding pattern (like HA155) to the active pocket but with better overall drug properties. Working around the potential stability issue due to presence of the double bond, we designed different linkers, introducing more rigidity. We also used the 3 5 substituted benzylic group as a hydrophobic lipid binding motif Our design and synthesis of novel ATX inhibitors focused on inhibitors with the benzoxaborole head moiety as the acidic headgroup, the substituted benzyl carbamate moiety as the lipophilic portion, and a rigid core spacer constituted of two saturated heterocyclic rings (Figure 4.). Our design and synthesis of novel ATX inhibitors focused on inhibitors with the benzoxaborole head moiety as the acidic headgroup, the substituted benzyl carbamate moiety as the lipophilic portion, and a rigid core spacer constituted of two saturated heterocyclic rings (Figure 4). used the 3,5-substituted benzylic group as a hydrophobic lipid binding motif. 1. Introduction Crystallographic studies by Hausman have demonstrated further insight for the boronic acid binding mode, in which Thr209 and Zinc atoms are in a complex with boronic acids [9]. This was illustrated by the ring strain generated in the ﬁve-membered oxaborole ring where the boron atom was in a neutral, trigonal-planar form [13]. Benzoxaboroles have physicochemical properties which are a consequence of the relatively strong Lewis acidic center on the boron atom and the presence of a free hydroxyl group [14]. The sp2 hybridized boron atom possesses an empty p-orbital which accepts electrons from the hydroxyl group of threonine 209 (Thr209) that explains adduct formation of boronic acids, as in the recent modelling of Lanier [12]. According to Lanier et al. [12], a boronic acid motif with this type of interaction may enhance binding aﬃnity up to 1000 fold. Similarly, we assumed that the binding mode and the mechanism of adduct formation in the ATX active site would resemble phenylboronic acids (Figure 3). 4 of 22 g adduct inding adduct Molecules 2019, 24, 3419 g affinity up to 1000 f According to Lanier affinity up to 1000 f (a) (b) Figure 3. Formation of an adduct of benzoxaborole in the ATX active site according to literatu [12]. (a) Nucleophilic attack on benzoxaboroles by catalytic threonine oxygen side chain; reversible covalent tetrahedral adduct Figure 3. Formation of an adduct of benzoxaborole in the ATX active site according to literature [12]. (a) Nucleophilic attack on benzoxaboroles by catalytic threonine oxygen side chain; (b) reversible covalent tetrahedral adduct. rmation in the ATX active site would resemble phenylboronic acids (Figure 3). (a) (b) Figure 3. Formation of an adduct of benzoxaborole in the ATX active site according to literatu [12]. (a) Nucleophilic attack on benzoxaboroles by catalytic threonine oxygen side chain; reversible covalent tetrahedral adduct (a) e 3 Fo atio of a addu t of b n the ATX active site would res (a) (b) th ATX ti it di ( g ) (b) (b) (b) (a) (a) Figure 3. Formation of an adduct of benzoxaborole in the ATX active site according to literat [12]. (a) Nucleophilic attack on benzoxaboroles by catalytic threonine oxygen side chain; reversible covalent tetrahedral adduct Figure 3. Formation of an adduct of benzoxaborole in the ATX active site according to literature [12]. (a) Nucleophilic attack on benzoxaboroles by catalytic threonine oxygen side chain; (b) reversible covalent tetrahedral adduct. 1. Introduction The design of the novel ATX inhibitors was also based on the available protein-ligand x-ray crystal structures in the PDB (Protein Data Bank) (Figure 6). Figure 6. Overlay of HA155 boronic acid (green) inhibitor in the binding site of ATX (Protein Data Bank (PDB) ID: 2XRG [9] and re-docked pose of HA155 (magenta) using Covalent Docking v1.3. [15]. Figure 6. Overlay of HA155 boronic acid (green) inhibitor in the binding site of ATX (Protein Data Bank (PDB) ID: 2XRG [9] and re-docked pose of HA155 (magenta) using Covalent Docking v1.3. [15]. Figure 6. Overlay of HA155 boronic acid (green) inhibitor in the binding site of ATX (Protein Data Bank (PDB) ID: 2XRG [9] and re-docked pose of HA155 (magenta) using Covalent Docking v1.3. [15]. Figure 6. Overlay of HA155 boronic acid (green) inhibitor in the binding site of ATX (Protein Data Bank (PDB) ID: 2XRG [9] and re-docked pose of HA155 (magenta) using Covalent Docking v1.3. [15]. ATX inhibitors [16] (Figure 7) target the hydrophobic lipid-binding pocket in the central catalytic phosphodiesterase (PDE) domain of ATX (Figure 8A). This is located underneath a shallow groove that accommodates lysophospholipids (Figure 8B). In the active site, the first Zn2+ ion is in the tetrahedral arrangement [17] and is coordinated by the catalytic Thr209 as well as the side chains of Asp171, Asp358, and His359. The second Zn2+ ion coordinates with Asp311, His315 and His474, and, usually, a solvent molecule or counter-ion. Furthermore, an open tunnel (or ‘channel’; Figure 6C), which is partially hydrophobic in nature, is located in close proximity where a variety of molecules can be accommodated, forming a ‘T-intersection’ with the shallow groove [18,19] ATX inhibitors [16] (Figure 7) target the hydrophobic lipid-binding pocket in the central catalytic phosphodiesterase (PDE) domain of ATX (Figure 8A). This is located underneath a shallow groove that accommodates lysophospholipids (Figure 8B). In the active site, the ﬁrst Zn2+ ion is in the tetrahedral arrangement [17] and is coordinated by the catalytic Thr209 as well as the side chains of Asp171, Asp358, and His359. The second Zn2+ ion coordinates with Asp311, His315 and His474, and, usually, a solvent molecule or counter-ion. Furthermore, an open tunnel (or ‘channel’; Figure 6C), which is partially hydrophobic in nature, is located in close proximity where a variety of molecules can be accommodated, forming a ‘T-intersection’ with the shallow groove [18,19]. 1. Introduction es 2019, 24, x FOR PEER REVIEW 6 of 22 In our hands, a standard docking protocol within Glide (with and without H-bond/meta nstraints) failed to dock HA155 in the active site of ATX in the pose observed in the x-ray structur DB ID: 2XRG [9]) This pose has the characteristic interaction of Thr209 with the boron atom. Base natural bond orbital (NBO) calculations [20], the existence of polar character of Thr209 and boro om of HA155 hybridized to sp3 character, we applied a covalent docking approach. Using covalen cking, HA155 was successfully docked in the conformation observed in the crystal structur MSD (heavy atoms) = 0.68 Å, Figure 6). This same approach was therefore applied to ou mpounds in this paper. (Figure 7). ure 7. Compound 37 docked in the binding site of ATX using covalent docking. The covalent bond ween the oxygen atom of Thr209 and the boron atom of boronate is labelled in green (d(O–B) = Figure 7. Compound 37 docked in the binding site of ATX using covalent docking. The covalent bond between the oxygen atom of Thr209 and the boron atom of boronate is labelled in green (d(O–B) = 1.48 Å). 7. Compound 37 docked in the binding site of ATX using covalent docking. The covalent bond en the oxygen atom of Thr209 and the boron atom of boronate is labelled in green (d(O–B) = Figure 7. Compound 37 docked in the binding site of ATX using covalent docking. The covalent bond between the oxygen atom of Thr209 and the boron atom of boronate is labelled in green (d(O–B) = 1.48 Å). 48 Å). he dative covalent bond between the boron atom and Thr209 is required for these inhibitors nd docking poses thus generated are in line with the previously reported co-crystalized ures of ATX (Figure 8a). In addition, this reversible covalent bond places the hydroxyl moiety boronate between both zinc ions in the ATX active site (Figure 7). 1. Introduction Our design and synthesis of novel ATX inhibitors focused on inhibitors with the benzoxaborole head moiety as the acidic headgroup, the substituted benzyl carbamate moiety as the lipophilic portion, and a rigid core spacer constituted of two saturated heterocyclic rings (Figure 4.). Figure 4. General structure of novel ATX inhibitors. Figure 4 General structure of novel ATX inhibitors Figure 4. General structure of novel ATX inhibitors. R REVIEW Figure 4. General structure of novel ATX inhibitors. Figure 4 General structure of novel ATX inhibitors Figure 4. General structure of novel ATX inhibitors. REVIEW Figure 4. General structure of novel ATX inhibitors. Figure 4 General structure of novel ATX inhibitors Figure 4. General structure of novel ATX inhibitors. REVIEW The structures of the warheads and spacer groups to be combined together are shown in Figure 5 The structures of the warheads and spacer groups to be combined together are shown in Figure O O H B O O O H B O O O H B O W1 W2 W3 W4 CS1 CS2 CS3 N N N N N N N N N O O H O B O O B O H O O H B O O O H B O Figure 5. Structures of the warheads (W) and the core spacers (CS). Figure 5. Structures of the warheads (W) and the core spacers (CS). W1 O O H B O W3 O O B O H W4 O O H O B O H W1 W3 W2 W1 CS1 N N N W4 W2 CS1 N N N CS2 N N N CS3 N N N CS1 CS1 CS3 CS2 Figure 5. Structures of the warheads (W) and the core spacers (CS). Figure 5. Structures of the warheads (W) and the core spacers (CS). 5 of 22 Molecules 2019, 24, 3419 The design of the novel ATX inhibitors was also based on the available protein-ligand x-ray crystal structures in the PDB (Protein Data Bank) (Figure 6). The design of the novel ATX inhibitors was also based on the available protein-ligand x-ray crystal structures in the PDB (Protein Data Bank) (Figure 6) The design of the novel ATX inhibitors was also based on the available protein-ligand x-ray crystal structures in the PDB (Protein Data Bank) (Figure 6). 1. Introduction The docking studies also d that the other oxygen atom of the boronate is involved in an interactions with the amides of 9 (d(O…H–NThr209) = 2.6 Å) and/or Asn230 Thr209 (d(O…H–NAsn230) = 2.3 Å), which are in proximity The binding of the rest of the molecule is mainly driven by hydrophobic interactions In our hands, a standard docking protocol within Glide (with and without H-bond/metal constraints) failed to dock HA155 in the active site of ATX in the pose observed in the x-ray structure (PDB ID: 2XRG [9]) This pose has the characteristic interaction of Thr209 with the boron atom. Based on natural bond orbital (NBO) calculations [20], the existence of polar character of Thr209 and boron atom of HA155 hybridized to sp3 character, we applied a covalent docking approach. Using covalent docking, HA155 was successfully docked in the conformation observed in the crystal structure (RMSD (heavy atoms) = 0.68 Å, Figure 6). This same approach was therefore applied to our compounds in this paper. (Figure 7). 6 of 22 7 of 22 Molecules 2019, 24, 3419 Molecules 2019, 24, x FOR Figure 8. Compound 37 docked in the binding site of ATX using covalent docking and overlapped with ligands from x-ray structures: (a) HA155 boronic acid inhibitor (green, PDB ID: 2XRG) [9]; (b) PF-8380 (magenta, PDB ID: 5L0K [21]). Figure 8. Compound 37 docked in the binding site of ATX using covalent docking and overlapped with ligands from x-ray structures: (a) HA155 boronic acid inhibitor (green, PDB ID: 2XRG) [9]; (b) PF-8380 (magenta, PDB ID: 5L0K [21]). Figure 8. Compound 37 docked in the binding site of ATX using covalent docking and overlapped with ligands from x-ray structures: (a) HA155 boronic acid inhibitor (green, PDB ID: 2XRG) [9]; (b) PF-8380 (magenta, PDB ID: 5L0K [21]). Figure 8. Compound 37 docked in the binding site of ATX using covalent docking and overlapped with ligands from x-ray structures: (a) HA155 boronic acid inhibitor (green, PDB ID: 2XRG) [9]; (b) PF-8380 (magenta, PDB ID: 5L0K [21]). As previously suggested for SAR transfer between series of active compounds, targeting this hydrogen bond can be useful in the modification of this class of benzoxaboroles [22]. The lipophilic tail of the molecule was designed according to data published for the PF-8380 molecule. This is where the carbonyl group creates a hydrogen bond with the acceptor of a Trp275 [9]. 1. Introduction To reduce the degrees of conformational freedom, the rigidity of the molecule increased by the introduction of several acyclic rings. These rings would bridge the between warhead benzoxaborole and carbamate moiety (Figure 8). During the final preparation of the manuscript, the Kang group analyzed the topological water network in the binding pocket of ATX. Using this pharmacophoric insight, new molecules were synthesized and tested for their ATX inhibitory activities [23]. 2. Results The dative covalent bond between the boron atom and Thr209 is required for these inhibitors [11], and docking poses thus generated are in line with the previously reported co-crystalized structures of ATX (Figure 8a). In addition, this reversible covalent bond places the hydroxyl moiety of the boronate between both zinc ions in the ATX active site (Figure 7). The docking studies also showed that the other oxygen atom of the boronate is involved in an interactions with the amides of Thr209 (d(O . . . H–NThr209) = 2.6 Å) and/or Asn230 Thr209 (d(O . . . H–NAsn230) = 2.3 Å), which are in close proximity. The binding of the rest of the molecule is mainly driven by hydrophobic interactions in similar way as other ligands with similar ‘tails’—such as PF-8380 (PDB ID: 5L0K [21], Figure 8b). The aromatic ring at the end of the ‘tail,’ a hydrophobic 3,5-dichlorobenzene, points directly into a deep hydrophobic pocket. The hydrogen bond between the carbonyl group pf the carbamate and the Trp 275 amide has been observed in the generated docking poses for some compounds described in this paper, similar to what is revealed in the co-crystalized structure with PF-8380 (d (O . . . H–NTrp275) = 3.1 Å, Figure 8b) and ATX. 2.1. ATX Inhibition All 21 new benzoxaboroles derivatives prepared herein were evaluated for ATX inhibition in vitro using a biochemical choline detection assay, combining elements of assays already described in the literature [24]. ATX activity was measured using Lyso PC (16:1) as a substrate and HA155 was used as the standard, control, compound. l P l l l t d f ll d i th P t ft [25] Ch l D As previously suggested for SAR transfer between series of active compounds, targeting this hydrogen bond can be useful in the modiﬁcation of this class of benzoxaboroles [22]. The lipophilic tail of the molecule was designed according to data published for the PF-8380 molecule. 2.1. ATX Inhibition All 21 new benzoxaboroles derivatives prepared herein were evaluated for ATX inhibition in vitro using a biochemical choline detection assay, combining elements of assays already described in the literature [24]. ATX activity was measured using Lyso PC (16:1) as a substrate and HA155 was used as the standard, control, compound. clogP values were calculated for all compounds using the Percepta software [25]. Chrom logD was determined experimentally using a procedure described in the supplementary data. Furthermore, the correlation between calculated clogP values and experimentally determined Chrom log D values were explored for molecules containing boron functional groups such as boronic acids and benzoxaboroles. The results are presented in Table 1. Table 1. Structures, inhibitory activities, calculated logP and determined Chrom logD values. Compound Structure a IC50 Value (µM) b clog P c Chrom logD Warhead Core Lipophilic Tail W CS R 30 W 1 CS 1 Cl 0.18 ± 0.04 3.82 4.52 31 W 4 CS 1 Cl 7.5 ± 5.60 3.61 4.63 32 W 2 CS 1 Cl 0.37± 0.35 3.82 4.38 33 W 3 CS 1 Cl 8.0 ± 2.58 3.61 5.40 34 W 1 CS 2 Cl 0.92 ± 0.82 4.02 5.49 35 W 2 CS 2 Cl 1.8 ± 1.75 4.02 4.58 36 W 4 CS 2 Cl 1.8 ± 1.52 3.99 4.58 37 W 1 CS 3 Cl 0.13 ± 0.12 3.60 4.58 38 W 2 CS 3 Cl 2.0 ± 1.73 3.60 4.46 39 W 1 CS 1 H >50 ± 4.20 2.75 3.05 40 W 2 CS 1 H >50 ± 3.08 2.75 2.90 41 W 1 CS 3 H 23 ± 1.18 2.40 3.07 42 W 2 CS 3 H >50 ± 32.84 2.40 2.90 43 W 1 CS 1 CH3 2.7 ± 2.11 3.17 4.13 44 W 2 CS 1 CH3 4.7 ± 1.14 3.17 3.99 45 W 1 CS 3 CH3 1.0 ± 1.15 2.90 4.17 46 W 2 CS 3 CH3 5.1 ± 2.75 2.90 4.01 47 W 1 CS 1 OCH3 14 ± 5.50 2.51 3.19 48 W 2 CS 1 OCH3 15 ±6.07 2.51 3.03 49 W 1 CS 3 OCH3 11 ± 4.59 2.48 3.21 50 W 2 CS 3 OCH3 13 ± 3.26 2.48 3.07 HA155 Molecules 2019, 24, x FOR PEER REVIEW 8 of Table 1. Structures, inhibitory activities, calculated logP and determined Chrom logD values. 1. Introduction This is where the carbonyl group creates a hydrogen bond with the acceptor of a Trp275 [9]. To reduce the degrees of conformational freedom, the rigidity of the molecule increased by the introduction of several acyclic rings. These rings would bridge the between warhead benzoxaborole and carbamate moiety (Figure 8). clogP values were calculated for all compounds using the Percepta software [25]. Chrom logD was determined experimentally using a procedure described in the supplementary data. Furthermore, the correlation between calculated clogP values and experimentally determined Chrom log D values were explored for molecules containing boron functional groups such as boronic acids During the ﬁnal preparation of the manuscript, the Kang group analyzed the topological water network in the binding pocket of ATX. Using this pharmacophoric insight, new molecules were synthesized and tested for their ATX inhibitory activities [23]. 7 of 22 7 of 22 Molecules 2019, 24, 3419 2. Results 2.1. ATX Inhibition a IC50 values determined in choline release assay using LPC C16:0 as a substrate. The results are shown as the mean standard deviation from duplicate experiments. b calculated log P was the octanol/water partition coefficient calculated using Percepta. c Chrom logD was determined at pH 7 4 using Luna C18 (50 × 3 mm i d 5 µm) column d IC50 values taken a IC50 values determined in choline release assay using LPC C16:0 as a substrate. The results are shown as the mean standard deviation from duplicate experiments. b calculated log P was the octanol/water partition coeﬃcient calculated using Percepta. c Chrom logD was determined at pH 7.4 using Luna C18 (50 × 3 mm i.d., 5 µm) column. d IC50 values taken from literature [26]. 3. Discussion The modiﬁcation of several regions had dramatic eﬀects on the inhibition of ATX. Thus, the hydrophobic part of the inhibitors showed sensitivity to phenyl substitution—the unsubstituted phenyl compound (41) being almost inactive (23 µM). The 3,5 dimethoxyphenyl analogue (49) was a slightly better (11 µM). Ten times better potency was achieved when comparing to 41 with 3,5-dimethyl substitution on phenyl ring for compound 45. The original 3,5-dichlorosubstitution (already described for PF-8380) remained the most favorable substitution pattern for the majority of the compounds prepared. SAR around the core spacer showed that the piperazine–azetidine bicycle was the best spacer. The position of the boron atom on the aromatic ring (acidic headgroup) was also very important and diﬀered to that observed with HA155. In our case, the most active compound was the 6-amido substituted benzoxaborole (compound 37), whilst 4-amido or 7-amido substitution was not tolerated. Among 21 tested compounds, four compounds showed ATX inhibition in the nanomolar range of activity. The IC50 for the most active compound (37) was 130 nM. In our LPC choline released assay, HA155 also showed nanomolar activity (88 nM) but was less potent then reported in published data (5.7 nM) [11]. Keeping in mind that the analogue of HA155 with tetrahydroisoquinoline as a rigid core spacer was signiﬁcantly less potent (ten times) then HA155, our molecule 36 with space core rigidity was only two times less potent that HA155. The kinetic solubility in plasma bovine serum (PBS) at pH 7.4, determined by turbidimetric method, was >100 µM for the majority of tested compounds. The most active compound 36 had a solubility of 30–100 µM, while HA155 suﬀered from very poor solubility (1–3 µM). Results regarding metabolic stability in rat and human liver microsomes suggest that the majority of tested compounds would be classiﬁed a high-predicted in vivo hepatic clearance (expressed as percentage of liver blood ﬂow, %LBF). Two exceptions include compounds 37 and 45, which had a moderate predicted in vivo clearance (62% and 45%, respectively) in rats. However, in human liver microsomes, all compounds showed high-predicted in vivo clearance, whereas the comparator compound showed a moderate predicted in vivo clearance. The results of plasma stability which were measured at four hours indicated a good stability of compound 37 that remained 71.2% of its initial concentration in rat and 79.1% of its initial concentration in human plasma. 2.1. ATX Inhibition Compound Structure a IC50 value (µM) b clog P c Chrom log Warhead Core Lipophilic tail W CS R 30 W 1 CS 1 Cl 0.18 ± 0.04 3.82 4.52 31 W 4 CS 1 Cl 7.5 ± 5.60 3.61 4.63 32 W 2 CS 1 Cl 0.37± 0.35 3.82 4.38 33 W 3 CS 1 Cl 8.0 ± 2.58 3.61 5.40 34 W 1 CS 2 Cl 0.92 ± 0.82 4.02 5.49 35 W 2 CS 2 Cl 1.8 ± 1.75 4.02 4.58 36 W 4 CS 2 Cl 1.8 ± 1.52 3.99 4.58 37 W 1 CS 3 Cl 0.13 ± 0.12 3.60 4.58 38 W 2 CS 3 Cl 2.0 ± 1.73 3.60 4.46 39 W 1 CS 1 H >50 ± 4.20 2.75 3.05 40 W 2 CS 1 H >50 ± 3.08 2.75 2.90 41 W 1 CS 3 H 23 ± 1.18 2.40 3.07 42 W 2 CS 3 H >50 ± 32.84 2.40 2.90 43 W 1 CS 1 CH3 2.7 ± 2.11 3.17 4.13 44 W 2 CS 1 CH3 4.7 ± 1.14 3.17 3.99 45 W 1 CS 3 CH3 1.0 ± 1.15 2.90 4.17 46 W 2 CS 3 CH3 5.1 ± 2.75 2.90 4.01 47 W 1 CS 1 OCH3 14 ± 5.50 2.51 3.19 48 W 2 CS 1 OCH3 15 ±6.07 2.51 3.03 49 W 1 CS 3 OCH3 11 ± 4.59 2.48 3.21 50 W 2 CS 3 OCH3 13 ± 3.26 2.48 3.07 HA155 0.088 ± 0.04 0.0057 d 3.76 NA a IC50 values determined in choline release assay using LPC C16:0 as a substrate. The results are shown as the mean standard deviation from duplicate experiments. b calculated log P 0.088 ± 0.04 0.0057 d 3.76 NA a IC50 values determined in choline release assay using LPC C16:0 as a substrate. The results are shown as the mean standard deviation from duplicate experiments. b calculated log P was the octanol/water partition coeﬃcient calculated using Percepta. c Chrom logD was determined at pH 7.4 using Luna C18 (50 × 3 mm i.d., 5 µm) column. Table 1. Structures, inhibitory activities, calculated logP and determined Chrom logD values. 8 of 22 Molecules 2019, 24, 3419 2.2. ADME Properties Eight of the most active compounds, together with HA155, were selected for characterization of in vitro ADME properties such as solubility, microsomal metabolic stability, plasma stability and plasma protein binding (PPB). Results are presented in Table 2. Eight of the most active compounds, together with HA155, were selected for characterization of in vitro ADME properties such as solubility, microsomal metabolic stability, plasma stability and plasma protein binding (PPB). Results are presented in Table 2. Table 2. In vitro ADME proﬁle of selected compounds. Compound a Solubility b PS (rat/human) c LMS (rat/human) d PPB (rat/human) 30 >100 64.5/66.5 86/82 98.8/98.4 31 >100 67.9/71.6 86/85 NA e/97.8 34 >100 61.6/70.6 77/89 NA e/99.2 35 >100 63.0/59.2 86/89 NA e/98.3 37 30–100 72.2/79.1 62/85 98.4/97.9 38 30–100 60.1/79.1 82/79 NA e 43 >100 59.5/79.2 80/85 96.1/97.8 45 >100 68.6/72.8 61/84 94.4/95.9 HA155 3–10 f 6.8 65.7/42.9 <30/54 99.9/>99.9 a Kinetic solubility range after 1h [µM]. b Plasma stability after 4 h [%remaining]. c LMS: Metabolic stability in human/rat liver microsomes -> predicted in vivo hepatic clearance expressed as %LBF. d PPB: Plasma protein binding in human/rat -> fraction bound [%]. e recovery issue. f solubility value taken from literature [7]. Table 2. In vitro ADME proﬁle of selected compounds. a Kinetic solubility range after 1h [µM]. b Plasma stability after 4 h [%remaining]. c LMS: Metabolic stability in human/rat liver microsomes -> predicted in vivo hepatic clearance expressed as %LBF. d PPB: Plasma protein binding in human/rat -> fraction bound [%]. e recovery issue. f solubility value taken from literature [7]. 3. Discussion However, HA155 was less stable with only 65.7% of its initial concentration in rat and 42.9% of its initial concentration in rat and human plasma, 9 of 22 Molecules 2019, 24, 3419 respectively, remaining after four hours. The binding to plasma proteins (PPB) of newly synthesized compounds was signiﬁcantly lower when compared with binding of standard compounds that showed a very high PPB of >99.9% in both tested species. That would impact eﬃcacy of 37 in comparison to HA155 since their free fractions are diﬀerent more than 20 times assuming that both molecules have the same binding kinetics kon/koﬀ. 4.1. Materials and Instruments All chemicals, solvents, and chemical and biochemical reagents were of analytical grade and purchased from commercial sources (Merck, Merck KGaA, Darmstadt, Germany, Fluka Sigma-Aldrich Laborchemikalien GmbH, Hannover, Germany, Alfa Aesar, Karlsruhe, Germany and Sigma, St. Louis, MO, USA, Combi-blocks, Combi-blocks Inc.San Diego, USA, TCI, TCI-Europe, Zwijndrecht, Belgium, Kemika, Kemika d.d., Zagreb, Croatia). All starting materials were obtained from commercial sources (Merck, Merck KGaA, Darmstadt, Germany, Fluka Sigma-Aldrich Laborchemikalien GmbH, Hannover, Germany, Alfa Aesar, Karlsruhe, Germany, and Sigma, St. Louis, MO, USA) and used without further puriﬁcation. The progress of all reactions was checked by UPLC-MS/UV Waters system (Waters, Waters Corporation, Milford, USA) and thin layer chromatography (TLC) using precoated Silica Gel 60F254 sheets (Merck, Darmstadt, Germany). The spots on plates were visualized under UV light (254 nm). Column chromatography was performed by an Interchim Puriﬂash 450 system or by the Waters Mass Directed AutoPuriﬁcation system. The 1H and 13C nucleic magnetic resonance (NMR) spectra were recorded at 400 MHz on a Bruker 400 spectrometer (Bruker Analytische Messtechnik GmbH, Rheinstetten, Germany). Chemical shifts were determined relatively to the signals of residual protons of the deuterated solvent (DMSO). Chemical shifts are reported in delta (δ) units in parts per million (ppm), and splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet and br, broad. Coupling constants were recorded in Hertz (Hz). High resolution mass spectra were determined on an Agilent 1100 series. 4.2. Synthesis 4.2. Synthesis 6N aqueous HCl solution. The white precipitate was collected by filtratio benzoxaborole acids 4 7 4.2.2. General Procedure for the Preparation of Carbamate Intermediates benzoxaborole acids 4 7. methyl 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2a [28] 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 2b [29] methyl 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2c [22] methyl 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2d, Yield: 53%, 1H NMR (DMSO- d6, 400 MHz, δ ppm): 7.84–7.88 (m, 1H), 7.49–7.57 (m, 2H), 3.97 (s, 3H), 2.49 (s, 3H), 1.47 (s, 12H) 13C The ﬁrst step in the synthetic pathway was the reduction of an appropriate carboxylic acid (8e–g) by LiAlH4. The entire reaction was performed under an argon atmosphere using a syringe septa technique. To a solution of lithium aluminum hydride (0.063 mol, 1.2 eq) in THF (15 mL) that was cooled in an ice bath, a solution of the substituted benzoic acid (8e–g) (0.052 mol, 1 eq) in THF (65 mL) was added dropwise during 30 minutes. The resulting mixture was stirred at ambient temperature for 2 h. To the reaction mixture were added: Water (3 mL), a 1M solution of NaOH (3 mL), and then water (10 mL) whilst stirring continued for a further 90 minutes at ambient temperature. The mixture was extracted with EtOAc (3 × 30 mL). The organic layers were combined, dried over Na2SO4/MgSO4, ﬁltered and evaporated in vacuo to give a crude product which was puriﬁed by Interchim Puriﬂash 450 with a gradient of EtOAc in cyclohexane. The appropriate fractions were combined and evaporated in vacuo to give corresponding alcohols (9e–g). The resultant alcohols (0.039 mol, 1 eq) and CDI (0.039 mol, 1 eq) were dissolved in DMF (100 mL) at ambient temperature, and the resulting mixture was treated with the Boc protected piperazine (0.035 mol, 0.9 eq), and stirring was continued for 16 h at ambient temperature. Solvent was evaporated in vacuo. Water (50 mL) was added to the residue. Extraction with dichloromethane (DCM) (3 × 30 mL) followed. The organic layers were combined, dried over Na2SO4/MgSO4, ﬁltered oﬀ, end evaporated in vacuo to give raw material which was puriﬁed by Interchim Puriﬂash 450 in the gradient of MeOH in DCM: 0%–3% in 20 CV. The appropriate fractions were combined and evaporated in vacuo to give corresponding Boc protected piperazine carbamates (10e–h). To a solution of Boc protected piperazine carbamate (10e–h) (0.035 mol, 1 eq) in DCM (200 mL), cooled in an ice bath, TFA (0.353 mol, 10 eq) was added. The resulting mixture was stirred at ambient temperature for 30 minutes. 4.2.1. General Procedure for the Preparation of Benzoxaborolic Acids The solution was cooled to 0 °C, and pH was adjusted to 2 with 1-hydroxy-3H-2,1-benzoxaborole-4-carboxylic acid 7, Yield: 58%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.28 (s, 1H), 8.02 (m, 1H), 7.96 (m, 1H), 7.50 (m, 1H), 5.21 (s, 2H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 167.2, 134.9, 132.1, 127.4, 71.3 HRMS: m/z calculated for C8H7BO4 [M −H]−176.0401; found 176.0397. 6N aqueous HCl solution. The white precipitate was collected by filtratio benzoxaborole acids 4 7 4.2.2. General Procedure for the Preparation of Carbamate Intermediates 4.2.1. General Procedure for the Preparation of Benzoxaborolic Acids The appropriate fractions were combined and evaporated in vacuo to give corresponding intermediates 2a–d. [27] To a solution of corresponding intermediates 2a–d (7.243 mmol) in benzene (40 ml), NBS (7.605 mmol, 1.05 eq) and AIBN (0.362 mmol, 0.05 eq) were added and the reaction mixture stirred at reflux. four hours later HRMS: m/z calculated for C15H21BO4 [M + H]+ 276.1642; found 276.1639. 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 4 [30] 1-hydroxy-3H-2,1-benzoxaborole-5-carboxylic acid 5 [30] 1-hydroxy-3H-2,1-benzoxaborole-7-carboxylic acid 6 [31] 1-hydroxy-3H-2,1-benzoxaborole-4-carboxylic acid 7, Yield: 58%, 1H NMR (DMSO-d6, 400 MHz, δ ) 9 28 ( 1H) 8 02 ( 1H) 7 96 ( 1H) 7 50 ( 1H) 5 21 ( 2H) 13C NMR (DMSO d6 dichloromethane (DCM): 0% 5% in 20 column volumes. T and evaporated in vacuo to give corresponding inter 1-hydroxy-3H-2,1-benzoxaborole-5-carboxylic acid 5 [30] d evaporated in vacuo to give corresponding inter rresponding intermediates 2a–d (7 243 mmol) in benzen 1-hydroxy-3H-2,1-benzoxaborole-7-carboxylic acid 6 [31] corresponding intermediates 2a d (7.243 mmol) in benzene (40 ml), NBS (7.605 mmol, 1.05 eq) and AIBN (0.362 mmol, 0.05 eq) were added and the reaction mixture stirred at reflux. four hours later solvent had evaporated in vacuo, and Et2O was added to the residue (40 ml). The resulting precipitate was filtered off, and the filtrate got extracted with 20% KOH (3 x 30 ml). The aqueous layers were combined and stirred at rt 90 min. The solution was cooled to 0 °C, and pH was adjusted to 2 with 1-hydroxy-3H-2,1-benzoxaborole-4-carboxylic acid 7, Yield: 58%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.28 (s, 1H), 8.02 (m, 1H), 7.96 (m, 1H), 7.50 (m, 1H), 5.21 (s, 2H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 167.2, 134.9, 132.1, 127.4, 71.3 HRMS: m/z calculated for C8H7BO4 [M −H]−176.0401; found 176.0397. corresponding intermediates 2a d (7.243 mmol) in benzene (40 ml), NBS (7.605 mmol, 1.05 eq) and AIBN (0.362 mmol, 0.05 eq) were added and the reaction mixture stirred at reflux. four hours later solvent had evaporated in vacuo, and Et2O was added to the residue (40 ml). The resulting precipitate was filtered off, and the filtrate got extracted with 20% KOH (3 x 30 ml). The aqueous layers were combined and stirred at rt 90 min. 4.2.1. General Procedure for the Preparation of Benzoxaborolic Acids Reactions were performed under argon atmosphere using syringe sep mide 1a–d (8.731 mmol), pinacol diboron (12.223 mmol, 1.4 eq) and KOAc methyl 4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2a [28] 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 2b [29] h l 2 h l 3 (4 4 h l 1 3 2 di b l 2 l)b 2 [ ] Reactions were performed under argon atmosphere using syringe sep mide 1a–d (8 731 mmol) pinacol diboron (12 223 mmol 1 4 eq) and KOAc methyl 4 methyl 3 (4,4,5,5 tetramethyl 1,3,2 dioxaborolan 2 yl)benzoate 2a [28] 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 2b [29] omide 1a d (8.731 mmol), pinacol diboron (12.223 mmol, 1.4 eq) and KOAc (26.193 m ere dissolved in dioxane (180 ml) at rt, and the resulting mixture was bubbled w Cl2(dppf) (0 873 mol 0 1 eq) was added and stirring was continued for 16 hours at methyl 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2c [22] methyl 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2d, Yield: 53% PdCl2(dppf) (0.873 mol, 0.1 eq) was added and stirring was continued for 16 hours at 90 °C. The reaction mixture was cooled to rt, quenched with NH4Cl (70 ml), and extracted with Et2O (3 × 70 ml). The organic layers were combined, dried over Na2SO4/MgSO4, filtered off end evaporated in vacuo t i t i l hi h b ifi d b I t hi P ifl h 450 i th di t f M OH i methyl 3-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 2d, Yield: 53%, H NMR (DMSO-d6, 400 MHz, δ ppm): 7.84–7.88 (m, 1H), 7.49–7.57 (m, 2H), 3.97 (s, 3H), 2.49 (s, 3H), 1.47 (s, 12H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 167.7, 140.8, 133.6, 132.6, 128.8, 125.7, 83.4, 52.3, 25.0, 21.0 HRMS: m/z calculated for C15H21BO4 [M + H]+ 276.1642; found 276.1639. PdCl2(dppf) (0.873 mol, 0.1 eq) was added and stirring was continued for 16 hours at 90 C. The reaction mixture was cooled to rt, quenched with NH4Cl (70 ml), and extracted with Et2O (3 × 70 ml). The organic layers were combined dried over Na2SO4/MgSO4 filtered off end evaporated in vacuo y y y y (DMSO-d6, 400 MHz, δ ppm): 7.84–7.88 (m, 1H), 7.49–7.57 (m, 2H), 3.97 (s, 3H), 2.49 (s, 3H), 1.47 (s, 12H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 167.7, 140.8, 133.6, 132.6, 128.8, 125.7, 83.4, 52.3, 25.0, 21.0 g y g p to give raw material which was been purified by Interchim Puriflash 450 in the gradient of MeOH in dichloromethane (DCM): 0%–5% in 20 column volumes. 4.2.1. General Procedure for the Preparation of Benzoxaborolic Acids Reactions were performed under argon atmosphere using syringe septa technique. Tolyl bromide 1a–d (8.731 mmol), pinacol diboron (12.223 mmol, 1.4 eq) and KOAc (26.193 mmol, 3 eq) were dissolved in dioxane (180 mL) at rt, and the resulting mixture was bubbled with argon. PdCl2(dppf) (0.873 mol, 0.1 eq) was added and stirring was continued for 16 h at 90 ◦C. The reaction mixture was cooled to rt, quenched with NH4Cl (70 mL), and extracted with Et2O (3 × 70 mL). The organic layers were combined, dried over Na2SO4/MgSO4, ﬁltered oﬀend evaporated in vacuo to give raw material which was been puriﬁed by Interchim Puriﬂash 450 in the gradient of MeOH in dichloromethane (DCM): 0%–5% in 20 column volumes. The appropriate fractions were combined and evaporated in vacuo to give corresponding intermediates 2a–d. [27] To a solution of corresponding intermediates 2a–d (7.243 mmol) in benzene (40 mL), NBS (7.605 mmol, 1.05 eq) and AIBN (0.362 mmol, 0.05 eq) were added and the reaction mixture stirred at reﬂux. four hours later solvent had evaporated in vacuo, and Et2O was added to the residue (40 mL). The resulting precipitate was ﬁltered oﬀ, and the ﬁltrate got extracted with 20% KOH (3 × 30 mL). The aqueous layers were combined and stirred at rt 90 min. The solution was cooled to 0 ◦C, and pH was adjusted to 2 with 6N aqueous HCl solution. The white precipitate was collected by ﬁltration to aﬀord corresponding benzoxaborole acids 4–7 (Scheme 1). Molecules 2019, 24, 3419 10 of 22 10 of 22 i ii iii,iv 4-7 3a-d 2a-d 1a-d Scheme I. Reagents and conditions: (i) Pinacol diborane, PdCl2(dppf), KOAc, dioxane, reflux; (ii) N- bromosuccinimide (NBS), azobisisobutyronitrile (AIBN), CCl4, reflux; (iii) Et2O, 20% KOH, rt; (ix) 0 °C, 6N HCl. Scheme 1. Reagents and conditions: (i) Pinacol diborane, PdCl2(dppf), KOAc, dioxane, reﬂux; (ii) N-bromosuccinimide (NBS), azobisisobutyronitrile (AIBN), CCl4, reﬂux; (iii) Et2O, 20% KOH, rt; (ix) 0 ◦C, 6N HCl. ii iii,iv 4-7 3a-d 4-7 3a-d 2a-d 2a-d 1a-d Scheme I. Reagents and conditions: (i) Pinacol diborane, PdCl2(dppf), KOAc, dioxane, reflux; (ii) N- bromosuccinimide (NBS), azobisisobutyronitrile (AIBN), CCl4, reflux; (iii) Et2O, 20% KOH, rt; (ix) 0 °C, 6N HCl. Scheme 1. Reagents and conditions: (i) Pinacol diborane, PdCl2(dppf), KOAc, dioxane, reﬂux; (ii) N-bromosuccinimide (NBS), azobisisobutyronitrile (AIBN), CCl4, reﬂux; (iii) Et2O, 20% KOH, rt; (ix) 0 ◦C, 6N HCl. ambient temperature, and the resulting mixture was treat mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] ambient temperature, and the resulting mixture was treat mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] ambient temperature, and the resulting mixture was treate mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] , q), g p p in vacuo. Water (50ml) was added to the residue. Extraction with dichloromethane (DCM) (3 × 30 mL) followed. The organic layers were combined, dried over Na2SO4/MgSO4, filtered off, end evaporated in vacuo to give raw material which was purified by Interchim Puriflash 450 in the gradient of MeOH in DCM: 0%–3% in 20 CV. The appropriate fractions were combined and (3,5-dimethylphenyl)methyl piperazine-1-carboxylate 11f, Yield: 98%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 8.19 (br. s, 1H), 6.93 (m, 3H), 5.00 (s, 2H), 2.62–2.70 (m, 2H), 2.25 (s, 6H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.7, 179.5, 163.2, 161.0, 137.4, 129.3, 129.2, 125.5, 125.4, 70.0, 66.5, 66.2, 45.1, 20.8 HRMS: m/z calculated for C14H20N2O2 [M + H]+ 249.1598; found 249.1599. , q), g p p in vacuo. Water (50ml) was added to the residue. Extraction with dichloromethane (DCM) (3 × 30 mL) followed. The organic layers were combined, dried over Na2SO4/MgSO4, filtered off, end evaporated in vacuo to give raw material which was purified by Interchim Puriflash 450 in the gradient of MeOH in DCM: 0%–3% in 20 CV. The appropriate fractions were combined and (3,5-dimethylphenyl)methyl piperazine-1-carboxylate 11f, Yield: 98%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 8.19 (br. s, 1H), 6.93 (m, 3H), 5.00 (s, 2H), 2.62–2.70 (m, 2H), 2.25 (s, 6H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.7, 179.5, 163.2, 161.0, 137.4, 129.3, 129.2, 125.5, 125.4, 70.0, 66.5, 66.2, 45.1, 20.8 HRMS: m/z calculated for C14H20N2O2 [M + H]+ 249.1598; found 249.1599. evaporated in vacuo to give corresponding Boc protected piperazine carbamates (10e–h). To a solution of Boc protected piperazine carbamate (10e–h) (0.035 mol, 1 eq) in DCM (200 ml), cooled in an ice bath, TFA (0.353 mol, 10 eq) was added. The resulting mixture was stirred at ambient temperature for 30 minutes. The first step in the synthetic pathway was the reduction of an approp O4-tert-butyl O1-[(3,5-dichlorophenyl)methyl] piperazine-1,4-dicarboxylate [32] g) by LiAlH4. The entire reaction was performed under an argon atmosphere using a syringe septa technique. To a solution of lithium aluminum hydride (0.063 mol, 1.2 eq) in THF (15 mL) that was cooled in an ice bath, a solution of the substituted benzoic acid (8e–g) (0.052 mol, 1 eq) in THF (65 mL) was added dropwise during 30 minutes. The resulting mixture was stirred at ambient f h T h dd d W (3 L) 1M l f N OH O4-tert-butyl O1-[(3,5-dimethylphenyl)methyl] piperazine-1,4-dicarboxylate 10f, Yield: 86%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 6.94 (s, 3H), 4.99 (s, 2H), 3.32–3.39 (m, 2H), 2.25 (s, 6H), 1.39 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.3, 154.0, 153.7, 137.4, 136.5, 129.5, 125.3, 79.1, 66.4, 43.2, 28.0, 20.8 HRMS: m/z calculated for C15H21BO4 [M + H-100]+ 276.1642; found 276.1641. temperature for 2 hours. To the reaction mixture were added: Water (3 mL), a 1M solution of NaOH (3 ml), and then water (10 ml) whilst stirring continued for a further 90 minutes at ambient temperature. The mixture was extracted with EtOAc (3 × 30 mL). The organic layers were combined, dried over Na2SO4/MgSO4, filtered and evaporated in vacuo to give a crude product which was purified by Interchim Puriflash 450 with a gradient of EtOAc in cyclohexane. The appropriate fractions were combined and evaporated in vacuo to give corresponding alcohols (9e–g) The O4-tert-butyl O1-[(3,5-dimethoxyphenyl)methyl] piperazine-1,4-dicarboxylate 10g, Yield: 83%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.84–7.88 (m, 2H), 7.49–7.57 (m, 1H), 5.02 (s, 2H), 3.72 (s, 6H), 3.32–3.39 (m, 2H), 1.47 (s, 12H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.7, 179.5, 154.3, 153.7, 139.0, 105.3, 99.4, 79.1, 66.1, 55.1, 27.9 HRMS: m/z calculated for C19H28N2O6 [M + Na]+ 403.1840; found 403.1825. p g resultant alcohols (0.039 mol, 1 eq) and CDI (0.039 mol, 1 b d h l O1-benzyl O4-tert-butyl piperazine-1,4-dicarboxylate 10h [33] 6N aqueous HCl solution. The white precipitate was collected by filtratio benzoxaborole acids 4 7 4.2.2. General Procedure for the Preparation of Carbamate Intermediates The pH of the reaction mixture was adjusted to 8 with 6N aqueous solution of NaOH and layers were separated. The aqueous layer was washed with DCM (2 × 100 mL). The organic layers were combined, dried over Na2SO4/MgSO4, ﬁltered oﬀand evaporated in vacuo to give corresponding piperazine carbamates intermediates (11e–h). The piperazine carbamate intermediates (11e–h) (0.010 mol, 1 eq) and corresponding N-Boc protected cyclic ketone (0.010 mol, 1 11 of 22 Molecules 2019, 24, 3419 1-hydroxy-3H-2,1-ben eq) were dissolved in DCM (45 mL) at rt, and to the resulting solution NaBH(OAc)3 (0.013 mol, 1.3 eq) was added in portions before stirring was continued for 16 h at rt. The reaction mixture was quenched with a saturated solution of NaHCO3 (30 mL). The layers were separated, and the aqueous layer was washed with DCM (2 × 20 mL). The organic layers were combined, dried over Na2SO4/MgSO4, ﬁltered oﬀand evaporated in vacuo to give crude product which was puriﬁed by Interchim Puriﬂash 450, in a gradient of MeOH in DCM: 0%–5% in 20 column volumes. The appropriate fractions were combined and evaporated in vacuo to give corresponding Boc protected intermediates (12–20). Finally, the Boc protecting group was cleaved with TFA to obtain carbamate intermediates (21–29) (Scheme 2). 1 hydroxy 3H 2,1 benzoxaborole 5 carboxylic acid 5 [30] 1-hydroxy-3H-2,1-benzoxaborole-7-carboxylic acid 6 [31] 1-hydroxy-3H-2,1-benzoxaborole-4-carboxylic acid 7, Yield: 58%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.28 (s, 1H), 8.02 (m, 1H), 7.96 (m, 1H), 7.50 (m, 1H), 5.21 (s, 2H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 167.2, 134.9, 132.1, 127.4, 71.3 HRMS: m/z calculated for C8H7BO4 [M–H]- 176.0401; found 176.0397. 4.2.2. General Procedure for the Preparation of Carbamate Intermediates i ii iii iv v 21-29 10e-h 12-20 8e-g 9e-h 11e-h e - Cl, f - Me, g - OMe, h - H Scheme II. Reagents and conditions: (i) LiAlH4, THF, 0 °C; (ii) tert-butyl piperazine-1-carboxylate, CDI, DMF, rt; (iii) TFA, dichloromethane (DCM), 0 °C to rt; (iv) Boc protected 4-, 5-, or 6-membered ketone NaBH(OAc)3 DCM 0 °C to rt; (v) TFA DCM 0 °C to rt Scheme 2. Reagents and conditions: (i) LiAlH4, THF, 0 ◦C; (ii) tert-butyl piperazine-1-carboxylate, CDI, DMF, rt; (iii) TFA, dichloromethane (DCM), 0 ◦C to rt; (iv) Boc protected 4-, 5-, or 6-membered ketone, NaBH(OAc)3, DCM, 0 ◦C to rt; (v) TFA, DCM, 0 ◦C to rt. 6N aqueous HCl solution. The white precipitate was collected by filtratio benzoxaborole acids 4 7 4.2.2. General Procedure for the Preparation of Carbamate Intermediates i ii iii 10e-h 8e-g 9e-h 11e-h 11e-h 9e-h iv v 21-29 12-20 e - Cl, f - Me, g - OMe, h - e - Cl, f - Me, g - OMe, h - H 12-20 21-29 Scheme II. Reagents and conditions: (i) LiAlH4, THF, 0 °C; (ii) tert-butyl piperazine-1-carboxylate, CDI, DMF, rt; (iii) TFA, dichloromethane (DCM), 0 °C to rt; (iv) Boc protected 4-, 5-, or 6-membered ketone NaBH(OAc)3 DCM 0 °C to rt; (v) TFA DCM 0 °C to rt Scheme 2. Reagents and conditions: (i) LiAlH4, THF, 0 ◦C; (ii) tert-butyl piperazine-1-carboxylate, CDI, DMF, rt; (iii) TFA, dichloromethane (DCM), 0 ◦C to rt; (iv) Boc protected 4-, 5-, or 6-membered ketone, NaBH(OAc)3, DCM, 0 ◦C to rt; (v) TFA, DCM, 0 ◦C to rt. The first step in the synthetic pathway was the reduction of an approp O4-tert-butyl O1-[(3,5-dichlorophenyl)methyl] piperazine-1,4-dicarboxylate [32] ambient temperature, and the resulting mixture was treat mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] (3,5-dichlorophenyl)methyl 4-(1-tert-butoxycarbonyl-4-piperidyl)piperazine-1-carboxylate 14, Yield: 77%, 1H NMR (DMSO-d6, 400 MHz δ ppm) 7.60 (s, 1H), 7.41–7.51 (m, 2H), 5.11 (s, 2H), 3.92–4.04 (m, 2H), 3.39–3.49 (m, 2H), 2.69–2.76 (m, 2H), 2.38–2.52 (m, 2H), 1.69–1.71 (m, 2H), 1.43 (s, 9H), 1.20–1.35 (m, 2H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 154.0, 153.8, 141.3, 134.0, 127.4, 126.1, 78.5, 64.6, 60.8, 48.3, 43.9, 28.0, 27.6 HRMS: m/z calculated for C22H31Cl2N3O4 [M + H]+ 472.1764; found 472.1774. (3,5-dimethylphenyl)methyl 4-(1-tert-butoxycarbonylazetidin-3-yl)piperazine-1-carboxylate 15, Yield: 67%, 1H NMR (DMSO-d6, 400 MHz δ ppm) 7.89–7.96 (m, 3H), 4.97 (s, 2H), 3.75–3.83 (m, 2H), 3.59–3.68 (m, 2H), 3.35–3.39 (m, 2H), 2.97–3.06 (m,1H), 2.24 (s, 6H), 2.17–2.23 (m, 2H), 1.47 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.4, 155.6, 154.4, 137.4, 136.6, 129.2, 125.5, 125.4, 78.6, 66.3, 53.0, 48.7, 43.2, 28.0, 20.8 HRMS: m/z calculated for C22H33N3O4 [M + H]+ 404.2544; found 404.2561. (3,5-dimethylphenyl)methyl 4-(1-tert-butoxycarbonylpyrrolidin-3-yl)piperazine-1-carboxylate 16, Yield: 59%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 7.01 (s, 3H), 5.08 (s, 2H), 3.38–3.44 (m, 4H), 3.11–3.26 (m, 1H), 2.91–3.03 (m, 1H), 2.71–2.87 (m, 1H), 2.39–2.49 (m, 1H), 2.30–2.39 (m, 1H), 2.29 (s, 6H), 1.61–1.75 (m, 1H), 1.42 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.0, 154.3, 153.4, 137.4, 136.6, 129.2, 125.4, 78.3, 66.2, 63.5, 62.7, 50.8, 49.3, 49.2, 44.6, 44.4, 43.3, 28.9, 28.1, 20.8 HRMS: m/z calculated for C23H36N3O4 [M + H]+ 418.2711; found 418.2718. (3,5-dimethoxyphenyl)methyl 4-(1-tert-butoxycarbonylazetidin-3-yl)piperazine-1-carboxylate 17, Yield: 74%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 6.45–6.51 (m, 2H), 6.42 (s, 1H), 4.99 (s, 2H), 3.75–3.87 (m, 2H), 3.72 (s, 6H), 3.59–3.68 (m, 2H), 3.34–3.46 (m, 4H), 2.98–3.07 (m, 1H), 1.36 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.1, 160.5, 155.5, 154.3, 139.2, 105.2, 99.3, 78.6, 66.0, 55.2, 55.1, 53.0, 48.7, 43.2, 28.0 HRMS: m/z calculated for C22H33N3O6 [M + H]+ 436.2442; found 436.2428. (3,5-dimethoxyphenyl)methyl 4-(1-tert-butoxycarbonylpyrrolidin-3-yl)piperazine-1-carboxylate 18, Yield: 55%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 6.46–6.49 (m, 2H), 6.40–6.45 (m, 1H), 4.99 (s, 2H), 3.72 (s, 6H), 3.45–3.53 (m, 1H), 3.35–3.42 (m, 4H), 2.87–2.98 (m, 1H), 2.61–2.69 (m, 1H), 2.34–2.43 (m, 1H), 2.27–2.35 (m, 1H), 1.94–2.06 (m, 1H), 1.37 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.5, 160.5, 105.2, 99.3, 65.9, 55.2 28.1 HRMS: m/z calculated for C22H35N3O6 [M + H]+ 450.2599 found 450.2641. ambient temperature, and the resulting mixture was treat mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] The pH of the reaction mixture was adjusted to 8 with 6N aqueous (3,5-dimethoxyphenyl)methyl piperazine-1-carboxylate 11g, Yield: 97%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 6.39–6.45 (m, 3H), 4.99 (s, 2H), 3.72 (s, 6H), 2.58–2.68 (m, 2H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.2, 160.5, 160.4, 154.4, 139.3, 105.3, 105.1, 99.3, 65.8, 55.2, 55.1, 45.3, 44.5 HRMS: m/z calculated for C14H20N2O4 [M + H]+ 281.1496; found 281.1522. benzyl piperazine-1-carboxylate 11h [35] 12 of 22 12 of 22 Molecules 2019, 24, 3419 (3,5-dichlorophenyl)methyl 4-(1-tert-butoxycarbonylazetidin-3-yl)piperazine-1-carboxylate 12, Yield: 72%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.74–7.85 (m, 1H), 7.5–7.72 (m, 2H), 5.30 (s, 2H), 3.99–4.12 (m., 2H), 3.80–3.94 (m, 2H), 3.52–3.71 (m, 4H), 1.60 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 155.6, 154.1, 141.2, 134.1, 127.7, 126.1, 78.6, 64.7, 53.0, 48.6, 43.2, 28.0 HRMS: m/z calculated for C20H27Cl2N3O4 [M + H]+ 444.1451; found 444.1450. (3,5-dichlorophenyl)methyl 4-(1-tert-butoxycarbonylazetidin-3-yl)piperazine-1-carboxylate 12, Yield: 72%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.74–7.85 (m, 1H), 7.5–7.72 (m, 2H), 5.30 (s, 2H), 3.99–4.12 (m., 2H), 3.80–3.94 (m, 2H), 3.52–3.71 (m, 4H), 1.60 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 155.6, 154.1, 141.2, 134.1, 127.7, 126.1, 78.6, 64.7, 53.0, 48.6, 43.2, 28.0 HRMS: m/z calculated for C20H27Cl2N3O4 [M + H]+ 444.1451; found 444.1450. (3,5-dichlorophenyl)methyl 4-(1-tert-butoxycarbonylazetidin-3-yl)piperazine-1-carboxylate 12, Yield: 72%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.74–7.85 (m, 1H), 7.5–7.72 (m, 2H), 5.30 (s, 2H), 3.99–4.12 (m., 2H), 3.80–3.94 (m, 2H), 3.52–3.71 (m, 4H), 1.60 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 155.6, 154.1, 141.2, 134.1, 127.7, 126.1, 78.6, 64.7, 53.0, 48.6, 43.2, 28.0 HRMS: m/z calculated for C20H27Cl2N3O4 [M + H]+ 444.1451; found 444.1450. (3,5-dichlorophenyl)methyl 4-(1-tert-butoxycarbonylpyrrolidin-3-yl)piperazine-1-carboxylate 13, Yield: 60%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 7.52–7.60 (m, 1H), 7.36–7.47 (m, 2H), 5.03 (s, 2H), 3.37–3.56 (m, 4H), 3.09–3.21 (m, 1H), 3.00–3.07 (m, 1H), 2.84–2.99 (m, 1H), 2.64–2.87 (m, 1H), 2.37–2.45 (m, 1H), 1.89–1.92 (m., 1H), 1.37 (s, 9H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 153.9, 141.2, 134.1, 127.4, 126.3, 126.2, 115.3, 64.8, 64.7, 64.4, 64.7, 50.8, 49.2, 44.7, 28.9 HRMS: m/z calculated for C21H29Cl2N3O4 [M + H]+ 458.1608; found 458.1625. ambient temperature, and the resulting mixture was treat mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] benzyl 4-(1-tert-butoxycarbonylazetidin-3-yl)piperazine-1-carboxylate 19 [36] benzyl 4-(1-tert-butoxycarbonylpyrrolidin-3-yl)piperazine-1-carboxylate 20 [37] (3,5-dichlorophenyl)methyl 4-(azetidin-3-yl)piperazine-1-carboxylate 21, Yield: 72%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.51–7.62 (m, 1H), 7.38–7.42 (m, 2H), 5.06 (s, 2H), 4.07–4.16 (m., 2H), 3.78–3.14 (m, 2H), 3.52–3.71 (m, 2H), 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.1, 161.1, 154.1, 141.2, 134.1, 127.5, 126.2, 64.8, 54.8, 53.0, 50.5, 48.6, 43.2 HRMS: m/z calculated for C15H19Cl2N3O2 [M + H]+ 344.0922; found 344.0931. 3,5-dichlorophenyl)methyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 22, Yield: 99%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 7.52–7.60 (m, 1H), 7.36–7.47 (m, 2H), 5.03 (s, 2H), 3.00–3.07 (m, 1H), 2.84–2.99 (m, 13 of 22 13 of 22 Molecules 2019, 24, 3419 1H), 2.75–2.83 (m, 1H), 2.64–2.87 (m, 1H), 2.59–2.64 (m, 1H), 1.87–1.89 (m, 1H), 1.55–1.68 (m, 1H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 154.0, 141.2, 134.1, 127.4, 126.2, 126.1, 64.7, 64.1, 51.0, 48.6, 44.7, 43.4, 28.5 HRMS: m/z calculated for C16H21Cl2N3O2 [M + H]+ 458.1608; found 458.1625. (3,5-dichlorophenyl)methyl 4-(4-piperidyl)piperazine-1-carboxylate 23, Yield: 99%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 7.52–7.60 (m, 1H), 7.36–7.47 (m, 2H), 5.03 (s, 2H), 3.92–4.04 (m, 2H), 3.39–3.49 (m, 2H), 2.69–2.76 (m, 2H), 2.38–2.52 (m, 2H), 1.69–1.71 (m, 2H), 1.20–1.35 (m, 2H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 154.0, 153.8, 141.3, 134.0, 127.4, 126.1, 64.6, 48.3, 43.9 HRMS: m/z calculated for C17H23Cl2N3O2 [M + H]+ 372.124; found 372.1237. (3,5-dimethylphenyl)methyl 4-(azetidin-3-yl)piperazine-1-carboxylate 24, Yield: 99%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 6.94–7.96 (m, 3H), 4.99 (s, 2H), 3.72–3.79 (m, 2H), 3.64–3.71 (m, 2H), 2.25 (s, 6H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.5, 165.4, 154.4, 137.4, 136.6, 129.3, 125.4, 66.3, 56.0, 54.7, 49.4, 48.4, 20.9, 20.8 HRMS: m/z calculated for C22H33N3O4 [M + H]+ 404.2544; found 404.2561. (3,5-dimethylphenyl)methyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 25, Yield: 99%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 6.89–7.05 (m, 3H), 5.01 (s, 2H), 3.73 (m, 1H), 3.31–3.46 (m, 1H), 3.11–3.26 (m, 1H), 2.91–3.03 (m, 1H), 2.71–2.87 (m, 1H), 2.39–2.49 (m, 1H), 2.30–2.39 (m, 1H), 2.29 (s, 6H), 1.61–1.75 (m, 1H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.5, 154.4, 137.4, 136.6, 129.3, 125.4, 66.3, 62.8, 50.7, 47.2, 44.0, 43.3, 27.4, 20.9 HRMS: m/z calculated for C18H27N3O2 [M + H]+ 318.2181; found 318.2186. ambient temperature, and the resulting mixture was treat mol, 0.9 eq), and stirring was continued for 16 hours at am (3,5-dichlorophenyl)methyl piperazine-1-carboxylate 11e [34] (3,5-dimethoxyphenyl)methyl 4-(azetidin-3-yl)piperazine-1-carboxylate 26, Yield: 95%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 6.45–6.51 (m, 2H), 6.42 (s, 1H), 4.99 (s, 2H), 3.75–3.87 (m, 2H), 3.72 (s, 6H), 3.59–3.68 (m, 2H), 3.34–3.46 (m, 2H), 2.98–3.07 (m, 1H), 2.24 (m, 4H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.6, 160.5, 154.3, 139.2, 105.2, 99.3, 66.0, 56.0, 55.2, 49.2, 48.4, 43.1 HRMS: m/z calculated for C17H25N3O4 [M + H]+ 336.1918; found 336.1939. Molecules 2019, 24, x FOR PEER REVIEW 14 of 22 1H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.5, 154.4, 137.4, 136.6, 129.3, 125.4, 66.3, 62.8, 50.7, 47.2, 44.0, 43.3, 27.4, 20.9 HRMS: m/z calculated for C18H27N3O2 [M+H]+ 318.2181; found 318.2186. (3,5-dimethoxycyclohexa-1,5-dien-1-yl)methyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 27, Yield: 93%, 1H NMR (DMSO-d6, 400 MHz, δ ppm) 6.46–6.49 (m, 2H), 6.40–6.45 (m, 1H), 4.99 (s, 2H), 3.72 (s, 6H), 3.00–3.18 (m, 2H), 2.87–2.98 (m, 1H), 2.70–2.83 (m, 1H), 2.59–2.63 (m, 1H), 2.37–2.47 (m, 2H), 1.87–1.96 (m, 1H), 1.54–1.67 (m, 1H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.5, 160.5, 154.2, 139.2, 150.2, 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H27N3O4 [M + H]+ 350.2074; found 350.2089. (3,5-dimethoxyphenyl)methyl 4-(azetidin-3-yl)piperazine-1-carboxylate 26, Yield: 95%, 1H NMR (DMSO- d6, 400 MHz, δ ppm) 6.45–6.51 (m, 2H), 6.42 (s, 1H), 4.99 (s, 2H), 3.75–3.87 (m, 2H), 3.72 (s, 6H), 3.59– 3.68 (m, 2H), 3.34–3.46 (m, 2H), 2.98–3.07 (m, 1H), 2.24 (m, 4H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.6, 160.5, 154.3, 139.2, 105.2, 99.3, 66.0, 56.0, 55.2, 49.2, 48.4, 43.1 HRMS: m/z calculated for C17H25N3O4 [M+H]+ 336.1918; found 336.1939. (3,5-dimethoxycyclohexa-1,5-dien-1-yl)methyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 27, Yield: 93%, 1H benzyl 4-(azetidin-3-yl)piperazine-1-carboxylate 28 [28] benzyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 29 [28] NMR (DMSO-d6, 400 MHz, δ ppm) 6.46–6.49 (m, 2H), 6.40–6.45 (m, 1H), 4.99 (s, 2H), 3.72 (s, 3.00–3.18 (m, 2H), 2.87–2.98 (m, 1H), 2.70–2.83 (m, 1H), 2.59–2.63 (m, 1H), 2.37–2.47 (m, 2H), 1.87 (m, 1H), 1.54–1.67 (m, 1H) 13C NMR (DMSO-d6, 100 MHz, δ ppm): 179.5, 160.5, 154.2, 139.2, 1 benzyl 4-(azetidin-3-yl)piperazine-1-carboxylate 28 [28] NMR (DMSO-d6, 400 MHz, δ ppm) 6.46–6.49 (m, 2H 3 00 3 18 ( 2H) 2 87 2 98 ( 1H) 2 70 2 83 ( 1H) benzyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 29 [28] 3.00–3.18 (m, 2H), 2.87–2.98 (m, 1H), 2.70–2.83 (m, 1H) (m, 1H), 1.54–1.67 (m, 1H) 13C NMR (DMSO-d6, 100 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 30, Yield: 79%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.55 (s, 1H), 7.35–7.48 (m, 3H), 5.05 (s, 2H), 5.01 (s, 2H), 3.71–3.80 (m, 2H), 3.58–3.67 (m, 2H), 3.50–3.58 (m, 2H), 2.84–2.94 (m, 2H), 2.73–2.84 (m, 2H), 2.34–2.47 (m, 2H), 2.08–2.18 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.6, 141.2, 134.1, 129.7, 129.3, 127.5, 126.2, 121.3, 69.9, 67.9, 63.7, 62.4, 54.3, 25.9 HREI-MS: m/z calculated for C24H26BCl2N3O5 [M]+ 517.1452; found 517.1448. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 30, Yield: 79%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.55 (s, 1H), 7.35–7.48 (m, 3H), 5.05 (s, 2H), 5.01 (s, 2H), 3.71–3.80 (m, 2H), 3.58–3.67 (m, 2H), 3.50–3.58 (m, 2H), 2.84–2.94 (m, 2H), 2.73–2.84 (m, 2H), 2.34–2.47 (m, 2H), 2.08–2.18 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.6, 141.2, 134.1, 129.7, 129.3, 127.5, 126.2, 121.3, 69.9, 67.9, 63.7, 62.4, 54.3, 25.9 HREI-MS: m/z calculated for C24H26BCl2N3O5 [M]+ 517.1452; found 517.1448. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-7-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 31, Yield: 53%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.28 (br.s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.69–7.73 (m, 1H),7.56 (s, 1H) 7.48–7.51 (m, 1H), 7.39–7.43 (m, 1H), 5.05 (s, 2H), 5.01 (s, 2H), 3.70–3.82 (m, 2H), 3.52–3.63 (m, 2H), 3.26–3.42 (m, 4H), 3.18–3.29 (m, 1H), 2.34–2.43 (m, 4H), 1.99–2.15 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.6, 141.2, 134.1, 129.7, 129.3, 127.5, 126.2, 121.3, 69.9, 67.9, 63.7, 62.4, 54.3, 25.9, HREI-MS: m/z calculated for C24H26BCl2N3O5 [M]+ 517.1452; found 517.1446. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 32, Yield: 68%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.33 (br.s, 1H), 7.66–7.88 (m, 1H), 7.47–7.59 (m, 2H), 7.35–7.46 (m, 3H), 5.04 (s, 2H), 5.00 (s, 2H), 3.70–3.76 (m, 2H), 3.48–3.62 (m, 2H), 2.85–2.92 (m, 4H), 2.06–2.17 (m, 4H), 1.95–2.06 (m, 2H) 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.4, 134.1, 131.9, 129.9, 127.4, 126.2, 125.8, 120.0, 99.8, 98.6, 62.0, 25.2, 19.7, 8.1, HREI-MS: m/z calculated for C24H26BCl2N3O5 [M]+ 517.1452; found 517.1451. 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H27 found 350 2089 To a solution of benzoxaborolic acid (4–7) (0.232 mmol, 1 eq), the carboxylate intermediate (21–29) (0.232 mmol) in DCM (1 mL), EDAC (0.372 mmol, 1.6 eq) and DMAP (0.009 mmol, 0.04 eq) were added at ambient temperature, and the resulting mixture was stirred for 16 h. The solvent was evaporated in vacuo, and the raw material was puriﬁed by a Waters Mass Directed AutoPuriﬁcation system, giving corresponding novel autotaxin inhibitors (30–50) (Scheme 3). found 350.2089. benzyl 4-(azetidin-3-yl)piperazine-1-carboxylate 28 [28] benzyl 4-pyrrolidin-3-ylpiperazine-1-carboxylate 29 [28] 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors i 21-29 30-50 4-7 R= H, Cl, Me, OMe Scheme III. Reagents and conditions: (i) N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC), 4-dimethylaminopyridine (DMAP), DCM, rt. Scheme 3. Reagents and conditions: (i) N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC), 4-dimethylaminopyridine (DMAP), DCM, rt. i 21-29 30-50 R= H, Cl, Me, OMe 4-7 R= H, Cl, Me, OMe Scheme III. Reagents and conditions: (i) N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC), 4-dimethylaminopyridine (DMAP), DCM, rt. Scheme 3. Reagents and conditions: (i) N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC), 4-dimethylaminopyridine (DMAP), DCM, rt. Scheme III. Reagents and conditions: (i) N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC), 4-dimethylaminopyridine (DMAP), DCM, rt. Scheme 3. Reagents and conditions: (i) N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC), 4-dimethylaminopyridine (DMAP), DCM, rt. 14 of 22 14 of 22 Molecules 2019, 24, 3419 (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 30, Yield: 79%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.55 (s, 1H), 7.35–7.48 (m, 3H), 5.05 (s, 2H), 5.01 (s, 2H), 3.71–3.80 (m, 2H), 3.58–3.67 (m, 2H), 3.50–3.58 (m, 2H), 2.84–2.94 (m, 2H), 2.73–2.84 (m, 2H), 2.34–2.47 (m, 2H), 2.08–2.18 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.6, 141.2, 134.1, 129.7, 129.3, 127.5, 126.2, 121.3, 69.9, 67.9, 63.7, 62.4, 54.3, 25.9 HREI-MS: m/z calculated for C24H26BCl2N3O5 [M]+ 517.1452; found 517.1448. 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 15 of 22 15 of 22 Molecules 2019, 24, 3419 (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 37, Yield: 83%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s., 1H), 8.00–8.02 (m, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.55 (s, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 1.9 Hz, 2H), 5.06 (s, 2H), 5.02 (s, 2H), 4.03–4.14 (m, 2H), 3.84–3.90 (m, 1H), 3.36–3.48 (m, 4H), 3.13–3.21 (m, 1H), 2.25–2.36 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.2, 156.7, 154.0, 141.3, 134.0, 131.7, 129.9, 127.5, 126.2, 121.6, 70.2, 64.8, 53.9, 48.9, 43.2, HREI-MS: m/z calculated for C23H24BCl2N3O5 [M]+ 503.1295; found 503.1299. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 37, Yield: 83%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s., 1H), 8.00–8.02 (m, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.55 (s, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 1.9 Hz, 2H), 5.06 (s, 2H), 5.02 (s, 2H), 4.03–4.14 (m, 2H), 3.84–3.90 (m, 1H), 3.36–3.48 (m, 4H), 3.13–3.21 (m, 1H), 2.25–2.36 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.2, 156.7, 154.0, 141.3, 134.0, 131.7, 129.9, 127.5, 126.2, 121.6, 70.2, 64.8, 53.9, 48.9, 43.2, HREI-MS: m/z calculated for C23H24BCl2N3O5 [M]+ 503.1295; found 503.1299. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 38, Yield: 77%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.33 (br.s., 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.62–7.64 (m, 1H), 7.54–7.58 (m, 2H), 7.41 (d, J = 1.9 Hz, 2H), 5.06 (s, 2H), 5.00 (s, 2H), 4.20–4.28 (m,2H), 3.99–4.12 (m, 2H), 3.09–3.20 (m, 1H), 2.24–2.32 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.2, 156.7, 154.0, 141.3, 134.0, 131.7, 129.9, 127.5, 126.2, 121.6, 70.2, 64.8, 53.9, 48.9, 43.2, HREI-MS: m/z calculated for C23H24BCl2N3O5 [M]+ 503.1295; found 503.1301. benzyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 39, Yield: 82%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.26 (br.s, 1H), 7.70–7.80 (m, 1H), 7.54–7.70 (m, 1H), 7.41–7.49 (m, 1H), 7.24–7.41 (m, 5H), 5.06 (s, 2H), 5.01 (s, 2H), 3.71–3.79 (m, 2H), 3.58–3.67 (m, 2H), 3.49–3.57 (m, 2H), 2.84–2.92 (m, 2H), 2.74–2.83 (m, 2H), 2.34–2.43 (m, 2H), 2.07–2.17 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.5, 155.5, 154.2, 136.8, 135.4, 129.6, 129.2, 128.4, 127.9, 121.3, 69.9, 66.2, 63.7, 62.2, 52.5, 50.7, 47.9, 43.3, HREI-MS: m/z calculated for C24H28BN3O5 [M]+ 449.2231; found 449.2240. 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-4-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate) 33, Yield: 59%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.32 (br.s, 1H), 7.82–7.86 (m, 1H), 7.71–7.74 (m, 1H), 7.62–7.58 (m, 1H), 7.34–7.42 (m, 2H),7.41–7.51 (m, 1H), 5.03 (s, 2H), 5.00 (s, 2H), 3.70–3.82 (m, 2H), 3.37–3.54 (m, 2H), 3.23–3.32 (m, 5H), 2.15–2.25 (m, 4H), 1.96–2.25 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.4, 134.1, 131.9, 129.9, 127.4, 126.2, 125.8, 120.0, 99.8, 98.6, 62.0, 25.2, 19.7, 8.1 HREI-MS: m/z calculated for C24H26BCl2N3O5 [M]+ 517.1452; found 517.1448. 3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)-4-piperidyl]piperazine-1- carboxylate 34, Yield: 81%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.31 (br.s., 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.45–7.47 (m, 2H) 7.38–7.42 (m, 2H), 5.05 (s, 2H), 5.00 (s, 2H), 4.41–4.53 (m, 1H), 3.54–3.65 (m, 1H), 2.93–3.07 (m, 1H), 2.70–2.82 (m, 1H), 2.50–2.54 (m, 1H), 2.44–2.54 (m, 4H), 2.43–2.47 (m, 4H), 1.77–1.87 (m, 1H), 1.63–1.74 (m, 1H), 1.29–1.42 (m, 2H) 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.1, 154.9, 154.0, 141.2, 134.9, 134.0, 129.2, 128.7, 127.4, 126.1, 121.4, 69.9, 64.7, 60.8, 48.4, 43.9, HREI-MS: m/z calculated for C25H28BCl2N3O5 [M]+ 531.1608; found 531.1606. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)-4-piperidyl]piperazine-1- carboxylate 35, Yield: 73%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.32 (br.s., 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.5–7.42 (m, 2H), 7.29–7.34 (m, 1H), 5.05 (s, 2H), 4.99 (s, 2H), 4.20–4.42 (m, 1H), 3.50–3.60 (m, 2H), 2.94–3.05 (m, 2H), 2.70–2.80 (m, 1H), 2.50–2.54 (m, 1H), 2.44–2.54 (m, 4H), 2.43–2.47 (m, 4H), 1.77–1.86 (m, 1H), 1.60–1.69 (m, 1H) 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.8, 154.0, 141.5, 138.4, 134.0, 130.5, 127.3, 126.3, 125.1, 119.5, 69.9, 64.8, 60.7, 48.3, 43.9, 28.4, HREI-MS: m/z calculated for C25H28BCl2N3O5 [M]+ 531.1608; found 531.1607. (3,5-dichlorophenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-4-carbonyl)-4-piperidyl]piperazine-1- carboxylate 36, Yield: 59%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.93 (s, 1H), 7.52–7.62 (m, 2H), 7.41 (s, 1H), 7.20–7.34 (m, 2H), 7.15–7.18 (m, 1H), 5.06 (s, 4H), 4.30–4.41 (m, 1H), 3.87–4.00 (m, 2H), 2.91–3.05 (m, 2H), 2.67–2.80 (m, 1H), 2.57–2.66 (m, 4H), 2.50–2.54 (m, 1H), 1.62–1.83 (m, 1H) 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.8, 154.0, 141.5, 138.4, 134.0, 130.5, 127.3, 126.3, 125.1, 119.5, 69.9, 64.8, 60.7, 48.3, 43.9, 28.4 HREI-MS: m/z calculated for C25H28BCl2N3O5 [M]+ 531.1608; found 531.1605. 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 (3,5-dimethylphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 44, Yield: 52%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.34 (br.s, 1H), 7.76–(d, J = 7.5 Hz, 1H), 7.56 (s, 1H), 7.47–7.55 (m, 1H), 6.88–6.97 (m, 3H), 4.92–5.03 (m, 4H), 3.70–3.82 (m, 2H), 3.37–3.45 (m, 2H), 3.23–3.32 (m, 5H), 2.24 (s, 6H), 2.15–2.25 (m, 4H), 1.96–2.17 (m, 2H), 13C NMR (DMSO-d6, 16 of 22 16 of 22 Molecules 2019, 24, 3419 400 MHz, δ ppm): 168.4, 154.5, 153.7, 137.5, 136.6, 130.5, 129.3, 125.7, 125.4, 120.0, 63.7, 62.4, 20.8, HREI-MS: m/z calculated for C26H32BN3O5 [M]+ 477.2544; found 477.2547. 400 MHz, δ ppm): 168.4, 154.5, 153.7, 137.5, 136.6, 130.5, 129.3, 125.7, 125.4, 120.0, 63.7, 62.4, 20.8, HREI-MS: m/z calculated for C26H32BN3O5 [M]+ 477.2544; found 477.2547. 400 MHz, δ ppm): 168.4, 154.5, 153.7, 137.5, 136.6, 130.5, 129.3, 125.7, 125.4, 120.0, 63.7, 62.4, 20.8, HREI-MS: m/z calculated for C26H32BN3O5 [M]+ 477.2544; found 477.2547. (3,5-dimethylphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 45, Yield: 78%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s., 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.73 (d, J = 7.8 Hz,1H), 7.47 (d, J = 7.7 Hz, 1H), 6.88–6.98 (m, 3H), 5.02 (s, 2H), 4.97 (s, 2H), 4.03–4.23 (m, 2H), 3.82–3.90 (m, 2H), 3.36–3.44 (m, 4H), 3.12–3.20 (m, 1H), 2.26–2.34 (m, 4H), 2.24 (s, 6H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.3, 156.3, 154.5, 137.4, 136.5, 131.6, 130.1, 129.2, 128.0, 125.4, 124.2, 121.5, 70.0, 66.3, 62.9, 53.6, 48.7, 20.8, HREI-MS: m/z calculated for C25H30BN3O5 [M]+ 463.2388; found 463.2390. (3,5-dimethylphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 46, Yield: 77%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.32 (br.s., 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.56 (d, J = 7.6 Hz,1H), 6.88–6.98 (m, 3H), 5.00 (s, 2H), 4.97 (s, 2H), 4.26–4.38 (m, 2H), 3.83–3.4.02 (m, 2H), 3.35–3.42 (m, 4H), 3.11–3.18 (m, 1H), 2.25–2.33 (m, 4H), 2.24 (s, 6H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.3, 156.3, 154.5, 137.4, 136.5, 131.6, 130.1, 129.2, 128.0, 125.4, 124.2, 121.5, 70.0, 66.3, 62.9, 53.6, 48.7, 20.8 HREI-MS: m/z calculated for C25H30BN3O5 [M]+ 463.2388; found 463.2391. 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 (3,5-dimethoxyphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6- carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 47, Yield: 71%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.29 (br.s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.63 (s, 1H), 7.56 (d, J = 7.6 Hz, 1H), 6.38–6.54 (m, 3H), 4.93–5.06 (m, 4H), 4.26–4.40 (m, 4H), 3.83–4.02 (m, 2H),3.73 (s, 6H), 3.35–3.42 (m, 4H), 3.11–3.18 (m, 1H), 2.25–2.33 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 160.6, 129.4, 121.6, 105.6, 99.5, 69.9, 64.0, 55.4, 50.8, HREI-MS: m/z calculated for C26H32BN3O7 [M]+ 509.2442; found 509.2444. (3,5-dimethoxyphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5- carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 48, Yield: 66%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.27 (br.s, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.64 (s, 1H), 7.46–7.63 (m, 1H), 6.45–6.53 (m, 2H), 6.43 (s, 1H), 4.93–5.08 (m, 4H), 3.71–3.75 (m, 2H), 3.70 (s,6H), 3.46–3.68 (m, 6H), 3.18–3.29 (m, 1H), 2.33–2.46 (m, 4H), 1.97–2.18 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 160.3, 130.7, 130.3, 125.6, 125.4, 120.0, 106.8, 105.3, 99.3, 55.1, HREI-MS: m/z calculated for C26H32BN3O7 [M]+ 509.2442; found 509.2444. (3,5-dimethoxyphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 49, Yield: 83%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.27 (br.s., 1H), 7.99–8.01 (m, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 6.41–6.50 (m, 3H), 5.00 (s, 2H), 4.99 (s, 2H), 4.26–4.33 (m, 4H), 3.84–3.91 (m, 2H), 3.72 (s, 6H), 3.35–3.48 (m, 4H), 3.12–3.20 (m, 1H), 2.25–2.35 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 160.6, 156.4, 154.5, 139.3, 134.3, 131.8, 130.1, 130.0, 129.5, 129.1, 121.5, 105.2, 99.4, 55.2, 54.4, 53.5, HREI-MS: m/z calculated for C25H30BN3O7 [M]+ 495.2286; found 495.2285. ((3,5-dimethoxyphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5- carbonyl)azetidin-3-yl]piperazine-1- carboxylate 50, Yield: 73%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.34 (br.s., 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.65 (s, 1H), 7.59 (d, J = 7.4 Hz, 1H), 6.42–6.51 (m, 3H), 5.00 (s, 2H), 4.99 (s, 2H), 4.23–4.31 (m, 2H), 3.83–3.91 (m, 2H), 3.72 (s, 6H), 3.34–3.46 (m, 4H), 3.14–3.20 (m, 1H), 2.23–2.36 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.2, 160.5, 154.3, 139.4, 134.9, 130.6, 126.2, 120.7, 106.7, 105.8, 99.3, 69.9, 66.0, 55.1, 53.6, 48.7, 43.3, HREI-MS: m/z calculated for C25H30BN3O7 [M]+ 495.2286; found 495.2286. 4.2.3. General Procedure for the Preparation of Novel Autotaxin Inhibitors 105.1, 99.3, 66.0, 64.3, 55.2, 41.1, 48.3, 28.5 HRMS: m/z calculated for C18H2 found 350 2089 benzyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 40, Yield: 71%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.30 (br.s, 1H), 7.71–7.81 (m, 1H), 7.48–7.53 (m, 1H), 7.40–7.47 (m, 1H), 7.27–7.38 (m, 5H), 5.05 (s, 2H), 5.00 (s, 2H), 3.72–3.79 (m, 2H), 3.58–3.67 (m, 2H), 3.45–3.53 (m, 4H), 3.38–3.44 (m, 4H), 3.19–3.29 (m, 1H), 2.06–2.28 (m, 2H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.4, 154.5, 154.0, 138.6, 137.0, 132.0, 128.5, 127.8, 127.5, 125.6, 120.0, 69.9, 66.1, 63.6, 62.3, 50.7, 43.4, HREI-MS: m/z calculated for C24H28BN3O5 [M]+ 449.2231; found 449.2243. benzyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 41, Yield: 87%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.31 (br.s., 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.65 (s, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.29–7.39 (m, 5H), 5.07 (s, 2H), 5.03 (s, 2H), 4.03–4.15 (m, 2H), 3.85–3.91 (m, 1H), 3.39–3.46 (m, 4H), 3.14–3.21 (m, 1H), 2.25–2.34 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.5, 156.5, 154.5, 137.1 132.0, 130.2, 128.5, 127.8, 127.6, 121.5, 69.9, 66.1, 57.1, 53.5, 52.9, 48.7, 43.1, HREI-MS: m/z calculated for C23H26BN3O5 [M]+ 435.2075; found 435.2079. benzyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-5-carbonyl)azetidin-3-yl]piperazine-1- carboxylate 42, Yield: 53%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.33 (br.s., 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.65 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.29–7.39 (m, 5H), 5.07 (s, 2H), 5.03 (s, 2H), 4.04–4.14 (m, 2H), 3.85–3.90 (m, 2H), 3.37–3.45 (m, 4H), 3.12–3.19 (m, 1H), 2.24–2.35 (m, 4H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 169.1, 154.5, 153.9, 136.8, 135.0, 134.4, 130.5, 128.5, 127.9, 126.4, 120.4, 69.9, 66.2, 56.5, 53.6, 52.2, 48.7, 43.1, HREI-MS: m/z calculated for C23H26BN3O5 [M]+ 435.2075; found 435.2078. (3,5-dimethylphenyl)methyl 4-[1-(1-hydroxy-3H-2,1-benzoxaborole-6-carbonyl)pyrrolidin-3-yl]piperazine-1- carboxylate 43, Yield: 79%, 1H NMR (DMSO-d6, 400 MHz, δ ppm): 9.32 (br.s, 1H), 7.79–7.92 (m, 1H), 7.57–7.68 (m, 1H), 7.45 (d, J = 7.8 Hz, 1H), 6.91–7.02 (m, 3H), 5.01 (s, 2H), 4.97 (s, 2H), 3.69–3.82 (m, 2H), 3.54–3.67 (m, 4H), 3.32–3.51 (m, 2H), 2.17–2.38 (m, 1H), 1.97–2.06 (m, 1H), 13C NMR (DMSO-d6, 400 MHz, δ ppm): 168.5, 155.4, 137.5, 136.6, 129.6, 129.3, 125.5, 121.3, 63.8, 62.3, HREI-MS: m/z calculated for C26H32BN3O5 [M]+ 477.2544; found 477.2546. 4.4. Biochemical Enzyme Assay Benzoxaborole derivatives were tested in the choline detection assay for ATX inhibition measuring ATX activity using LysoPC (16:1) as a substrate (Figure 9). Mother plates with serial dilutions of compounds in pure DMSO were prepared from 10 mM DMSO stock solutions. Storplate-384-deep-well-V plates (Perkin Elmer) were also used for mother plate preparation. Compounds were diluted in a DMSO in a ratio of 1:3. An aliquot of 200 nL of compound solution was transferred from the mother plate to the test plate with a Mosquito nanoliter liquid handling system (TTP labtech). The assay was performed in Spectra-Plate-384 TC, transparent, (Perkin Elmer, Cat#6007658) using a 20 µL assay volume. The total ﬁnal percentage of DMSO in the test media was 1%. Compounds were tested at 11 consecutive three-fold dilutions starting from 50 µM (ﬁnal concentrations) in duplicate, covering range of 0.0008–50 µM to determine IC50 values. The control compound (HA 155) was tested on every test plate. After dispensing of compounds directly to the dry plate, we added 10 µL of recombinant ATX (2 ng/mL) in a Tris–HCl buﬀer (140 mM NaCl, 1mM MgCl2, 5 mM KCl, 3 mM CaCl2, 1 mg/ml bovin serum albumin (BSA, fatty acid free) and 100 mM Tris–HCl, pH 9). Finally, 10 µL of 200 µM LysoPC (16:1) in a Tris–HCl buﬀer was added to each well, while the plate was incubated 90 min at 37 ◦C. The above-described solution with dimethyl sulfoxide but without the test compound was used as a control. LysoPC without ATX was used as a control for the autohydrolysis of LysoPC. After 1.5 h of incubation, 20 µL of stop solution and developer was added {Choline oxidase (1.2 U/mL), Peroxidase (1.2 U/mL), TOOS (2.7 mM), 4-Aminoantipyrine (2.7 mM), and EDTA (50 mM)} to the reaction mixture, and absorbance was measured after 30 min of incubation. The absorbance was measured by using Perkin-Elmer EnVision plate reader (λ = 555 nm). Data were analyzed using GraphPad Prism software. The calculation of IC50 data, curves and quality control (QC) analysis was made by using Excel tools and GraphPadPrism software. Brieﬂy, individual concentration–eﬀect curves were generated by plotting the logarithm of the tested concentration of tested compounds (X) vs. corresponding percentage inhibition values (Y) using a least squares (ordinary) ﬁt. Best ﬁt IC50 values were calculated using the Log (inhibitor) vs. response – variable slope (four parameters) equation, where Y = 100/(1 + 10ˆ((LogIC50–X)*HillSlope)). 4.3. Molecular Modelling Studies All visualizations and molecular modelling studies were done using Schrodinger Suite 2018-1 [19]. The co-crystal structure of ATX in complex with the HA155 boronic acid inhibitor (PDB ID: 2XRG) [9] was used as a template for the docking studies due to the similarity between HA155 and examined molecules. The protein was prepared (modelled) using Protein Preparation Wizard (with Epik v4.3, Impact v7.8, and Prime v5.1) using default settings. Missing hydrogen atoms and sidechains were Molecules 2019, 24, 3419 17 of 22 added, the hydrogen bond network was optimized, and the structure was minimized using the OPLS-2005 force ﬁeld [38]. The initial neutral 3D structures of ligands were generated by LigPrep and OPLS2005 force-ﬁeld was used. Water molecules were removed from the protein structure, and zinc ions of the active site were retained within the structure for docking studies. Docking was performed using Covalent Docking v1.3 [15] within Glide v7.8, where Thr209 of ATX was selected as a reactive residue in the formation of covalent bond in the reaction of boron acid addition [20]. Default values were used for the receptor grid generation around HA155 ligand in the binding site. Performance of the covalent docking was tested by re-docking of HA155 into ATX (Figure 5). This veriﬁed protocol was used for the docking of boronate compounds in this paper. concentrations dose respons 4.5. Lipophilicity Determination 4.5. Lipophilicity Determination Chrom logD values were determined from the following equation: Chrom logD = 0.0857 x CHI – 2 [39]. Chromatographic hydrophobic index (CHI) values were determined from the gradient retention times (RT). These values approximately corresponded to the volume percentage of organic component in the mobile phase when the compound elutes. CHI values were determined at pH 7.4. Chromatograms were obtained using on Agilent 1100 Series liquid chromatography system with an HPLC diode-array detector (DAD) coupled with a Micromass Quattro micro atmosphere pressure ionization (API) mass spectrometer. The column used for analyses was Phenomenex Luna C18 (50 x 3 mm, 5 µm, 100 Å). The column was maintained at 25 °C. The flow rate was 1.0 ml/min. The mobile phase was composed of 50 mM ammonium acetate in water at pH 7.4 (solvent A) and acetonitrile (solvent B). The solvent gradient condition was initially 0.5% B, and then it increased linearly to 100% Chrom logD values were determined from the following equation: Chrom logD = 0.0857 × CHI −2 [39]. Chromatographic hydrophobic index (CHI) values were determined from the gradient retention times (RT). These values approximately corresponded to the volume percentage of organic component in the mobile phase when the compound elutes. CHI values were determined at pH 7.4. Chromatograms were obtained using on Agilent 1100 Series liquid chromatography system with an HPLC diode-array detector (DAD) coupled with a Micromass Quattro micro atmosphere pressure ionization (API) mass spectrometer. The column used for analyses was Phenomenex Luna C18 (50 × 3 mm, 5 µm, 100 Å). The column was maintained at 25 ◦C. The ﬂow rate was 1.0 mL/min. The mobile phase was composed of 50 mM ammonium acetate in water at pH 7.4 (solvent A) and acetonitrile (solvent B). The solvent gradient condition was initially 0.5% B, and then it increased linearly to 100% B over 3.0 min, remaining at 100% B for 0.5 min, then decreasing linearly to 0% B over 0.2 min followed by an equilibration period of 1.3 min prior to the next injection. The total experimental duration was 5 min. Injection volume was 2.0 µl. Samples temperature was maintained at 15 ◦C in the autosampler chamber. A group of ten standard compounds having diﬀerent CHI values was used for calibration of RP-HPLC [40]. 4.4. Biochemical Enzyme Assay QC criteria parameters (Z,’ S:B, ∆mP) were checked for every plate. 18 of 22 pe)). QC Molecules 2019, 24, 3419 – variable slope (fo it i t Figure 9. ATX inhibition results of biochemical testing of new benzoxaborole inhibitors. 11— concentrations dose–response curves, as average of duplicates, are presented Figure 9. ATX inhibition results of biochemical testing of new benzoxaborole inhibitors. 11—concentrations dose–response curves, as average of duplicates, are presented. Figure 9. ATX inhibition results of biochemical testing of new benzoxaborole inhibitors. 11— concentrations dose response curves as average of duplicates are presented Figure 9. ATX inhibition results of biochemical testing of new benzoxaborole inhibitors. 11—concentrations dose–response curves, as average of duplicates, are presented. 4.6. Kinetic Solubility Turbidimetric solubility allows for a rapid determination of kinetic solubility by using small amounts of the test compound. Assay controls were α-naphtoﬂavone (low solubility) and sulfaphenazole (expected solubility >100 µM). DMSO stock solutions (10 mM) of each compound were ﬁrst serially diluted 1 in 3 in DMSO and then spiked into an aqueous buﬀer (100 mM PBS), resulting in 5 following ﬁnal concentration: 100, 30, 10, 3 and 1 µM (each concentration was tested in duplicate). The prepared solutions were then incubated at 37 ◦C for 2 h with gentle shaking, followed by absorbance measurements on microplate reader (Inﬁnite F500, Tecan, CH) at 620 nm (at t = 0 and t = 2 h). Absorbance is proportionally increased with the concentration of insoluble particles as compound precipitates. Compound samples were compared to a solvent control in an aqueous buﬀer (DMSO 1% ﬁnal concentration), and a signiﬁcant increase of sample absorbance was considered to have occurred when the absorbance reached a 3-fold standard deviation of the average DMSO absorbance. Molecules 2019, 24, 3419 19 of 22 The calculation of solubility range data was made by Excel tool software. Results are expressed as an estimated solubility range. The calculation of solubility range data was made by Excel tool software. Results are expressed as an estimated solubility range. 4.8. In Vitro Metabolic Stability in Rat and Liver Human Microsomes 4.8. In Vitro Metabolic Stability in Rat and Liver Human Microsomes Metabolic stability was assessed in rat and human liver microsomes, with testosterone and propranolol as positive controls. All compounds, at a ﬁnal concentration of 1 µM (0.03% DMSO), were incubated in a phosphate buﬀer (50 mM, pH 7.4) for 60 min at 37 ◦C, together with liver microsomes in the absence and presence of NADPH generating system (nicotinamide adenine dinucleotide phosphate (NADP, 2 mM), glucose-6-phosphate (20 mM), glucose-6-phosphate dehydrogenase (6U) and magnesium chloride (2 mM)). Aliquots were taken at diﬀerent time points (0, 10, 20, 30, 45 and 60 min), followed by reaction termination by addition of CH3CN:CH3OH 2:1 v/v, containing diclofenac as internal standard (IS). Samples were then centrifuged (at 2000 rpm at 4 ◦C for 30 min), and resulting supernatants were subjected to LC/MS/MS analysis. The in vitro half-life was calculated in GraphPadPrism software from % remaining vs. time regression using non-linear regression ﬁt. In vitro intrinsic clearance, expressed as mL/min/g liver, was determined from in vitro half-life and normalized for the protein concentration in the incubation mixture, assuming a 52.5 mg/g liver. Finally, predicted in vivo hepatic clearance, expressed as %LBF (liver blood ﬂow), was determined from in vitro intrinsic clearance normalized for the weight over whole body weight (87.5 g/kg for mouse and 25.7 g/kg for human) and liver blood ﬂow (131 mL/min/kg for mouse and 21 mL/min/kg for human). 4.9. Plasma Protein Binding Assay Plasma protein binding was measured using the equilibrium dialysis technique. Positive controls included in this assay were: Acebutolol (low binding) and nicardipine (very high binding). The assay was performed in a 96-well Teﬂon dialysis unit, where each well consisted of two chambers separated by a dialysis membrane. Plasma (human and rat, commercially obtained from Seralab) spiked with each compound (ﬁnal concentration: 5 µM, 0.5% DMSO) were added to one chamber, and a blank buﬀer solution (60 mM Na2HPO4/14 mM KH2PO4/70 mM NaCl; pH 7.4) was added to the other side of the well. Each compound was analyzed in duplicate, for 4 h at 37 ◦C. After 4 h, both the plasma (free and bound fraction) and buﬀer (only free fraction) side of the well were sampled, matrix matched, then precipitated with a STOP solution (CH3CN:CH3OH 2:1 v/v, containing diclofenac as internal standard), and centrifuged at 2000 rpm for 30 min (+4 ◦C). The resulting supernatants were analyzed by LC-MS/MS. The fraction bound was calculated as the concentration diﬀerence (analyte vs. internal standard peak area ratio) in the plasma and buﬀer sides divided by the total concentration in the plasma side. Sampling was also performed from spiked plasma at t = 0 min in order to determine recovery, i.e., % of compound detected at t = 4 h in both the plasma and buﬀer chambers divided by the compound detected at t = 0 min. 4.7. Plasma Stability The in vitro plasma stability of the test compounds was studied in human and rat plasma with propranolol, benﬂorex, and eucatropine as control substances. All compounds at ﬁnal concentrations of 5 µM (0.5% DMSO) were incubated in a phosphate buﬀer (50 mM, pH 7.4) for 4 h at 37 ◦C together with plasma. Aliquots were taken at diﬀerent time points (0, 30, 120 min and 4 h), followed by reaction termination by the addition of CH3CN:CH3OH 2:1 v/v., which contained diclofenac and warfarin as internal standards (IS). Samples were then centrifuged (at 1600 rpm at 4 ◦C for 30 min), and the resulting supernatants were subjected to LC/MS/MS analysis. Results are expressed as % remaining. 5. Conclusions Our strategy was to target the Thr209 and zinc atoms in the binding pocket of ATX, with the boronic acid isostere: Borinic acid. As well as similar enzyme potencies, we expected better ADME properties of such modiﬁed compounds. The group occupying the hydrophobic lipid-binding pocket 20 of 22 Molecules 2019, 24, 3419 was optimally that of the Pﬁzer molecule PF-8380. As the core spacer, we used saturated bicyclic systems (piperazino–piperidine, piperazino–pyrolidine and piperazino azetidine). This overall design was ratiﬁed by docking experiments, and the molecules were then synthesized (compounds 30–50). ATX inhibition in vitro and ADME properties were measured aiming for better overall proﬁle than that possessed by HA130 and HA155. The compounds HA130 and HA155 were reported to be active in the in vivo study, lowering LPA signiﬁcantly but with a very short half-life (less than ﬁve min) [10]. The four new compounds designed, synthesized and tested in this work (30, 32, 34, and 37) showed ATX inhibition in the nanomolar range with the IC50 value for the most active compound 37 being 130 nM, which is only two times less potent than HA155. Compound 37 has a core spacer more rigid than in HA155, designed for better stability. Most of ADME parameters for tested compounds were better than for HA155. The plasma stability for compound 37 showed 71.2% and 79.1% remaining in rat and human plasma, respectively, after four hours. HA155 showed 65.7% and 42.9% in rat and human plasma, respectively, also measured at four hours. Furthermore, the PPB of 37 was signiﬁcantly lower than for HA155 (fraction unbound for 37 was 1.6% in rat and 2.1% in human plasma, while for HA155 was 0.4% and less than 0.1%, respectively). Based on our ﬁndings, we have identiﬁed potent ATX inhibitors with in vitro enzyme inhibition potencies similar to that of HA155 but with an improved ADME proﬁle. 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https://openalex.org/W4288453046	https://ejournals.epublishing.ekt.gr/index.php/psychology/article/download/30686/23716	Greek, Modern	null	Η κατανόηση της πυκνής διάταξης των ρητών από μαθητές Β΄ Λυκείου: ένα διδακτικό πείραμα	Psychologia: to periodiko tīs Ellīnikīs Psychologikīs Etaireias/Psychologia. To periodiko tīs Ellīnikīs Psychologikīs Etaireias	2,022	cc-by-sa	10,997	Psychology: the Journal of the Hellenic Psychological Society Vol 27, No 1 (2022) Special Section: Learning Counter-intuitive Explanations from a Conceptual Change Perspective How 10th graders understand the dense ordering of rational numbers: a teaching experiment Dimitris Fokas, Xenia Vamvakoussi doi: 10.12681/psyhps.30686 Copyright © 2022, Δημήτρης Φωκάς, Ξένια Βαμβακούση This work is licensed under a Creative Commons Attribution-ShareAlike 4.0. Fokas, D., & Vamvakoussi, X. (2022). How 10th graders understand the dense ordering of rational numbers: a teaching experiment . Psychology: The Journal of the Hellenic Psychological Society, 27(1), 48–65. https://doi.org/10.12681/psyhps.30686 ΣΤΟΙΧΕΙΑ ΕΠΙΚΟΙΝΩΝΙΑΣ Ξένια Βαμβακούση, Πανεπιστήμιο Ιωαννίνων Παιδαγωγικό Τμήμα Νηπιαγωγών, Μεταβατικό Κτήριο, 45110, Πανεπιστημιούπολη Ιωάννινα xvamvak@uoi.gr Δημήτρης ΦΩΚΑΣ1, Ξένια ΒΑΜΒΑΚΟΥΣΗ2 Δημήτρης ΦΩΚΑΣ1, Ξένια ΒΑΜΒΑΚΟΥΣΗ2 1 Παιδαγωγικό Τμήμα Δημοτικής Εκπαίδευσης, Πανεπιστήμιο Δυτικής Μακεδονίας, Φλώρινα, Ελλάδα 2 Παιδαγωγικό Τμήμα Νηπιαγωγών, Πανεπιστήμιο Ιωαννίνων, Ιωάννινα, Ελλάδα ΠΕΡΙΛΗΨΗ Η μετάβαση από τους φυσικούς στους ρητούς αριθμούς παρουσιάζει δυσκολίες για τους μαθητές, μέρος των οποίων οφείλεται στην καταχρηστική μεταφορά γνώσης για τους φυσικούς στους ρητούς. Μια ιδιότητα των φυσικών αριθμών που μεταφέρεται καταχρηστικά στους ρητούς είναι η διακριτότητα: Αντίθετα με το σύνολο των ρητών αριθμών, το σύνολο των φυσικών αριθμών είναι διακριτά διατεταγμένο, δηλαδή, για κάθε φυσικό αριθμό ορίζεται ο επόμενός του. Οι ρητοί αριθμοί είναι πυκνά διατεταγμένοι, δηλαδή, δεν ορίζεται ο επόμενος για κανέναν αριθμό στο σύνολο αυτό. Η προσέγγιση της θεωρίας πλαισίου στην εννοιολογική αλλαγή προβλέπει ότι οι θεμελιώδεις παραδοχές των μαθητών για τον αριθμό ως φυσικό δεν αίρονται δια μιάς, με την ιδέα της διακριτότητας να είναι ιδιαίτερα ανθεκτική. Στην εργασία παρουσιάζεται ένα διδακτικό πείραμα με 15 μαθητές Λυκείου. Εξετάσαμε την υπόθεση ότι διαφορετικές πτυχές της πυκνότητας των ρητών αριθμών (συγκεκριμένα, η απειρία των ρητών σε οποιοδήποτε διάστημα και η μη ύπαρξη του επόμενου), παρότι μαθηματικά ισοδύναμες, παρουσιάζουν διαφορετικές δυσκολίες για τους μαθητές. Η αρχική κατανόηση των μαθητών για την πυκνότητα ελέγχθηκε ατομικά. Κάθε μαθητής ξεχωριστά συμμετείχε σε μικρής διάρκειας παρέμβαση, στην οποία εισήχθη η ιδέα του αριθμητικού μέσου ενός διαστήματος ως εργαλείο που δυνητικά μπορεί να οδηγήσει στην κατανόηση και των δύο πτυχών της πυκνής διάταξης. Τέλος, κάθε μαθητής επανεξέτασε τις απαντήσεις του στα έργα του προελέγχου και παρακινήθηκε να αξιοποιήσει την ιδέα του αριθμητικού μέσου. Τα αποτελέσματα δείχνουν ότι οι μαθητές συνέχισαν να θεωρούν ότι υπάρχει ο επόμενος στους ρητούς, ακόμα και όταν συμπέραναν την απειρία των ενδιάμεσων. Υπό το πρίσμα της θεωρίας που υιοθετήσαμε, η αντίληψη αυτή είναι συνθετική, καθώς οι μαθητές αλλάζουν μερικώς την αντίληψή τους για τη διάταξη των ρητών, χωρίς να αίρεται η αρχή του επόμενου αριθμού. Ρητοί αριθμοί, πυκνή διάταξη, εννοιολογική αλλαγή, συνθετικά μοντέλα To cite this article: Fokas, D., & Vamvakoussi, X. (2022). How 10th graders understand the dense ordering of rational numbers: a teaching experiment . Psychology: The Journal of the Hellenic Psychological Society, 27(1), 48–65. https://doi.org/10.12681/psyhps.30686 https://epublishing.ekt.gr \| e-Publisher: EKT \| Downloaded at: 24/10/2024 08:17:17 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 © 2022, Δημήτρης Φωκάς, Ξένια Βαμβακούση Άδεια CC-BY-SA 4.0 Η προσέγγιση της θεωρίας πλαισίου στην εννοιολογική αλλαγή Η βασική υπόθεση της προσέγγισης της θεωρίας πλαισίου στην εννοιολογική αλλαγή είναι ότι τα μικρά παιδιά ερμηνεύουν και οργανώνουν τις εμπειρίες τους στο πλαίσιο του φυσικού και κοινωνικο-πολιτισμικού τους περιβάλλοντος σε λίγα, σχετικά συνεκτικά, επεξηγηματικά πλαίσια, τις λεγόμενες «θεωρίες πλαισίου». Μια τέτοια θεωρία πλαισίου αφορά τις πρώιμες εμπειρίες ποσοτικοποίησης των παιδιών οι οποίες, κατά κανόνα, αφορούν το διακριτό μέγεθος του πλήθους και άρα συνδέονται από νωρίς με τις διακριτές ποσότητες και τους φυσικούς αριθμούς (Vamvakoussi & Vosniadou, 2010· Vosniadou et al., 2008· Βαμβακούση, 2019). Πράγματι, πριν ακόμα ξεκινήσουν τη σχολική τους εκπαίδευση, τα παιδιά έχουν ήδη εκτεθεί σε συμβολικά (λεκτικά ή μη) εργαλεία (π.χ., αριθμολέξεις, αριθμητικά σύμβολα), δραστηριότητες (π.χ., σύγκριση και καταμέτρηση διακριτών συλλογών αντικειμένων) και πρακτικές (π.χ., χρήση των δακτύλων στην καταμέτρηση) που αφορούν τους φυσικούς αριθμούς και αναδεικνύουν τη διακριτή τους φύση. Οι αρχικές αυτές εμπειρίες για τον αριθμό εμπλουτίζονται και συστηματικοποιούνται περαιτέρω στα πρώτα χρόνια της σχολικής εκπαίδευσης, που δίνει μεγάλη έμφαση στους φυσικούς αριθμούς, τις ιδιότητές τους, τις σχέσεις τους (π.χ., σχέσεις διάταξης και κυρίως προσθετικές σχέσεις) και τις αριθμητικές πράξεις που τους αφορούν. Έτσι, πριν την εισαγωγή των μη φυσικών αριθμών στη διδασκαλία, συνήθως με τη μορφή κλασμάτων, έχουν ήδη εγκαθιδρυθεί αρχικές θεωρίες για τον αριθμό, στο πλαίσιο των οποίων ο αριθμός λειτουργεί ως φυσικός αριθμός. Θεμελιώδες χαρακτηριστικό των αριθμών στο πλαίσιο αυτό είναι η διακριτότητα. Το χαρακτηριστικό αυτό συνάδει με όλες τις εμπειρίες των παιδιών σχετικά με την πληθικότητα διακριτών συλλογών αντικειμένων και, κυρίως, με τα συμβολικά εργαλεία που αφορούν τους αριθμούς, όπως η ακολουθία των αριθμολέξεων (ένα, δύο, τρία,…) και η αριθμητική ακολουθία (1, 2, 3,...). Η ιδέα της διακριτότητας των αριθμών θεωρείται ως θεμελιώδης παραδοχή των αρχικών θεωριών των παιδιών για τον αριθμό (Vamvakoussi & Vosniadou, 2010). Η προσέγγιση της θεωρίας πλαισίου στην εννοιολογική αλλαγή υποθέτει ότι οι μαθητές βασίζονται στις αρχικές τους θεωρίες για τον αριθμό προκειμένου να ερμηνεύσουν τις νέες πληροφορίες για τους ρητούς, στις οποίες εκτίθενται κατά τη διδασκαλία. Πολλές από τις πληροφορίες αυτές δεν είναι συμβατές με την αρχική τους θεωρία για τον αριθμό. Παρόμοια με άλλες προσεγγίσεις στην εννοιολογική αλλαγή, η προσέγγιση της θεωρίας πλαισίου προβλέπει συστηματικά λάθη στις περιπτώσεις αυτές. Επιπλέον, η προσέγγιση της θεωρίας πλαισίου επιχειρεί να περιγράψει τη διαδικασία της εξέλιξης των αρχικών θεωριών πλαισίου των παιδιών για τον αριθμό, υπό την επίδραση της διδασκαλίας (Βαμβακούση, 2019). ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) Παράλληλα, οι ρητοί αριθμοί προκαλούν τεράστιες δυσκολίες στους μαθητές, σε όλες τις εκπαιδευτικές βαθμίδες. Συστηματικά λάθη των μαθητών έχουν τεκμηριωθεί όσον αφορά τη σύγκριση, τη διάταξη, τις πράξεις και την ερμηνεία των αναπαραστάσεων των ρητών αριθμών, καθώς και την επίλυση προβλημάτων σε καταστάσεις όπου εμπλέκονται ρητοί. Ένα μεγάλο μέρος αυτών των δυσκολιών αποδίδεται στην καταχρηστική μεταφορά της προϋπάρχουσας γνώσης για τους φυσικούς αριθμούς στο πλαίσιο των ρητών αριθμών (Moss, 2005). Πράγματι, μια μεγάλη ποικιλία συστηματικών λαθών παρατηρείται στα σημεία εκείνα στα οποία οι διαφορές μεταξύ των φυσικών και των ρητών καθιστούν την εφαρμογή της προϋπάρχουσας γνώσης για τους φυσικούς προβληματική. To φαινόμενο αυτό είναι τόσο εκτεταμένο, ώστε του έχει αποδοθεί ο όρος προκατάληψη του ακεραίου (Ni & Zhou, 2005) ή του φυσικού αριθμού (Vamvakoussi et al., 2012). Στο άρθρο αυτό εστιάζουμε στην κατανόηση της πυκνής διάταξης των ρητών αριθμών, μιας ιδιότητας που αντανακλά μια θεμελιώδη διαφορά μεταξύ των φυσικών και των ρητών αριθμών. Υιοθετώντας την οπτική της προσέγγισης της «θεωρίας πλαισίου» στην εννοιολογική αλλαγή (Vosniadou et al., 2008· Vosniadou, 2013), πραγματοποιήσαμε ένα διδακτικό πείραμα για να εξετάσουμε τον τρόπο με τον οποίο μαθητές της Β΄ Λυκείου αντιλαμβάνονται τη διάταξη των ρητών αριθμών, πριν και αφού εκτεθούν στην ιδέα του αριθμητικού μέσου σε ένα διάστημα με άκρα ρητούς αριθμούς καθώς και στη διαδικασία υπολογισμού του. Εισαγωγή Η χρονική στιγμή στην οποία εισάγονται οι ρητοί αριθμοί στο σχολείο είναι ένα ορόσημο για τη μελλοντική πορεία των παιδιών στα σχολικά μαθηματικά. Οι ρητοί αποτελούν μέρος ενός εκτεταμένου και πολύπλοκου δικτύου μαθηματικών εννοιών και σχέσεων που περιλαμβάνει τον πολλαπλασιασμό και τη διαίρεση, το λόγο και την αναλογία, τις γραμμικές συναρτήσεις, τη διαστατική ανάλυση και δεν εξαντλείται σε αυτά (Vergnaud, 1996). Ως εκ τούτου, η κατανόηση των ρητών με τις διάφορες αναπαραστάσεις τους, καθώς και των καταστάσεων στις οποίες βρίσκουν εφαρμογές αφορά ένα μεγάλο μέρος του περιεχομένου των σχολικών μαθηματικών σε διάφορες περιοχές, όπως για παράδειγμα τα στοχαστικά μαθηματικά, τη γεωμετρία και την άλγεβρα. Δεν αποτελεί έκπληξη, επομένως, το γεγονός ότι η επίδοση στους ρητούς προβλέπει τη γενικότερη επίδοση στα μαθηματικά (Bailey et al., 2012· Booth & Newton, 2012· Torbeyns et al., 2015). © 2022, Δημήτρης Φωκάς, Ξένια Βαμβακούση Άδεια CC-BY-SA 4.0 © 2022, Δημήτρης Φωκάς, Ξένια Βαμβακούση Άδεια CC-BY-SA 4.0 Η πυκνή διάταξη των ρητών Η διάταξη των φυσικών και, γενικότερα, των ακεραίων, αριθμών είναι διακριτή. Ανάμεσα σε δύο διαφορετικούς φυσικούς αριθμούς υπάρχει πεπερασμένο πλήθος φυσικών αριθμών. Διαφορετικά, για κάθε φυσικό αριθμό ορίζεται ο (μοναδικός) επόμενός του, ο οποίος είναι κατά μία μονάδα μεγαλύτερος από το δεδομένο φυσικό. Η ιδιότητα αυτή είναι γνωστή και ως «αρχή του επόμενου». Η αρχή του επόμενου γίνεται αντιληπτή από νωρίς. Ακόμα και παιδιά της πρωτοσχολικής ηλικίας είναι σε θέση να την αξιοποιήσουν για να καταλήξουν στο συμπέρασμα ότι δεν υπάρχει «ο μεγαλύτερος φυσικός αριθμός» (Hartnett & Gelman, 1998). Αντίθετα με το σύνολο των φυσικών, το σύνολο των ρητών αριθμών είναι πυκνά διατεταγμένο. Ανάμεσα σε οποιουσδήποτε δύο διαφορετικούς ρητούς αριθμούς υπάρχει πάντα ένας, άρα τελικά άπειροι αριθμοί. Ισοδύναμα, στο σύνολο των ρητών αριθμών παύει να ισχύει η αρχή του επόμενου. Η πυκνότητα είναι, θεωρητικά, προσπελάσιμη με μαθηματικές γνώσεις και εργαλεία που είναι διαθέσιμα στους μαθητές ήδη από την πρωτοβάθμια εκπαίδευση. Για παράδειγμα, μπορεί κανείς να συμπεράνει ότι ανάμεσα σε δύο δεκαδικούς (π.χ., 0,5 και 0,6) υπάρχουν κι άλλοι αριθμοί, προσθέτοντας ένα μηδενικό στο δεκαδικό τους μέρος (π.χ., 0,50 και 0,60). Παρόμοια, μπορεί κανείς να συμπεράνει ότι ανάμεσα σε δύο κλάσματα (π.χ., 2/5 και 3/5) υπάρχουν κι άλλοι αριθμοί, μετατρέποντάς τα σε ισοδύναμα κλάσματα με μεγαλύτερους όρους (π.χ., 4/10 και 6/10). Η διαδικασία αυτή μπορεί να επαναληφθεί για οποιουσδήποτε από τους ενδιάμεσους αριθμούς που προκύπτουν και να συνεχιστεί επ’ άπειρο, παράγοντας άπειρους ενδιάμεσους στους αρχικούς αριθμούς. Στην πραγματικότητα, ωστόσο, η κατανόηση της πυκνής διάταξης των ρητών είναι ιδιαίτερα απαιτητική. Πλήθος ερευνών τεκμηριώνουν ότι η διακριτότητα --θεμελιώδης ιδιότητα των φυσικών αριθμών-- αποδίδεται καταχρηστικά στους ρητούς (και τους πραγματικούς) αριθμούς από μαθητές στην πρωτοβάθμια και τη δευτεροβάθμια εκπαίδευση (Malara, 2001· McMullen et al., 2015· Merenluoto & Lehtinen, 2002· Neumann, 1998· Hannula et al., 2006· Smith, 1995· Smith et al., 2005), από φοιτητές (Vamvakoussi et al., 2012), ακόμα και από φοιτητές σε τμήματα Μαθηματικών (Giannakoulias et al., 2007), καθώς και από μελλοντικούς εκπαιδευτικούς (Depaepe et al., 2015· Tirosh et al., 1999). Υιοθετώντας την προσέγγιση της θεωρίας πλαισίου στην εννοιολογική αλλαγή, οι Vamvakoussi και Vosniadou (2004, 2007, 2010) υπέθεσαν ότι η ιδέα της διακριτότητας δεν αίρεται δια μιας και προέβλεψαν την ύπαρξη ενδιάμεσων επιπέδων κατανόησης της πυκνής διάταξης των ρητών, τα οποία και τεκμηρίωσαν δείχνοντας ότι η κατανόηση της απειρίας των ενδιάμεσων σε ένα διάστημα με άκρα ρητούς αριθμούς εξαρτάται από το είδος των άκρων. ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 οι νέες πληροφορίες είναι ασύμβατες με την προϋπάρχουσα δομή, η αφομοίωσή τους διασπά τη συνοχή της δομής και προκαλεί εσωτερική ασυνέπεια και παρανοήσεις. Κατά τη διάρκεια αυτής της αργής και σταδιακής διαδικασίας, εμφανίζεται ένας ιδιαίτερος τύπος παρανοήσεων, οι συνθετικές αντιλήψεις ή συνθετικά μοντέλα. Αυτού του είδους οι παρανοήσεις αντανακλούν την αφομοίωση της νέας πληροφορίας, χωρίς την άρση των αρχών και των παραδοχών της αρχικής θεωρίας πλαισίου. Η υπόθεση αυτή έχει οδηγήσει στη διατύπωση νέων προβλέψεων για την ανάπτυξη της κατανόησης για τους ρητούς και, ειδικότερα, για τη διάταξή τους (Βαμβακούση, 2019). οι νέες πληροφορίες είναι ασύμβατες με την προϋπάρχουσα δομή, η αφομοίωσή τους διασπά τη συνοχή της δομής και προκαλεί εσωτερική ασυνέπεια και παρανοήσεις. Κατά τη διάρκεια αυτής της αργής και σταδιακής διαδικασίας, εμφανίζεται ένας ιδιαίτερος τύπος παρανοήσεων, οι συνθετικές αντιλήψεις ή συνθετικά μοντέλα. Αυτού του είδους οι παρανοήσεις αντανακλούν την αφομοίωση της νέας πληροφορίας, χωρίς την άρση των αρχών και των παραδοχών της αρχικής θεωρίας πλαισίου. Η υπόθεση αυτή έχει οδηγήσει στη διατύπωση νέων προβλέψεων για την ανάπτυξη της κατανόησης για τους ρητούς και, ειδικότερα, για τη διάταξή τους (Βαμβακούση, 2019). Η προσέγγιση της θεωρίας πλαισίου στην εννοιολογική αλλαγή Πιο συγκεκριμένα, υποθέτει ότι οι μαθητές εμπλουτίζουν τις αρχικές τους θεωρίες πλαισίου για τον αριθμό με νέες πληροφορίες για τους μη- φυσικούς, χρησιμοποιώντας συνήθως προσθετικούς μηχανισμούς μάθησης, όπως η αφομοίωση. Όταν, ωστόσο, 49 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Η πυκνή διάταξη των ρητών Πράγματι, φαίνεται ότι οι μαθητές της δευτεροβάθμιας αντιλαμβάνονται την απειρία των ενδιάμεσων πρώτα ανάμεσα σε δύο φυσικούς αριθμούς, στη συνέχεια ανάμεσα σε δύο δεκαδικούς, και τέλος ανάμεσα σε δύο κλάσματα. Επιπλέον, οι μαθητές δυσκολεύονται να αποδεχτούν ότι ανάμεσα σε δύο δεκαδικούς μπορούν να βρίσκονται κλάσματα και ότι ανάμεσα σε δύο κλάσματα μπορούν να βρεθούν δεκαδικοί, ακόμα και όταν απαντούν ότι υπάρχουν άπειροι ενδιάμεσοι. Τέλος υπάρχουν και μαθητές οι οποίοι συστηματικά διαφοροποιούν τα κλάσματα και τους δεκαδικούς ως προς τη διάταξη, θεωρώντας ότι οι δεκαδικοί είναι πυκνά διατεταγμένοι, ενώ η διάταξη των κλασμάτων είναι διακριτή, ή αντίστροφα. Οι Vamvakoussi και Vosniadou θεώρησαν ότι τα ευρήματα αυτά δείχνουν ότι οι μαθητές έχουν διαμορφώσει ένα συνθετικό μοντέλο για το σύνολο των ρητών. Στο συνθετικό αυτό μοντέλο, το σύνολο των ρητών αναπαρίσταται ως μια συλλογή ξένων μεταξύ τους συνόλων (φυσικοί, δεκαδικοί, κλάσματα) που προκύπτει καθώς οι μαθητές αφομοιώνουν πληροφορίες για τους μη φυσικούς αριθμούς στην προϋπάρχουσα θεωρία πλαισίου για τον αριθμό, χωρίς να αντιλαμβάνονται ότι οι (ρητοί) δεκαδικοί και τα κλάσματα είναι διαφορετικές συμβολικές αναπαραστάσεις των 50 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ίδιων μαθηματικών αντικειμένων (και όχι διαφορετικά είδη αριθμών) και χωρίς να ανα-κατηγοριοποιούν τους φυσικούς ως ειδική περίπτωση κλασμάτων ή δεκαδικών. Οι Vamvakoussi και Vosniadou (2012) υπέθεσαν ότι υπάρχει και μια διαφορετική συνθετική αντίληψη των μαθητών για την πυκνή διάταξη. Συγκεκριμένα, υπέθεσαν ότι, παρά το γεγονός ότι η «απειρία των ενδιάμεσων» και η «μη ύπαρξη του επόμενου» είναι ισοδύναμες όψεις της πυκνότητας από μαθηματική άποψη, δε συμβαίνει το ίδιο και από την πλευρά των μαθητών. Παρατήρησαν ότι στη σχετική βιβλιογραφία, όταν οι μαθητές είχαν εξεταστεί και για την «απειρία των ενδιάμεσων» και για τη «μη ύπαρξη του επόμενου», η επίδοσή τους ήταν συστηματικά καλύτερη στην πρώτη περίπτωση. Οι Vamvakoussi και Vosniadou έδωσαν μια πιθανή ερμηνεία αυτού του φαινομένου επικαλούμενες τη διάκριση ανάμεσα στο δυνάμει άπειρο και το ενεργεία άπειρο. Η πιο προσιτή μορφή του απείρου είναι το δυνάμει άπειρο, με τη μορφή μιας ατέρμονης διαδικασίας (βλ. και Hartnett & Gelman, 1998· Núñez & Lakoff, 2005· Singer & Voica, 2008). Για παράδειγμα, η συνειδητοποίηση ότι για κάθε φυσικό είναι δυνατόν να βρεθεί ο επόμενός του προσθέτοντας μια μονάδα, κι επόμενος του επόμενου με τον ίδιο τρόπο, και ούτω καθεξής, μπορεί να οδηγήσει στο συμπέρασμα ότι οι φυσικοί αριθμοί δεν τελειώνουν ποτέ, άρα είναι άπειροι στο πλήθος. Η πυκνή διάταξη των ρητών Σε παρόμοιες επαναλήψιμες διαδικασίες, με διακριτά βήματα, σε κάθε ένα από τα οποία παράγεται ένα πεπερασμένο πλήθος αριθμών, μπορούν να εξελιχθούν οι μετασχηματισμοί δύο δεδομένων δεκαδικών (προσθέτοντας μηδενικά στο τέλος του δεκαδικού τους μέρους) ή κλασμάτων (μετατρέποντας σε ισοδύναμα με μεγαλύτερους όρους) που περιγράφτηκαν παραπάνω. Από τη στιγμή που θα γίνει αντιληπτό ότι η διαδικασία αυτή μπορεί να επαναλαμβάνεται επ’ άπειρο, είναι εφικτό το συμπέρασμα ότι οι ενδιάμεσοι αριθμοί είναι άπειροι. Ωστόσο, αυτό δε συνεπάγεται απαραίτητα ότι γίνεται παράλληλα κατανοητό ότι είναι πυκνοί, με την έννοια ότι για κανέναν δεν ορίζεται επόμενος αριθμός. Οι Vamvakoussi και Vosniadou (2012), στο πρώτο πείραμα που παρουσιάζεται στο άρθρο, έδωσαν έργα κλειστού τύπου για την απειρία των ενδιάμεσων και έργα για τη μη ύπαρξη του επόμενου με τη μορφή δηλώσεων ενταγμένων σε ένα σενάριο με τις οποίες μαθητές Γυμνασίου και Λυκείου έπρεπε να διαφωνήσουν ή να συμφωνήσουν και να εξηγήσουν τις απαντήσεις τους. Τα αποτελέσματα έδειξαν ότι τα έργα για τον επόμενο ήταν πιο απαιτητικά από τα έργα για την απειρία των ενδιάμεσων και ότι ακόμα και μαθητές που απαντούσαν συστηματικά ότι το πλήθος των ενδιάμεσων είναι άπειρο, δεν απέρριπταν το ενδεχόμενο να υπάρχει ο επόμενος. Ωστόσο, η έρευνα των Vamvakoussi και Vosniadou (2012) ήταν ποσοτική, με πρόσβαση μόνο στις γραπτές εξηγήσεις των μαθητών, γεγονός που αφήνει ερωτήματα σχετικά με τον τρόπο με τον οποίο σκέφτονται οι μαθητές για τους δύο διαφορετικούς τύπους έργων και, συνακόλουθα, για τις δύο όψεις της πυκνής διάταξης των ρητών. Ειδικότερα, όπως επισημαίνουν οι ερευνήτριες δεν είναι ξεκάθαρο πώς ερμηνεύουν οι μαθητές τον όρο «άπειρο» (Vamvakoussi & Vosniadou, 2012). Είναι πιθανόν κάποιοι μαθητές, όταν απαντούν «άπειροι αριθμοί» να εννοούν ένα πολύ μεγάλο, αλλά πεπερασμένο, πλήθος αριθμών (μπορεί κανείς να σκεφτεί, για παράδειγμα, το πλήθος των κόκκων της άμμου σε μια έρημο). Από την άλλη μεριά, από τις απαντήσεις των μαθητών σε έργα για τον επόμενο δεν είναι σαφές αν πιστεύουν ότι ο επόμενος υπάρχει ή όχι. Για παράδειγμα, οι Vamvakoussi και Vosniadou (2012) αναφέρουν μαθητές που διαφώνησαν με τη δήλωση ότι το 2,002 είναι ο επόμενος του 2,001, επικαλούμενοι αντιπαραδείγματα, όπως το παρακάτω: Δεν είναι απαραίτητο το 2,002 να έρχεται αμέσως μετά το 2,001. Ας πούμε, υπάρχει και το 2,0015 και το 2,0012 (σελ. 277). Το ίδιο πρόβλημα υπάρχει και με απαντήσεις του τύπου «δεν μπορώ να προσδιορίσω τον επόμενο» ή «δεν μπορεί να βρεθεί ο επόμενος», στις οποίες δε διευκρινίζεται αν ο επόμενος αριθμός υπάρχει ή όχι. Η πυκνή διάταξη των ρητών Θα πρέπει να σημειωθεί, επίσης, ότι τα δεδομένα που συλλέγονται μέσω ερωτηματολογίων αποτυπώνουν ένα στιγμιότυπο της κατανόησης του κάθε μαθητή και δεν δίνουν πληροφορίες για τις διαδικασίες της αλλαγής της στο διάστημα κατά το οποίο συμβαίνει. Συμμετέχοντες Οι συμμετέχοντες ήταν 15 μαθητές της Β΄ τάξης του Λυκείου, αγόρια, από διάφορα σχολεία της κεντρικής και δυτικής Θεσσαλονίκης. Όλοι οι συμμετέχοντες είχαν επιλέξει τη Θετική Κατεύθυνση, ήταν μαθητές διαφορετικού επιπέδου στα μαθηματικά και παρακολουθούσαν φροντιστηριακά μαθήματα σε τάξη στην οποία δίδασκε ο πρώτος συγγραφέας. Η παρούσα έρευνα Στην παρούσα έρευνα εξετάσαμε την κατανόηση μαθητών της Β΄ Λυκείου σε έργα για την απειρία των ενδιάμεσων σε ένα διάστημα με άκρα ρητούς αριθμούς και σε έργα για τη μη ύπαρξη του επόμενου. Στόχος 51 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 μας ήταν να ελέγξουμε την υπόθεση ότι οι δύο όψεις της πυκνότητας, στις οποίες αντιστοιχούν τα έργα αυτά, δεν είναι ισοδύναμες από την πλευρά των μαθηματικών, με τη δεύτερη να είναι πιο απαιτητική. H μέθοδος που επιλέξαμε δανείζεται στοιχεία από το διδακτικό πείραμα (Steffe & Thompson, 2000) και τη μικρογενετική προσέγγιση στην ανάλυση της μάθησης (Siegler, 2006). Πιο συγκεκριμένα, αφού ελέγχθηκαν οι αρχικές τους απαντήσεις στα έργα, οι μαθητές εκτέθηκαν σε μια μικρής διάρκειας παρέμβαση για τον αριθμητικό μέσο δύο αριθμών και τον τρόπο υπολογισμού του στο πλαίσιο μια επαναλήψιμης διαδικασίας. Στη συνέχεια εξετάσαμε αν αξιοποίησαν αυθόρμητα ή μετά από βοηθητική νύξη το εργαλείο αυτό και τις αλλαγές που αυτό πυροδότησε τόσο όσον αφορά τις απαντήσεις των μαθητών, όσο και τις εξηγήσεις τους. Η συγκεκριμένη μέθοδος επιτρέπει να γίνουν ερωτήσεις στους μαθητές, ώστε να διευκρινιστεί τόσο η ερμηνεία που δίνουν στον όρο «άπειροι ενδιάμεσοι», όσο και το αν πιστεύουν ότι ο επόμενος υπάρχει ή όχι, κάτι που αποτελεί πρόβλημα σε έρευνες με ερωτηματολόγια (Vamvakoussi & Vosniadou, 2012). Επιπλέον, η παρέμβαση με την παροχή ενός εργαλείου, το οποίο δίνει μια νέα δυνατότητα στους μαθητές να αντιμετωπίσουν τα έργα για την πυκνότητα, αναμένεται να πυροδοτήσει μεταβολές στον τρόπο που αντιλαμβάνονται την πυκνή διάταξη των ρητών και να επιτρέψει την εξέτασή τους. Αξίζει να διευκρινιστεί ότι στο συγκεκριμένο σχεδιασμό δεν εξετάζεται η αποτελεσματικότητα της παρέμβασης (π.χ., σε σχέση με μια άλλη, βλ. Siegler, 2006). Ειδικότερα, στόχος είναι η μελέτη παραμέτρων της μεταβολής στον τρόπο σκέψης των μαθητών για το δεδομένο ζήτημα. Η οπτική αυτή είναι συμβατή με τις αρχές του διδακτικού πειράματος, κεντρικός στόχος του οποίου είναι η διερεύνηση υποθέσεων για τον τρόπο που σκέφτονται οι μαθητές (Steffe & Thompson, 2000). Η πρόβλεψή μας, συνεπής με την υπόθεσή μας, ήταν ότι οι μαθητές θα βελτιώνονταν στα έργα για την απειρία των ενδιάμεσων, αλλά δε θα έφταναν να αμφισβητήσουν την ύπαρξη του επόμενου αριθμού. Πίνακας 1 Πίνακας 1 Έργα για την απειρία των ενδιάμεσων σε ένα διάστημα με άκρα ρητούς αριθμούς Πίνακας 1 Έργα για την απειρία των ενδιάμεσων σε ένα διάστημα με άκρα ρητούς αριθμούς Ερευνητικά εργαλεία Για τις ανάγκες του προελέγχου και του μεταελέγχου χρησιμοποιήθηκαν δύο τύποι έργων σχετικά με την πυκνή διάταξη: έργα για το πλήθος των ενδιάμεσων αριθμών σε ένα διάστημα και έργα για τη (μη) ύπαρξη του επόμενου αριθμού στους ρητούς. Συγκεκριμένα, στον προέλεγχο δόθηκε στους μαθητές ερωτηματολόγιο με δύο τύπων έργα. Στο πρώτο, με συνολικά 7 δοκιμασίες, οι μαθητές ρωτήθηκαν πόσοι αριθμοί υπάρχουν ανάμεσα σε δύο ρητούς αριθμούς. Σε περίπτωση που η απάντησή τους ήταν «άπειροι», τους ζητήθηκε να την εξηγήσουν. Στον Πίνακα 1 παρουσιάζονται τα ζευγάρια των αριθμών που χρησιμοποιήθηκαν ως άκρα του διαστήματος, η σειρά με την οποία δόθηκαν στους μαθητές και οι κωδικοί με τους οποίους θα αναφέρονται στη συνέχεια. Ακολουθούσε ένα έργο με 2 δοκιμασίες για τη (μη) ύπαρξη του επόμενου ενός δεκαδικού (ΕπΔ) και ενός κλάσματος (ΕπΚ). Τα έργα αυτά παρουσιάστηκαν με τη μορφή δήλωσης ενός υποθετικού παιδιού, ως εξής: Ο αμέσως επόμενος αριθμός του 0,15 είναι ο 0,16, γιατί αμέσως μετά το 15 είναι το 16 και Το 3/7 είναι ο αμέσως επόμενος αριθμός του 2/7, γιατί το 3 έρχεται αμέσως μετά το 2. Οι μαθητές ρωτήθηκαν αν συμφωνούν ή διαφωνούν με την κάθε δήλωση. Σε περίπτωση διαφωνίας τους ζητήθηκε να εξηγήσουν γιατί και να διατυπώσουν τη δική τους άποψη. Στο μεταέλεγχο χρησιμοποιήθηκαν τα ίδια έργα, όπως στον προέλεγχο, και δύο επιπλέον έργα μεταφοράς (Μετ1, Μετ2) που εξέταζαν κατά πόσο οι μαθητές μπορούσαν να ανταποκριθούν σε ερωτήματα για την πυκνή 52 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) διάταξη σε ένα γενικευμένο πλαίσιο που, αντί συγκεκριμένων αριθμών, περιλάμβανε μεταβλητές. Στο Μετ1 οι μαθητές ρωτήθηκαν αν μπορούν να προσδιορίσουν πόσοι αριθμοί βρίσκονται ανάμεσα σε δύο οποιουσδήποτε πραγματικούς αριθμούς α και β. Στο Μετ2 ρωτήθηκαν αν μπορεί να προσδιοριστεί η πρώτη τιμή που θα πάρει η μεταβλητή x, αν το x κινείται στο ανοιχτό διάστημα(0,1). Πίνακας 1 Έργα για την απειρία των ενδιάμεσων σε ένα διάστημα με άκρα ρητούς α Α/Α Έργου Άκρα διαστήματος Κωδικός 1 1,512 – 1,513 Δ1 2 2/5 – 3/5 Κ1 3 0,1 – 0,2 Δ2 4 9/5 – 9/4 Κ2 5 0 – 1 Φ 6 9/5 – 7/3 Κ3 7 0,5 – 5/6 Δ-Κ Διαδικασία Η συλλογή των δεδομένων έγινε σε διάστημα 4 μηνών. Κάθε μαθητής συμμετείχε σε δύο ατομικές συνεντεύξεις, στο σπίτι του ή στο φροντιστήριο, οι οποίες μαγνητοφωνήθηκαν και απομαγνητοφωνήθηκαν για τις ανάγκες της έρευνας. Η κάθε συνάντηση είχε διάρκεια κατά μέσο όρο 30 - 40 λεπτά. Οι δύο συναντήσεις έγιναν με διαφορά δύο εβδομάδων περίπου η μία από την άλλη. Στην πρώτη συνάντηση δόθηκε σε κάθε μαθητή ατομικά το ερωτηματολόγιο του προέλεγχου. Ζητήθηκε προφορική απάντηση, εξήγηση της απάντησης και γραπτή καταγραφή της απάντησης. Στην περίπτωση που κάποιος μαθητής απαντούσε ότι υπάρχουν άπειροι ενδιάμεσοι, του γίνονταν διευκρινιστικές ερωτήσεις. Για παράδειγμα, «Τι εννοείς όταν λες «άπειροι»;», «Εννοείς 1.000; 1.000.000; Ή κάτι άλλο;», «Θα μπορούσε ένα υπολογιστής να τους βρει όλους;», «Μπορείς να δώσεις παραδείγματα τέτοιων αριθμών;». Στην περίπτωση που κάποιος μαθητής απαντούσε ότι δεν μπορεί να προσδιοριστεί ο επόμενος αριθμός, του γίνονταν διευκρινιστικές ερωτήσεις με στόχο να εκφράσει την άποψή του για τον αν υπάρχει ή όχι ο επόμενος. Για παράδειγμα, «Θα μπορέσει [το παιδί του σεναρίου] να τον βρει; Γιατί;» «Θα μπορούσε ένας υπολογιστής να τον βρει;», «Υπάρχει [ο επόμενος αριθμός], αλλά δεν μπορείς να πεις ποιος είναι;» Ακολούθησε η εισαγωγή του αριθμητικού μέσου. Στην δεύτερη συνάντηση δόθηκε σε κάθε μαθητή το ερωτηματολόγιο που είχαν συμπληρώσει στην πρώτη. Του ζητήθηκε να διαβάσει τις απαντήσεις του στις ερωτήσεις για το πλήθος των ενδιάμεσων αριθμών και ρωτήθηκε αν θέλει να αλλάξει κάτι σε αυτές. Την πρώτη φορά που ο μαθητής διατηρούσε μια απάντηση του τύπου «υπάρχει πεπερασμένο πλήθος ενδιάμεσων αριθμών», γινόταν μια πρώτη βοηθητική νύξη με την ερώτηση αν ανάμεσα στους δεδομένους, ή σε δύο από τους ενδιάμεσους που πρότεινε ο μαθητής δεν υπάρχει κανένας άλλος αριθμός. Αν επέμενε στην αρχική απάντηση, του γινόταν και μία δεύτερη νύξη με το ερώτημα αν μπορεί να βρει τον αριθμητικό μέσο αυτών των δύο αριθμών. Ανεξάρτητα από την τελική απάντηση του μαθητή και αν άλλαζε ή όχι την αρχική του απάντηση, δεν γινόταν άλλη νύξη για τον αριθμητικό μέσο για τις υπόλοιπες ερωτήσεις. Η διαδικασία αυτή επαναλήφθηκε και για τις ερωτήσεις για τον επόμενο. Στην πρώτη ερώτηση που οι μαθητές αναφέρθηκαν στον επόμενο του δοσμένου αριθμό, ρωτήθηκαν αν ανάμεσα σε αυτούς του δύο αριθμούς υπάρχει άλλος αριθμός. Διαδικασία Αν η απάντηση ήταν αρνητική, ακολουθούσε μία δεύτερη νύξη, με την οποία επαναδιατυπωνόταν το ερώτημα ως εξής: «Αν αυτός είναι ο επόμενος του δεδομένου αριθμού, τότε ανάμεσα σε αυτούς τους δύο αριθμούς δεν υπάρχει άλλος, σωστά;» Αν ο μαθητής συμφωνούσε, τότε ακολουθούσε το ερώτημα αν είναι δυνατόν να βρεθεί ο αριθμητικός μέσος αυτών των δύο αριθμών. Καμιά άλλη νύξη δεν έγινε στη συνέχεια. Οι ίδιες διευκρινιστικές ερωτήσεις που έγιναν στον προέλεγχο έγινα και στο μεταέλεγχο. Τέλος δόθηκαν τα έργα μεταφοράς (Μετ1, Μετ2). Στα έργα αυτά δεν έγινε καμία νύξη για τον αριθμητικό μέσο. Η εισαγωγή του αριθμητικού μέσου Για τις ανάγκες της έρευνας αξιοποιήθηκε η ιδέα του αριθμητικού μέσου δύο αριθμών: Δεδομένων οποιωνδήποτε δύο διαφορετικών αριθμών α, β, με α <β, ο αριθμητικός τους μέσος είναι το μισό του αθροίσματός τους και αντιστοιχεί στον αριθμό που βρίσκεται στο μέσο του διαστήματος που ορίζουν τα α, β. Η γνώση του αριθμητικού μέσου μπορεί να οδηγήσει στην εύρεση ενός ενδιάμεσου ανάμεσα σε δύο ψευδο- διαδοχικούς αριθμούς, όπως το 0,1 και το 0,2, ή το 3/5 και το 4/5. Αν η διαδικασία αυτή επαναληφθεί στο διάστημα με ένα από τα αρχικά άκρα και το μέσο, μπορεί να βρεθεί και άλλος ενδιάμεσος. Αν γίνει αντιληπτή η επ’άπειρο επαναληψιμότητα αυτής της διαδικασίας και εφόσον γενικευτεί για οποιαδήποτε άκρα, τότε μπορεί να οδηγήσει στην απειρία των ενδιάμεσων, αλλά και στη μη ύπαρξη του επόμενου. Η εισαγωγή του αριθμητικού μέσου έγινε ατομικά με κάθε μαθητή και διάρκεσε περίπου 40 λεπτά. Αρχικά, οι μαθητές ρωτήθηκαν αν θυμούνται τον ορισμό και τον τύπο του αριθμητικού μέσου. Όταν ήταν απαραίτητο, τους έγινε υπενθύμιση, τους δόθηκε ο σχετικός τύπος και η γεωμετρική αναπαράσταση του αριθμητικού μέσου ως το μέσο του διαστήματος μεταξύ δυο αριθμών στην αριθμογραμμή. Στη συνέχεια τους ζητήθηκε να βρουν τον αριθμητικό μέσο για τους αριθμούς 2 και 3, 0,1 και 0,2, 2/5 και 3/5. Για κάθε ζευγάρι, τους ζητήθηκε να βρουν τον αριθμητικό μέσο μεταξύ του πρώτου αριθμητικού μέσου και ενός από τους δύο αρχικούς αριθμούς, και η διαδικασία επαναλήφθηκε δύο φορές για τα νέα ζευγάρια αριθμών. Σε κάθε δοκιμασία επαναλαμβανόταν η ερώτηση «Μπορώ να συνεχίσω με τον ίδιο τρόπο;». Οι μαθητές είχαν στη διάθεσή τους κομπιουτεράκια, προκειμένου να μην επιβαρυνθούν με τις απαραίτητες πράξεις. Στόχος μας ήταν να εξοικειωθούν οι μαθητές με την ιδέα μιας επαναλήψιμης διαδικασίας και να συμπεράνουν ότι ανάμεσα σε δύο αριθμούς μπορεί να βρεθεί πάντα ένας ενδιάμεσος, με τον τύπο του αριθμητικού μέσου. Ωστόσο, δεν έγινε καμία ρητή σύνδεση με τα έργα για την απειρία των ενδιάμεσων και του επόμενου αριθμούς, τα οποία προηγήθηκαν κατά τον προέλεγχο, έτσι ώστε να ελεγχθεί κατά πόσο οι μαθητές θα αξιοποιούσαν αυθόρμητα την ιδέα του αριθμητικού μέσου για να αντιμετωπίσουν τα έργα για την πυκνότητα. 53 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Έργο για την απειρία του πλήθους των ενδιάμεσων αριθμών Κοινές δοκιμασίες προελέγχου-μεταελέγχου. Οι απαντήσεις των μαθητών στις δοκιμασίες για την απειρία κατηγοριοποιήθηκαν σε 4 κατηγορίες. Η πρώτη (ΚΕ) περιλαμβάνει τις απαντήσεις του τύπου «δεν υπάρχει κανένας ενδιάμεσος αριθμός». Η δεύτερη (ΠΠ-) περιλαμβάνει τις απαντήσεις του τύπου «υπάρχει πεπερασμένο πλήθος ενδιάμεσων», όπου αναφέρονται «λίγοι» ενδιάμεσοι αριθμοί, συνήθως δεκαδικοί με το πολύ τρία δεκαδικά ψηφία. Η τρίτη (ΠΠ+) περιλαμβάνει τις απαντήσεις του τύπου «υπάρχει πεπερασμένο πλήθος ενδιάμεσων», με το πλήθος των ενδιάμεσων να προσδιορίζεται με τη χρήση «μεγάλων» αριθμών (για παράδειγμα, «είναι δισεκατομμύρια»). Τέλος, η τέταρτη κατηγορία (ΑΠ) περιλαμβάνει τις απαντήσεις του τύπου «υπάρχουν άπειροι ενδιάμεσοι», στις οποίες συμπεριλαμβάνονται και περιγραφές όπως «είναι αμέτρητοι», «δεν τελειώνουν ποτέ». Στον Πίνακα 2 παρουσιάζεται η συχνότητα της κάθε κατηγορίας ανά έργο στον προέλεγχο (ΠροΕ) και στον μεταέλεγχο (ΜεταΕ). Από τον Πίνακα 2 φαίνεται ότι στον προέλεγχο η επικρατούσα απάντηση των μαθητών ήταν ότι υπάρχει πεπερασμένο πλήθος, συμπεριλαμβανομένου και του μηδενός, ενδιάμεσων αριθμών. Πράγματι, στο σύνολο 54 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) των απαντήσεων που δόθηκαν από τους 15 μαθητές στα 7 έργα (15x7=105), οι απαντήσεις (ΚΕ), (ΠΠ-) και (ΠΠ+) ήταν συνολικά 62 (59%). Αντίθετα, στο μεταέλεγχο, στο σύνολο των απαντήσεων σε όλα τα έργα (105), οι 28 (26,7%) ήταν του τύπου «πεπερασμένο πλήθος ενδιάμεσων», ενώ κανένας μαθητής δεν απάντησε ότι δεν υπάρχει κανένας ενδιάμεσος, σε κανένα έργο. Η επικρατούσα απάντηση στο μεταέλεγχο ήταν του τύπου «άπειροι ενδιάμεσοι αριθμοί» (Ν=77, 73,3%). Είναι σημαντικό να αναφερθεί, ωστόσο, ότι η απάντηση αυτού του τύπου δεν αντανακλά απαραίτητα την επαρκή κατανόηση για τη σημασία του όρου «άπειροι». Πράγματι, ορισμένοι μαθητές έδωσαν παραδείγματα ενδιάμεσων αριθμών, δείχνοντας ότι διέθεταν ένα μηχανισμό που παράγει άπειρους ενδιάμεσους. Για παράδειγμα, ο Μ11 έφερε ως παραδείγματα αριθμών ανάμεσα στους 2/5 και 3/5 τους 2,5/6, 2,56/6, … Ο Μ13 επεξήγησε τη χρήση του όρου «άπειροι» λέγοντας ότι «πάντα μπορούν να βρεθούν παραπάνω … δεν μπορεί να προσδιοριστεί». Αντίθετα, ο μαθητής Μ7 απάντησε σε κάποιες ερωτήσεις «είναι αμέτρητοι», αλλά σε άλλες προσδιόρισε τον όρο «αμέτρητοι» ως «δισεκατομμύρια». Παρόμοια, ο Μ2 απάντησε ότι υπάρχουν «άπειροι» σε όλες τις ερωτήσεις, αλλά όταν του ζητήθηκε να προσδιορίσει τι εννοούσε, περίγραψε το πλήθος ως «πολλοί, πάνω από 1.000», ή «αρκετοί τέλος πάντων, 2.000 και …» Πίνακας 2 Πίνακας 2 Έργα για την απειρία των ενδιάμεσων αριθμών: Συχνότητες των κατηγοριών απάντησης ανά έργο, στον προέλεγχο και το μεταέλεγχο Πίνακας 2 Έργα για την απειρία των ενδιάμεσων αριθμών: Συχνότητες των κατηγοριών απάντησης ανά έργο, στον προέλεγχο και το μεταέλεγχο Έργα ΚΕ ΠΠ- ΠΠ+ ΑΠ ΠροΕ ΜεταΕ ΠροΕ ΜεταΕ ΠροΕ ΜεταΕ ΠροΕ ΜεταΕ Φ 0 0 4 2 3 2 8 11 Δ1 5 0 5 2 0 1 5 12 Δ2 1 0 7 3 1 2 6 10 Κ1 2 0 7 3 1 1 5 11 Κ2 3 0 6 3 1 1 5 11 Κ3 1 0 7 2 0 2 7 11 Δ-Κ 0 0 7 4 1 0 7 11 Σύνολο 12 0 43 19 7 9 43 77 Για τον λόγο αυτόν, προκειμένου να γίνει μια πιο ακριβής καταγραφή της συνολικής εικόνας του κάθε μαθητή στον προέλεγχο και το μεταέλεγχο, εξετάστηκε πόσες φορές αναφέρθηκε κάθε μαθητής ρητά σε πεπερασμένο πλήθος ενδιάμεσων στο σύνολο των έργων. Στον Πίνακα 3 παρουσιάζονται συγκεντρωτικά τα σχετικά στοιχεία, έτσι ώστε να είναι ορατή η μετατόπιση του κάθε μαθητή από τον προέλεγχο στο μεταέλεγχο. Πιο συγκεκριμένα, στην πρώτη στήλη εμφανίζεται το πλήθος των ρητών αναφορών σε πεπερασμένο πλήθος ενδιάμεσων αριθμών (ΡΑΠΠ) που έδωσε κάθε μαθητής στον προέλεγχο (ΠροΕ), ενώ στην πρώτη γραμμή εμφανίζεται το αντίστοιχο πλήθος για το μεταέλεγχο (ΜεταΕ). Σε κάθε κελί αντιστοιχίζονται οι μαθητές ανάλογα με τις απαντήσεις που έδωσαν. Ένας μαθητής βρίσκεται στο κελί (κ, λ) αν έκανε κ ΡΑΠΠ στον προέλεγχο και λ ΡΑΠΠ στο μεταέλεγχο. Με έντονο χρώμα σημαίνονται οι μαθητές στους οποίους έγινε βοηθητική νύξη που παρέπεμπε στην παρέμβαση κατά το μεταέλεγχο (9 στους 15). Από τον Πίνακα 3 φαίνεται ότι στο μεταέλεγχο 11 μαθητές δεν έκαναν καμία ρητή αναφορά σε πεπερασμένο πλήθος ενδιάμεσων. Οι 4 από αυτούς (Μ3 Μ5, Μ11, Μ13, σκιασμένοι με γκρι χρώμα, εφεξής Ομάδα 1) είχαν απαντήσει ότι υπάρχει άπειρο πλήθος ενδιάμεσων αριθμών σε όλα τα έργα, ήδη από τον 55 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 προέλεγχο. Οι μαθητές αυτοί διατήρησαν τις απαντήσεις τους και στο μεταέλεγχο και δε χρειάστηκαν βοηθητική νύξη. Οι υπόλοιποι 7 μαθητές (Μ2, Μ4, Μ7, Μ10, Μ12, Μ14, Μ15, εφεξής, Ομάδα 2) είχαν απαντήσει ότι υπάρχει πεπερασμένο πλήθος ενδιάμεσων σε ένα έως επτά έργα του προελέγχου. Πίνακας 2 Από αυτούς τους μαθητές, ο Μ4 ήταν ο μόνος που επικαλέστηκε αυθόρμητα τον αριθμητικό μέσο σε όλα τα έργα κατά το μεταέλεγχο, αναφέροντας ότι «με αιτιολογία τον αριθμητικό μέσο, σε όλες τις ερωτήσεις θα υπάρχουν άπειροι ενδιάμεσα». Ο Μ14 χρειάστηκε μόνο μία νύξη, ενώ σε όλους τους υπόλοιπους χρειάστηκε και η δεύτερη νύξη, δηλαδή η ρητή αναφορά από τον ερευνητή στον αριθμητικό μέσο, προκειμένου να αλλάξουν τις αρχικές τους απαντήσεις. Οι υπόλοιποι 4 μαθητές (Μ1, Μ6, Μ8, Μ9, σκιασμένοι με μαύρο χρώμα, εφεξής Ομάδα 3) απάντησαν «πεπερασμένο πλήθος ενδιάμεσων αριθμών» σε τουλάχιστον έξι από τα έργα του προελέγχου και διατήρησαν αυτή την απάντηση σε τουλάχιστον 3 από τα 7 έργα του μεταελέγχου. Ενδιαφέρον παρουσιάζει το γεγονός ότι ο μαθητής Μ1 άλλαξε από πεπερασμένο σε άπειρο μόνο στο έργο στο οποίο του έγιναν οι δύο νύξεις και στις επόμενες διατήρησε τις αρχικές απαντήσεις του. Ο μαθητής Μ8 άλλαξε από πεπερασμένο σε άπειρο πλήθος μόνο στην περίπτωση των φυσικών αριθμών. Τέλος, ο μαθητής Μ6, σε όλες τις ερωτήσεις ακόμα και στο μεταέλεγχο συνεχίζει να αναφέρεται σε πεπερασμένο πλήθος αριθμών. Ωστόσο, πρέπει να αναφερθεί ότι αυτοί οι 4 μαθητές σημείωσαν μια μικρή βελτίωση σε σχέση με τις απαντήσεις τους στον προέλεγχο, καθώς μετακινήθηκαν από απαντήσεις του τύπου ΚΕ (κανείς ενδιάμεσος) σε απαντήσεις του τύπου ΠΠ- ή ΠΠ+. Πράγματι, όπως φαίνεται και από τον Πίνακα 1, στο μεταέλεγχο κανείς μαθητής δεν απάντησε ότι ανάμεσα σε κάποιο διάστημα δεν υπάρχει κανένας αριθμός. ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) Θα πρέπει να πάρουμε κάποιους αριθμούς στα α και β. Εεεεε…τελικά άπειροι γιατί θα μικραίνει ο αριθμός θα πάει 0,1 0,01, 0,001. (Μ9) Θα πρέπει να πάρουμε κάποιους αριθμούς στα α και β. Εεεεε…τελικά άπειροι γιατί θα μικραίνει ο αριθμός θα πάει 0,1 0,01, 0,001. (Μ9) Σημειώνουμε ότι όλοι αυτοί οι μαθητές έδειξαν να θεωρούν ότι με την απάντηση «άπειροι» δεν προσδιορίζεται το πλήθος των ενδιάμεσων αριθμών. Τυπικά οι απαντήσεις τους είχαν την μορφή «όχι, (δεν μπορεί να προσδιοριστεί), γιατί είναι άπειροι». Ο Μ15 εξέφρασε με αρκετή σαφήνεια αυτή την άποψη: Όχι, γιατί υπάρχουν άπειροι αριθμοί ανάμεσα τους. Αλλά το άπειρο είναι ένας αριθμός που δεν μπορούμε να προσδιορίσουμε. Είναι ένα κατασκεύασμα. (Μ15) ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) Έργο μεταφοράς 1. Με βάση τις απαντήσεις τους στο έργο Μετ1, οι μαθητές μπορούν καταρχήν να κατηγοριοποιηθούν σε 2 αδρές κατηγορίες --σε αυτούς που θεώρησαν ότι η απάντηση εξαρτάται από ποιες συγκεκριμένες τιμές θα πάρουν οι μεταβλητές α και β, και σε αυτούς που δεν έθεσαν αυτό τον περιορισμό. Στην πρώτη κατηγορία βρέθηκαν 3 μαθητές (Μ1, Μ6, Μ10). Και οι τρεις ανέφεραν ότι η απάντηση εξαρτάται από τις «μεταβλητές» ή τα «γράμματα». Οι μαθητές Μ6 και Μ10 έδωσαν παραδείγματα, δίνοντας συγκεκριμένες τιμές στις μεταβλητές και αναφερόμενοι σε πεπερασμένο πλήθος ενδιάμεσων αριθμών. Για παράδειγμα, ο μαθητής Μ6 ανέφερε ότι «αν έχω 3 με 100 είναι πολλοί, ενώ αν έχω 2 με 3 είναι λιγότεροι». Και οι δύο αυτοί μαθητές ανήκαν στην Ομάδα 3, δηλαδή, την ομάδα των μαθητών που μετακινήθηκαν ελάχιστα από τις αρχικές απαντήσεις τους («πεπερασμένο πλήθος ενδιάμεσων» σε όλα τα έργα του προελέγχου) στο μεταέλεγχο (βλ. Πίνακα 3). Αντίθετα, ο Μ10 ανήκε στην Ομάδα 2 και, μάλιστα, στο κελί (1, 0) του Πίνακα 2 (δηλ., έδωσε μόνο μία απάντηση του τύπου «πεπερασμένο πλήθος ενδιάμεσων» στον προέλεγχο και καμία στο μεταέλεγχο). Εντούτοις δεν έκανε το βήμα της γενίκευσης που απαιτείται για να απαντήσει ότι σε οποιοδήποτε διάστημα υπάρχουν άπειροι ενδιάμεσοι αριθμοί. Από τους υπόλοιπους 12 μαθητές, οι 2 (Μ7, Μ14), οι οποίοι ανήκαν στην Ομάδα 2, αναφέρθηκαν σε «πάρα πολλούς» ενδιάμεσους αριθμούς: Όχι, δεν μπορώ. Αν το βάλω στον υπολογιστή θα είναι αμέτρητοι… ξέρω κι εγώ… Αν μου ζητάτε και αριθμό να πούμε 1.000.000. Ένας άνθρωπος θα βαρεθεί να το κάνει. (Μ7) Όχι, δεν μπορώ. Αν το βάλω στον υπολογιστή θα είναι αμέτρητοι… ξέρω κι εγώ… Αν μου ζητάτε και αριθμό να πούμε 1.000.000. Ένας άνθρωπος θα βαρεθεί να το κάνει. (Μ7) Όχι θα υπάρχουν άπειροι - πάρα πάρα πολλοί. Θα σταματάει κάποτε αλλά δεν μπορούμε να προσδιορίσουμε πόσο πολλοί είναι αυτοί. (Μ14) Όχι θα υπάρχουν άπειροι - πάρα πάρα πολλοί. Θα σταματάει κάποτε αλλά δεν μπορούμε να προσδιορίσουμε πόσο πολλοί είναι αυτοί. (Μ14) Οι υπόλοιποι 10 μαθητές αναφέρθηκαν ρητά σε «άπειρους ενδιάμεσους αριθμούς». Ενδιαφέρον παρουσιάζει το γεγονός ότι ανάμεσα σε αυτούς τους μαθητές βρίσκονται οι Μ8, Μ9 (Ομάδα 3), οι οποίοι αναφέρθηκαν σε μια επαναλήψιμη διαδικασία: Οι υπόλοιποι 10 μαθητές αναφέρθηκαν ρητά σε «άπειρους ενδιάμεσους αριθμούς». Ενδιαφέρον παρουσιάζει το γεγονός ότι ανάμεσα σε αυτούς τους μαθητές βρίσκονται οι Μ8, Μ9 (Ομάδα 3), οι οποίοι αναφέρθηκαν σε μια επαναλήψιμη διαδικασία: Όχι, γιατί θα είναι πάρα πολλοί αριθμοί. Θα είναι άπειροι. Πίνακας 3 Πίνακας 3 Πλήθος ρητών αναφορών σε πεπερασμένο πλήθος ενδιάμεσων: Μετατοπίσεις των μαθητών από τον προέλεγχο στο μεταέλεγχο ΡΑΠΠ ΡΑΠΠ (ΜεταΕ) (ΠροΕ) 0 1 2 3 4 5 6 7 0 Μ3 Μ5 Μ11 Μ13 1 Μ10 2 3 Μ2 4 Μ12 5 Μ7 6 Μ15 Μ8 7 Μ4 Μ14 Μ9 Μ1 Μ6 * Σημείωση: ΡΑΠΠ: Πλήθος ρητών αναφορών σε πεπερασμένο πλήθος ενδιάμεσων αριθμών 56 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Κατηγορία Δ: Υπάρχει επόμενος, μπορεί να προσδιοριστεί. Στην κατηγορία αυτή εντάχθηκαν οι απαντήσεις των μαθητών που υποστήριξαν ότι ο επόμενος υπάρχει και μπορεί να προσδιοριστεί. Αντίθετα με τις απαντήσεις που εντάχθηκαν στην Κατηγορία Γ, στις απαντήσεις αυτές δεν προσδιορίζεται ο επόμενος (βλ. Παραδείγματα 9, 10, 11). Κατηγορία Ε: Υπάρχει ο επόμενος (δεν προσδιορίζεται). Στην κατηγορία αυτή εντάχθηκαν οι απαντήσεις των μαθητών που υποστήριξαν ότι ο επόμενος υπάρχει, αλλά δεν μπορεί να προσδιοριστεί με ακρίβεια, εξαιτίας της απειρίας των αριθμών (βλ. Παράδειγμα 12). Κατηγορία Ζ: Άλλη απάντηση. Σε αυτή την κατηγορία εντάχθηκαν απαντήσεις οι οποίες δεν μπορούσαν να ενταχθούν σε κάποια από τις προηγούμενες κατηγορίες. Πρόκειται για τις απαντήσεις δύο μαθητών (Μ6, Μ11), οι οποίοι απάντησαν ότι ο επόμενος αριθμός εξαρτάται από την επιθυμητή ακρίβεια (Παραδείγματα 13, 14). Έργο για τη (μη) ύπαρξη του επόμενου Κοινές δοκιμασίες προελέγχου και μεταελέγχου. Οι απαντήσεις των μαθητών στα έργα ΕπΔ και ΕπΚ ομαδοποιήθηκαν στις εξής κατηγορίες, για τις οποίες παρουσιάζονται ενδεικτικά παραδείγματα στον Πίνακα 4. Κατηγορία Α: Ο επόμενος είναι ο δεδομένος αριθμός. Στην κατηγορία αυτή εντάχθηκαν οι απαντήσεις των μαθητών που συμφώνησαν με τις δηλώσεις «ο επόμενος του 0,15 είναι ο 0,16» (ΕπΔ) και «ο επόμενος του 2/5 είναι ο 3/5» (ΕπΚ). Κατηγορία Β: Ο επόμενος είναι άλλος (πεπερασμένο πλήθος δεκαδικών ψηφίων). Στην κατηγορία αυτή εντάχθηκαν οι απαντήσεις των μαθητών που διαφώνησαν με τις παραπάνω δηλώσεις, και πρότειναν έναν εναλλακτικό «επόμενο». Στην περίπτωση του δεκαδικού, οι απαντήσεις τύπου Β προέκυψαν συνήθως προσθέτοντας δεκαδικά ψηφία στον αριθμό (Παράδειγμα 1), αλλά και προσθέτοντας μια υποδιαστολή ακόμα (Παραδείγματα 2, 3). Στην περίπτωση των κλασμάτων, οι απαντήσεις τύπου Β προέκυψαν πάλι είτε προσθέτοντας δεκαδικά ψηφία στον αριθμητή (Παράδειγμα 4), είτε με μετατροπή του κλάσματος σε δεκαδικό, συχνά λαμβάνοντας υπόψη μόνο ορισμένα αρχικά ψηφία της δεκαδικής μορφής (Παραδείγματα 5, 6). Κατηγορία Γ: Ο επόμενος είναι άλλος (άπειρα δεκαδικά ψηφία). Στην κατηγορία αυτή εντάχθηκαν οι απαντήσεις των μαθητών που αναφέρθηκαν σε ένα δεκαδικό με άπειρα δεκαδικά ψηφία ως τον επόμενο, είτε του 0,15 είτε του 2/7. Τέτοιες απαντήσεις παρουσιάζονται στα παραδείγματα 7, 8. 57 Σημείωση. Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί, Ζ: Άλλη Πίνακας 5 Πίνακας 5 Πίνακας 5 Συχνότητα των απαντήσεων των μαθητών ανά κατηγορία και ανά έργο, στον προέλεγχο (ΠροΕ) και στο μεταέλεγχο (ΜεταΕ) Κατηγορίες απαντήσεων Έργο ΕπΔ Έργο ΕπΚ ΠροΕ ΜεταΕ ΠροΕ ΜεταΕ Α 3 1 7 2 Β 8 0 4 2 Γ 1 1 2 1 Δ 0 5 0 3 Ε 2 6 1 5 Ζ 1 2 1 2 Σύνολο 15 15 15 15 Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη ς 5 Συχνότητα των απαντήσεων των μαθητών ανά κατηγορία και ανά έργο, στον προέλεγχο (ΠροΕ) και στο μεταέλεγχο (ΜεταΕ) Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη Όπως φαίνεται στους Πίνακες 6 και 7, από τους μαθητές της Ομάδας 1 κανείς δεν έδωσε απάντηση τύπου Α ή Β, ήδη από τον προέλεγχο. Ο Μ11 παρέκαμψε στην ουσία το πρόβλημα του επόμενου, λέγοντας ότι ο επόμενος εξαρτάται από την επιθυμητή ακρίβεια (βλ. Πίνακα 4, Παράδειγμα 13). Στο μεταέλεγχο διατήρησε αυτού του τύπου την απάντηση, αναγνωρίζοντας ταυτόχρονα την απειρία των ενδιάμεσων. Για παράδειγμα: Δεν συμφωνώ [ότι το 0,16 είναι ο επόμενος του 0,15]. Γιατί μπορεί να είναι πάλι άπειροι, 0,151 ή 0,1511, ανάλογα πόσα ψηφία θέλω. Θα μπορούσε να τον βρει, αν ο επόμενος αριθμός θέλω να έχει 3 ψηφία και τα λοιπά. Αν ήταν πιο συγκεκριμένη η εκφώνηση θα μπορούσε να τον βρει. Οι υπόλοιποι μαθητές της Ομάδας 1 έδωσαν απαντήσεις τύπου Γ ή Ε και στα δύο έργα στον προέλεγχο, τις οποίες διατήρησαν και στο μεταέλεγχο. Παρά τις νύξεις που τους έγιναν, δεν έφτασαν στο συμπέρασμα ότι δεν υπάρχει επόμενος, καθώς δεν μπορούσαν να φανταστούν με ποιο τρόπο ένας αριθμός που δεν μπορεί να προσδιοριστεί με ακρίβεια μπορεί να χρησιμοποιηθεί στον τύπο για να υπολογιστεί ο αριθμητικός μέσος. Δεν μπορώ να βρω τον αριθμητικό μέσο γιατί δεν ξέρω πόσα μηδενικά έχει το 0,15000…..01 και είναι ένας θεωρητικός αριθμός. Δεν θα τον βρει ακριβώς ποτέ. Θα μπορούσαμε να δώσουμε κάποιον αριθμό που θα πλησιάζει. […] Ούτε με τον υπολογιστή θα τον βρω. Δεν μπορεί να προσεγγιστεί εύκολα, γιατί έχω απροσδιόριστα μηδενικά […]. Πίνακας 4 Πίνακας 4 ς 4 Παραδείγματα απαντήσεων στα έργα ΕπΔ, ΕπΚ, ανά κατηγορία Παραδείγματα απαντήσεων στα έργα ΕπΔ, ΕπΚ, ανά κατηγορία Κατηγορία Παραδείγματα Β Ο επόμενος του 0,15 είναι ο: Ο επόμενος του 2/7 είναι ο: 1. 0,151 2. 0,15.1 3. 0,15 κόμμα κάτι 4. 2,1/7 5. 0,29 επειδή το 2/7 κάνει 0,28 6. 0,286 Γ 7. 0,15000…001 8. 0,2857142850…..001 Δ 9. Κάποιος θα υπάρχει. Θα είναι 0,155, αλλά μετά, αν συνεχίσει, θα βρει άπειρους, δεν θα σταματάνε. Θα τον βρει με δυσκολία. 10. Όχι το 0,151, θα είναι άλλος. Αν κάτσει και ψάξει θα το βρει, φυσικά και υπάρχει. 11. Υπάρχουν και άλλοι αριθμοί ανάμεσα τους. Θα τον βρει αν το κάνει με υπολογιστή, θα είναι πιο εύκολο για να κάνει τον τύπο που κάναμε με τις πράξεις. Ε 12. Υπάρχει επόμενος, αλλά δεν θα τον βρει γιατί, όπως πριν, θα βρίσκει συνέχεια αριθμούς και δεν θα σταματά ποτέ. Ζ 13. Ναι, υπάρχει, αλλά θα πρέπει να είναι πιο συγκεκριμένη η εκφώνηση στο πόσα ψηφία θέλει να έχει. 14. Σύμφωνα με τον αριθμητικό μέσο θα υπάρχουν κι άλλοι. Αλλά συμφωνώ [ότι το 0,16 είναι ο επόμενος του 0,15] γιατί ο Κώστας [το παιδί του σεναρίου] αλλάζει τα νούμερά του ανά μία μονάδα. Σημείωση. Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (ά λήθ δ δ ώ φί ) ά ό ί δ ί ά ό δ Σημείωση. Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί, Ζ: Άλλη Σημειώνουμε ότι στις απαντήσεις και των δύο πρώτων κατηγοριών (Α,Β), ο «επόμενος» των δεδομένων αριθμών αναζητείται σε ένα διακριτό, πεπερασμένο, σύνολο αριθμών που διατηρούν τη διάταξη των φυσικών αριθμών. Η ορθή απάντηση («Δεν υπάρχει επόμενος») δε δόθηκε από κανένα μαθητή, ούτε στο προέλεγχο, ούτε στο μεταέλεγχο και δεν εμφανίζεται στα αποτελέσματα. 58 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) Στον Πίνακα 5 παρουσιάζεται το πλήθος των απαντήσεων των μαθητών ανά κατηγορία και ανά έργο, στον προέλεγχο (ΠροΕ) και στο μεταέλεγχο (ΜεταΕ). Στον Πίνακα 5 παρουσιάζεται το πλήθος των απαντήσεων των μαθητών ανά κατηγορία και ανά έργο, στον προέλεγχο (ΠροΕ) και στο μεταέλεγχο (ΜεταΕ). Όπως φαίνεται από τον Πίνακα 5, στον προέλεγχο οι απαντήσεις που ανήκουν στην κατηγορία Α ή Β αποτελούν τις 11 από τις 15 απαντήσεις που δόθηκαν στον προέλεγχο, με την Α να υπερτερεί στην περίπτωση των κλασμάτων. Οι απαντήσεις αυτές μειώνονται στο μεταέλεγχο και αυξάνονται οι απαντήσεις του τύπου Ε, ενώ εμφανίζεται και η απάντηση τύπου Δ. Στους Πίνακες 6 και 7 καταγράφονται οι μετατοπίσεις των μαθητών ως προς την κατηγορία απάντησης που έδωσαν στον προέλεγχο και το μεταέλεγχο για τον επόμενο αριθμό ενός δεκαδικού και ενός κλάσματος. Οι βοηθητικές νύξεις έγιναν στο πρώτο έργο (ΕπΔ) και οι μαθητές που τις χρειάστηκαν σημαίνονται με έντονο χρώμα στον Πίνακα 6. Όπως φαίνεται, οι νύξεις ήταν απαραίτητες σε όλους τους μαθητές. Δηλαδή, κανένας μαθητής δεν άλλαξε αυθόρμητα την αρχική του απάντηση. Πίνακας 5 Υπάρχει, αλλά δεν μπορώ να τον βρω. Κατά προσέγγιση θα μπορεί να είναι ο επόμενος το 0,15000000.....01. (Μ13) 59 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Πίνακας 6 Μετατόπιση των μαθητών ως προς τις κατηγορίες απάντησης στο ΕπΔ («επόμενος» του 0,15) από τον προέλεγχο (ΠροΕ) στο μετάλεγχο (ΜεταΕ) ΠροΕ ΜεταΕ Α Β Γ Δ Ε Ζ (άλλη) Α Μ2 Μ10 Μ6 Β Μ1 Μ4 Μ7 Μ9 Μ12 Μ8 Μ14 Μ15 Γ Μ3 Δ Ε Μ5 Μ13 Ζ Μ11 Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) Οι μαθητές της Ομάδας 2 ξεκίνησαν στον προέλεγχο δίνοντας απαντήσεις τύπου Α ή Β και στα δύο έργα. Στο μεταέλεγχο, μετακινήθηκαν σε απαντήσεις τύπου Δ ή Ε στο ΕπΔ, με την εξαίρεση του Μ2 που παρέμεινε στην απάντηση Α, την οποία και διατήρησε και στο ΕπΚ. Δύο μαθητές της ομάδας αυτής (Μ12, Μ15) έδωσαν λιγότερο εκλεπτυσμένες απαντήσεις (τύπου Β) στο ΕπΚ, σε σύγκριση με το ΕπΔ. Παρόμοια, οι μαθητές της Ομάδας 3 ξεκίνησαν από απαντήσεις τύπου Α ή Β στο ΕπΔ στον προέλεγχο και μετακινήθηκαν προς τις κατηγορίες Δ και Ε. Εξαίρεση ήταν ο Μ6 ο οποίος, παρόμοια με τον Μ11 της Ομάδας 1, παρέκαμψε το πρόβλημα, λέγοντας ότι ο επόμενος εξαρτάται από την επιθυμητή ακρίβεια (βλ. Πίνακα 4, Παράδειγμα 14). Στο ΕπΚ, ένας μαθητής (Μ9) της ομάδας αυτής ξεκίνησε και παρέμεινε στην απάντηση τύπου Α, ενώ οι υπόλοιποι απάντησαν παρόμοια με το ΕπΔ δίνοντα απαντήσεις τύπου Δ και Ε. Στο μετάλεγχο, οι μαθητές της Ομάδας 2 και 3 που μετατοπίστηκαν προς πιο εκλεπτυσμένες απαντήσεις, χρησιμοποίησαν τον αριθμητικό μέσο και την ιδέα της επαναλήψιμης διαδικασίας προκειμένου να εξηγήσουν τις απαντήσεις τους. Για παράδειγμα: Μπορώ να βρω το μέσο [του 0,15 και του 0,151], ναι, υπάρχει, είναι ο 0,1505. Μπορεί να μη τον βρει [τον επόμενο του 0,15], γιατί αν κάνει συνέχεια την πράξη δε θα σταματήσει ποτέ. Δε θα τον βρει. Υπάρχει, αλλά δεν ξέρουμε ποιος είναι. (Μ8) Έργο μεταφοράς 2. Στο έργο Μετ2, οι μαθητές ρωτήθηκαν αν μπορούν να προσδιορίσουν την πρώτη τιμή που θα πάρει μια μεταβλητή x, η οποία κινείται στο διάστημα (0,1). Όλοι οι μαθητές της Ομάδας 1 αναφέρθηκαν σε έναν αριθμό με άπειρα δεκαδικά ψηφία και όλοι, εκτός από τον Μ5, πρότειναν συγκεκριμένα τον 0,00…01. Οι απαντήσεις αυτές συνάδουν με τις απαντήσεις τους στα έργα ΕπΔ, ΕκΚ ως προς την ύπαρξη του επόμενου και, μάλιστα, ακόμα και αυτοί που είχαν απαντήσει ότι ο επόμενος δεν μπορεί να προσδιοριστεί, φάνηκαν πιο πρόθυμοι να τον προσδιορίσουν ως ένα συγκεκριμένο δεκαδικό με άπειρα δεκαδικά ψηφία. Από τους μαθητές της Ομάδας 2, ο Μ4 πρότεινε τον αριθμό 0,100… που, στην ουσία, ταυτίζεται με τον 0,1. Οι Μ2, Μ7, Μ14 και Μ15 πρότειναν τον 0,00…01, ενώ οι Μ10 και Μ12 απάντησαν ότι η πρώτη τιμή, αν και υπάρχει, δεν μπορεί να προσδιοριστεί, γιατί «οι αριθμοί είναι άπειροι». ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) Όλοι οι μαθητές της Ομάδας 3 (Μ1, Μ6, Μ8, Μ9) προσδιόρισαν την πρώτη τιμή της μεταβλητής, αναφέροντας αριθμούς όπως τον 0,1, τον 0,01 και τον 0,001.Οι μαθητές αυτοί υπαναχώρησαν, σε σχέση με τις απαντήσεις τους στα έργα για τον επόμενο του μεταελέγχου, σε λιγότερο εκλεπτυσμένες απαντήσεις. Πίνακας 7 Πίνακας 7 Μετατόπιση των μαθητών ως προς τις κατηγορίες απάντησης στο ΕπΚ («επόμενος» του 2/5) από τον προέλεγχ (ΠροΕ) στο μετάλεγχο (ΜεταΕ) ΠροΕ ΜεταΕ Α Β Γ Δ Ε Ζ Α Μ2 Μ9 Μ1 Μ4 Μ7 Μ10 Μ6 Β Μ12 Μ15 Μ8 Μ14 Γ Μ3 Μ13 Δ Ε Μ5 Ζ Μ11 Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη Πίνακας 7 Μετατόπιση των μαθητών ως προς τις κατηγορίες απάντησης στο ΕπΚ («επόμενος» του 2/5) από τον προέλεγχο (ΠροΕ) στο μετάλεγχο (ΜεταΕ) ΠροΕ ΜεταΕ Α Β Γ Δ Ε Ζ Α Μ2 Μ9 Μ1 Μ4 Μ7 Μ10 Μ6 Β Μ12 Μ15 Μ8 Μ14 Γ Μ3 Μ13 Δ Ε Μ5 Ζ Μ11 Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη Πίνακας 7 Μετατόπιση των μαθητών ως προς τις κατηγορίες απάντησης στο ΕπΚ («επόμενος» του 2/5) από τον προέλεγχο (ΠροΕ) στο μετάλεγχο (ΜεταΕ) Ζ Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη *Σημείωση. Α: Ο δεδομένος είναι ο επόμενος αριθμός, Β. Υπάρχει άλλος επόμενος (πεπερασμένο πλήθος δεκαδικών ψηφίων), Γ. Υπάρχει άλλος επόμενος (άπειρο πλήθος δεκαδικών ψηφίων), Δ. Υπάρχει επόμενος, μπορεί να προσδιοριστεί, Ε. Υπάρχει επόμενος, δεν μπορεί να προσδιοριστεί., Ζ: Άλλη 60 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 ενδιάμεσων» που έδωσαν στο προέλεγχο, προέκυψαν τρεις αδρές ομάδες μαθητών. Η Ομάδα 1 περιελάμβανε τους μαθητές που απάντησαν ότι υπάρχουν άπειροι ενδιάμεσοι, ήδη από τον προέλεγχο. Οι μαθητές αυτοί είχαν ήδη στη διάθεσή τους μια επαναλήψιμη διαδικασία παραγωγής ενδιάμεσων, βασισμένη σε ιδιότητες των δεκαδικών. Ούτε αυτή, ούτε η διαδικασία με τον αριθμητικό μέσο τους οδήγησε στο συμπέρασμα ότι δεν υπάρχει ο επόμενος αριθμός. Η Ομάδα 2 περιελάμβανε τους μαθητές που έδωσαν απαντήσεις του τύπου «πεπερασμένο πλήθος ενδιάμεσων» στον προέλεγχο, αλλά όχι στο μεταέλεγχο. Με μία μόνο εξαίρεση, οι βοηθητικές νύξεις που παρέπεμπαν στον αριθμητικό μέσο ήταν απαραίτητες για τη μετατόπιση αυτή, η οποία, ωστόσο, δεν εξασφάλισε απαραίτητα εκλεπτυσμένες απαντήσεις σε όλα τα έργα που ακολούθησαν. Στον Πίνακα 7, για παράδειγμα, φαίνεται ότι 5 από τα 7 μέλη της ομάδας αυτής, στο μεταέλεγχο, έδωσαν απαντήσεις του τύπο Α, Β, ή Δ στο ΕπΚ. Δηλαδή, είτε πρότειναν έναν συγκεκριμένο αριθμό ως επόμενο του κλάσματος 2/5, είτε ισχυρίστηκαν ότι μπορεί να βρεθεί. Για την ακρίβεια, μια προσεκτική εξέταση των αποτελεσμάτων ανά άτομο, δείχνει ότι όλα τα μέλη αυτής της ομάδας, σε κάποια από τα έργα ΕπΔ, ΕπΚ, Μετ1, Μετ2 έδωσαν λιγότερο εκλεπτυσμένες απαντήσεις σε σχέση με τα υπόλοιπα. Οι μαθητές της Ομάδας 2 ενσωμάτωσαν τον αριθμητικό μέσο και την επαναλαμβανόμενη χρήση του ως στρατηγική και χρησιμοποίησαν εκφράσεις όπως «αμέτρητοι», «άπειροι», «δεν θα σταματάνε». Αυτό δε σημαίνει απαραίτητα ότι όλοι θεώρησαν ότι η διαδικασία θα συνεχίζεται πράγματι επ’άπειρο (όπως φαίνεται, για παράδειγμα, από τις απαντήσεις των Μ7 και Μ14 στο Μετ1) και, βέβαια, δεν οδηγήθηκαν στο συμπέρασμα ότι δεν υπάρχει ο επόμενος αριθμός. Τέλος, η Ομάδα 3 περιελάμβανε τους μαθητές που, τόσο στον προέλεγχο, όσο και στο μεταέλεγχο, έδωσαν απαντήσεις του τύπου «πεπερασμένο πλήθος ενδιάμεσων» σε τρία έως και εφτά από τα αντίστοιχα έργα, παρά τις βοηθητικές νύξεις. Παρόμοια με τους μαθητές της Ομάδας 2, κάποια από τα μέλη της ομάδας αυτής χρησιμοποίησαν την ιδέα του αριθμητικού μέσου και αντιμετώπισαν κάποια από τα επόμενα έργα με μεγαλύτερη επιτυχία απ’ όσο θα αναμενόταν από την επίδοσή τους στα έργα για την απειρία των ενδιάμεσων στο μεταέλεγχο (όπως, για παράδειγμα, οι Μ8 και Μ9 στο Μετ1, και όλα τα μέλη της ομάδας στο ΕπΔ –βλ. Πίνακα 6). Παρ’όλ’αυτά, όλοι οι μαθητές της Ομάδας 3 επανήλθαν στις λιγότερο εκλεπτυσμένες απαντήσεις στο Μετ2, προτείνοντας το 0,1, το 0,01 και το 0,001 ως την πρώτη τιμή που παίρνει η μεταβλητή x στο (0,1). Συμπεράσματα – Συζήτηση Στην εργασία αυτή εξετάσαμε τις αντιλήψεις μαθητών της Β΄ Λυκείου για την απειρία των αριθμών σε ένα διάστημα με άκρα ρητούς αριθμούς και την ύπαρξη (ή μη) του επόμενου αριθμού στους ρητούς. Οι μαθητές εξετάστηκαν πριν και μετά την εισαγωγή ενός μαθηματικού εργαλείου, συγκεκριμένα του αριθμητικού μέσου σε ένα διάστημα στο πλαίσιο μιας επαναλήψιμης διαδικασίας, το οποίο δυνητικά μπορεί να υποστηρίξει την κατανόηση και των δύο παραπάνω όψεων της πυκνής διάταξης των ρητών. Η μέθοδος που υιοθετήσαμε ανταποκρίθηκε σε δύο ζητούμενα με βάση τη βιβλιογραφία (π.χ., Vamvakoussi & Vosniadou, 2012): Επέτρεψε την ταυτοποίηση μαθητών που διαφοροποιούν την έκφραση «άπειροι αριθμοί» από εκφράσεις του τύπου «ένα πολύ μεγάλο πλήθος αριθμών». Ταυτόχρονα, επέτρεψε να διευκρινιστούν οι απαντήσεις του τύπου «δεν μπορώ να προσδιορίσω ποιος θα είναι ο επόμενος» ως προς τον αν ο επόμενος υπάρχει ή όχι. Επιπλέον, η παροχή ενός υποστηρικτικού εργαλείου και η παρώθηση των μαθητών να το χρησιμοποιήσουν πυροδότησε μεταβολές στον τρόπο που σκεφτόταν ο κάθε μαθητής για την πυκνή διάταξη (Siegler, 2006) και επέτρεψε την εξέτασή τους. Όπως αναμενόταν, πριν την παρέμβαση, η πλειοψηφία των απαντήσεων των μαθητών ήταν συμβατή με την αντίληψη ότι οι ρητοί αριθμοί είναι διακριτοί, όπως οι φυσικοί αριθμοί. Παρά το γεγονός ότι υπήρξαν διαβαθμίσεις ως προς το μέγεθος του πλήθους των ενδιάμεσων (από κανένας, μέχρι ένα πολύ μεγάλο πλήθος της τάξης του δισεκατομμυρίου), η επικρατούσα απάντηση ήταν το πεπερασμένο πλήθος ενδιάμεσων. Κατηγοριοποιώντας τους μαθητές ανάλογα με το πλήθος των απαντήσεων του τύπου «πεπερασμένο πλήθος 61 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) μαθητές της Ομάδας 2) δεν αμφισβήτησαν την ύπαρξη του επόμενου. Επομένως, το εύρημα αυτό συνηγορεί υπέρ της προβλεπτικής ισχύος της προσέγγισης της θεωρίας πλαισίου στην εννοιολογική αλλαγή, καθώς η δημιουργία συνθετικών αντιλήψεων αποτελεί κεντρική πρόβλεψη της συγκεκριμένης προσέγγισης (Vamvakoussi & Vosniadou, 2010· Vosniadou et. al., 2008· Βαμβακούση, 2019). Βιβλιογραφία Bailey, D. H., Hoard, M. K., Nugent, L., & Geary, D. C. (2012). Competence with fractions predicts gains in mathematics achievement. Journal of Experimental Child Psychology, 113, 447-455. http://dx.doi.org/10.1016/j.jecp.2012.06.004 Βαμβακούση, Ξ. (2019 ). Η προβλεπτική και επεξηγηματική ισχύς της προσέγγισης της θεωρίας πλαισίου στην εννοιολογική αλλαγή στα μαθηματικά: Η περίπτωση των ρητών αριθμών. Στο Ε. Σκοπελίτη & Ν. Κυριακοπούλου (Επιμ.), Νόηση και μάθηση υπό το πρίσμα της εννοιολογικής αλλαγής (σελ. 145-162). Εκδόσεις Gutenberg. Booth, J. L., & Newton, K. J. (2012). Fractions: Could they really be the gatekeeper’s doorman? Contemporary Educational Psychology, 37(4), 247-253. http://dx.doi.org/10.1016/j.cedpsych.2012.07.001 Booth, J. L., & Newton, K. J. (2012). Fractions: Could they really be the gatekeeper’s doorman? Contemporary Educational Psychology, 37(4), 247-253. http://dx.doi.org/10.1016/j.cedpsych.2012.07.001 Depaepe, F., Torbeyns, J., Vermeersch, N., Janssens, D., Janssen, R., Kelchtermans, G., Verschaffel, L., & Van Dooren, W. (2015). Teachers' content and pedagogical content knowledge on rational numbers: A comparison of prospective elementary and lower secondary school teachers. Teaching and Teacher Education, 47, 82-92. http://dx.doi.org/10.1016/j.tate.2014.12.009 Depaepe, F., Torbeyns, J., Vermeersch, N., Janssens, D., Janssen, R., Kelchtermans, G., Verschaffel, L., & Van Dooren, W. (2015). Teachers' content and pedagogical content knowledge on rational numbers: A comparison of prospective elementary and lower secondary school teachers. Teaching and Teacher Education, 47, 82-92. http://dx.doi.org/10.1016/j.tate.2014.12.009 Giannakoulias, E., Souyoul, A., & Zachariades, T. (2007). Students’ thinking about fundamental real numbers properties. In D. Pitta-Pantazi, & G. Philippou (Eds.), Proceedings of the Fifth Congress of the European Society for Research in Mathematics Education (pp. 426-425). ERME, Department of Education, University of Cyprus. http://erme.site/cerme-conferences/cerme-5/ Hannula, M. S., Pehkonen, E., Maijala, H., & Soro, R. (2006). Levels of students’ understanding on infinity.Teaching Mathematics and Computer Science, 4(2), 317–337. http://dx.doi.org/10.5485/ TMCS.2006.0129 Hannula, M. S., Pehkonen, E., Maijala, H., & Soro, R. (2006). Levels of students’ understanding on infinity.Teaching Mathematics and Computer Science, 4(2), 317–337. http://dx.doi.org/10.5485/ TMCS.2006.0129 Hartnett, P. M., & Gelman, R. (1998). Early understandings of number: Paths or barriers to the construction of new understandings? Learning and Instruction, 8, 341-374. http://dx.doi.org/10.1016/S0959- 4752(97)00026-1 Hartnett, P. M., & Gelman, R. (1998). Early understandings of number: Paths or barriers to the construction of new understandings? Learning and Instruction, 8, 341-374. http://dx.doi.org/10.1016/S0959- 4752(97)00026-1 Malara, N. (2001). From fractions to rational numbers in their structure: Outlines for an innovative didactical strategy and the question of density. In J. Novotná (Ed.), Proceedings of the 2nd Conference of the European Society for Research Mathematics Education II (pp. 35–46). ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Παρά τις διαφορές μεταξύ των μαθητών στις τρεις ομάδες, υπάρχει ένα κοινό σημείο: Κανένας μαθητής δεν αμφισβήτησε την αρχή του επόμενου στους ρητούς αριθμούς. Το εύρημα αυτό έχει ιδιαίτερο ενδιαφέρον όσον αφορά τους μαθητές της Ομάδας 1 που, αντίθετα με τους συμμαθητές τους των άλλων ομάδων, διέθεταν εξαρχής στρατηγικές που παράγουν ακολουθίες από άπειρους ενδιάμεσους σε ένα διάστημα, οικειοποιήθηκαν και τη στρατηγική του αριθμητικού μέσου και τις χρησιμοποίησαν όλες με συνέπεια στα διάφορα έργα. Τα παραδείγματά τους και οι εξηγήσεις τους δείχνουν ότι με τον όρο «άπειροι» αυτοί οι μαθητές αναφέρονταν στα προϊόντα μιας ατέρμονης διαδικασίας. Παρ’όλ’αυτά, συνέχισαν να πιστεύουν ότι ο επόμενος των δεδομένων αριθμών υπάρχει. Τα αποτελέσματα αυτά είναι σύμφωνα με ευρήματα πλήθους ερευνών που υποδεικνύουν ότι η πυκνή διάταξη των ρητών παρουσιάζει σημαντικές δυσκολίες για τους μαθητές διαφόρων ηλικιών και επιπέδων εκπαίδευσης (Depaepe et al., 2015· Giannakoulias et al., 2007· Malara, 2001· McMullen, et al., 2015· Merenluoto & Lehtinen, 2002· Neumann, 1998· Hannula et al., 2006· Smith, 1995· Smith et al., 2005· Tirosh et al., 1999· Vamvakoussi et al., 2012). Ειδικότερα, είναι συμβατά με την υπόθεση της προσέγγισης της θεωρίας πλαισίου για την εννοιολογική αλλαγή (Vosniadou et al., 2008) ότι η ιδέα της διακριτότητας, ως θεμελιώδης παραδοχή της αρχικής θεωρίας πλαισίου για τον αριθμό, δεν αίρεται διαμιάς. Όσον αφορά την απειρία των ενδιάμεσων, αυτό είναι εμφανές, για παράδειγμα, σε όλους τους μαθητές της Ομάδας 3, αλλά και σε μαθητές της Ομάδας 2 (π.χ., Μ10) που υπαναχώρησαν σε λιγότερο εκλεπτυσμένες απαντήσεις στο Μετ1. Ταυτόχρονα, τα αποτελέσματα ισχυροποιούν τον ισχυρισμό των Vamvakoussi και Vonsiadou (2012) ότι η αναθεώρηση της αρχής του επόμενου είναι πιο απαιτητική από την κατανόηση της απειρίας των ενδιάμεσων αριθμών. Επιπλέον, τεκμηριώνουν την ύπαρξη μιας συνθετικής αντίληψης για τη διάταξη των ρητών που συνδυάζει την απειρία των ενδιάμεσων αριθμών σε ένα διάστημα με την ύπαρξη του επόμενου, δείχνοντας ότι μαθητές που πράγματι συμπέραναν την απειρία των ενδιάμεσων (όλοι οι μαθητές της Ομάδας 1 αλλά και 62 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Bridging the gap: Fraction understanding is central to mathematics achievement in students from three different continents. Learning and Instruction, 37, 5- 13. http://dx.doi.org/10.1016/j.learninstruc.2014.03.002 Vamvakoussi, X., Van Dooren, W., & Verschaffel, L. (2012). Naturally biased? In search for reaction time evidence for a natural number bias in adults. 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Routledge. https://doi.org/10.4324/9781410602725 Steffe, L. P., & Thompson, P. W. (2000). Teaching experiment methodology: Underlying principles and essential elements. In A. E. Kelly & R. Lesh (Eds.), Handbook of research design in mathematics and science education (pp. 267-307). Routledge. https://doi.org/10.4324/9781410602725 Tirosh, D., Fischbein, E., Graeber, A. O., & Wilson, J. W. (1999). Prospective elementary teachers’ conceptions of rational numbers. Retrieved May 05, 2005 from http://jwilson.coe.uga.edu/Texts.Folder/ Tirosh/Pros.El.Tchrs.html Tirosh, D., Fischbein, E., Graeber, A. O., & Wilson, J. W. (1999). Prospective elementary teachers’ conceptions of rational numbers. Retrieved May 05, 2005 from http://jwilson.coe.uga.edu/Texts.Folder/ Tirosh/Pros.El.Tchrs.html Torbeyns, J., Schneider, M., Xin, Z., & Siegler, R. S. (2015). Bridging the gap: Fraction understanding is central to mathematics achievement in students from three different continents. Learning and Instruction, 37, 5- 13. http://dx.doi.org/10.1016/j.learninstruc.2014.03.002 Torbeyns, J., Schneider, M., Xin, Z., & Siegler, R. S. (2015). Βιβλιογραφία Univerzita Karlova v Praze, Pedagogická Faculta. http://erme.site/wp-content/uploads/2021/06/CERME2_proceedings.pdf Malara, N. (2001). From fractions to rational numbers in their structure: Outlines for an innovative didactical strategy and the question of density. In J. Novotná (Ed.), Proceedings of the 2nd Conference of the European Society for Research Mathematics Education II (pp. 35–46). Univerzita Karlova v Praze, Pedagogická Faculta. http://erme.site/wp-content/uploads/2021/06/CERME2_proceedings.pdf McMullen, J., Laakkonen, E., Hannula-Sormunen, M., & Lehtinen, E. (2015). Modeling the developmental trajectories of rational number concept(s). Learning and Instruction, 37, 14-20. http://dx.doi.org/10.1016/j.learninstruc.2013.12.004 McMullen, J., Laakkonen, E., Hannula-Sormunen, M., & Lehtinen, E. (2015). Modeling the developmental trajectories of rational number concept(s). Learning and Instruction, 37, 14-20. http://dx.doi.org/10.1016/j.learninstruc.2013.12.004 Merenluoto, K., & Lehtinen, E. (2002). Conceptual change in mathematics: Understanding the real numbers. In: M. Limon, & L. Mason (Eds.), Reconsidering conceptual change: Issues in theory and practice (pp. 233– 258). Kluwer. http://dx.doi.org/10.1007/0-306-47637-1_13 Merenluoto, K., & Lehtinen, E. (2002). Conceptual change in mathematics: Understanding the real numbers. In: M. Limon, & L. Mason (Eds.), Reconsidering conceptual change: Issues in theory and practice (pp. 233– 258). Kluwer. http://dx.doi.org/10.1007/0-306-47637-1_13 Moss, J. (2005). Pipes, tubes, and beakers: new approaches to teaching the rational-number system. In M. S. Donovan & J. D. Bransford (Eds.), How students learn: mathematics in the classroom (pp. 121–162). National Academic Press. https://nap.nationalacademies.org/read/11101/chapter/6 Moss, J. (2005). Pipes, tubes, and beakers: new approaches to teaching the rational-number system. In M. S. Donovan & J. D. Bransford (Eds.), How students learn: mathematics in the classroom (pp. 121–162). National Academic Press. https://nap.nationalacademies.org/read/11101/chapter/6 Neumann, R. (1998). Students’ ideas on the density of fractions. In H.G. Weigand, A. Peter-Koop, N.Neil, K.Reiss, G. Torner & B. Wollring (Eds.), Proceedings of the Annual Meeting of the Gesellschaft fur Didaktik der Mathematik (pp. 97-104). Retrieved June 13 2022 from Neumann, R. (1998). Students’ ideas on the density of fractions. In H.G. Weigand, A. Peter-Koop, N.Neil, K.Reiss, G. Torner & B. Wollring (Eds.), Proceedings of the Annual Meeting of the Gesellschaft fur Didaktik der Mathematik (pp. 97-104). Retrieved June 13 2022 from http://webdoc.gwdg.de/ebook/e/gdm/1998/neumann3.pdf der Mathematik (pp. 97-104). Retrieved June 13 2022 from http://webdoc.gwdg.de/ebook/e/gdm/1998/neumann3.pdf pp http://webdoc.gwdg.de/ebook/e/gdm/1998/neumann3.pdf 63 ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 ABSTRACT A major source of difficulty in the transition from natural to rational numbers is the interference of natural number knowledge in rational number learning. The framework theory approach to conceptual change assumes that students use their initial theories of numbers as natural numbers to make sense of rational numbers; and that the background assumptions of these theories are not easily revised, an example being the idea that numbers are discrete. Natural numbers are indeed discrete (i.e., given any natural number, it is always possible to define its successor in the natural numbers set). On the contrary, rational numbers are densely ordered (i.e., given any rational number, it is never possible to define its successor). The idea of discreteness stands in the way of students’ understanding of the density of rational numbers. We present a teaching experiment that investigated 15 11th graders’ understanding of two different aspects of density, namely the fact that there are infinitely many numbers in any rational number interval (“infinity of intermediates”); and that no rational number has a successor (“no successor principle”). We hypothesized that these aspects of density, although mathematically equivalent, are not equally challenging for students. First, students’ initial understandings of density were pre-tested in individual interviews; then each student was exposed to the idea of the arithmetic mean of two numbers that, in principle, could support understanding of both aspects of density; and, finally, each student revisited the tasks of the pretest and was prompted, when necessary, to use the idea of the arithmetic mean. Most students grasped the “infinity of intermediates” but all continued to assume the successor principle. From the perspective of the framework theory approach to conceptual change, this is a synthetic conception, since students accept the infinity of intermediates while retaining the successor principle. How 10th graders understand the dense ordering of rational numbers: a teaching experiment Dimitris FOKAS1, Xenia VAMVAKOUSSI2 1 Department of Primary Education, School of Humanities and Social Sciences, University of Western Macedonia 2 Department of Early Childhood Education, School of Education, University of Ioannina ΨΥΧΟΛΟΓΙΑ \| PSYCHOLOGY, 27 (1), 48-65 Lawrence Erlbaum Associates. http://dx.doi.org/10.4324/9780203154472 64 ΦΩΚΑΣ & ΒΑΜΒΑΚΟΥΣΗ (2022) ΕΜΠΕΙΡΙΚΗ ΕΡΓΑΣΙΑ \| RESEARCH PAPER Xenia Vamvakoussi, University of Ioannina, Department of Early Childhood Education, Metavatiko ktirio, 45110 University Campus, Ioannina xvamvak@uoi.gr CORRESPONDENCE © 2022, Dimitris Fokas, Xenia Vamvakoussi Licence CC-BY-SA 4.0 © 2022, Dimitris Fokas, Xenia Vamvakoussi Licence CC-BY-SA 4.0 65
https://openalex.org/W4284688662	https://www.frontiersin.org/articles/10.3389/fpls.2022.945471/pdf	English	null	SoyMAGIC: An Unprecedented Platform for Genetic Studies and Breeding Activities in Soybean	Frontiers in plant science	2,022	cc-by	8,891	ORIGINAL RESEARCH published: 07 July 2022 doi: 10.3389/fpls.2022.945471 ORIGINAL RESEARCH published: 07 July 2022 doi: 10.3389/fpls.2022.945471 SoyMAGIC: An Unprecedented Platform for Genetic Studies and Breeding Activities in Soybean Seyed Mohammad Hashemi , Gregory Perry , Istvan Rajcan and Milad Eskandari * Department of Plant Agriculture, Ontario Agriculture College, University of Guelph, Guelph, ON, Canada Multi-Parent Advanced Generation Inter-Cross (MAGIC) populations are emerging genetic platforms for high-resolution and fine mapping of quantitative traits, such as agronomic and seed composition traits in soybean (Glycine max L.). We have established an eight- parent MAGIC population, comprising 721 recombinant inbred lines (RILs), through conical inter-mating of eight soybean lines. The parental lines were genetically diverse elite cultivars carrying different agronomic and seed composition characteristics, including amino acids and fatty acids, as well as oil and protein concentrations. This study aimed to introduce soybean MAGIC (SoyMAGIC) population as an unprecedented platform for genotypic and phenotypic investigation of agronomic and seed quality traits in soybean. The RILs were evaluated for important seed composition traits using replicated field trials during 2020 and 2021. To measure the seed composition traits, near-infrared reflectance (NIR) was employed. The RILs were genotyped using genotyping-by-sequencing (GBS) method to decipher the genome and discover single-nucleotide polymorphic (SNP) markers among the RILs. A high-density linkage map was constructed through inclusive composite interval mapping (ICIM). The linkage map was 3,770.75 cM in length and contained 12,007 SNP markers. Chromosomes 11 and 18 were recorded as the shortest and longest linkage groups with 71.01 and 341.15 cM in length, respectively. Observed transgressive segregation of the selected traits and higher recombination frequency across the genome confirmed the capability of MAGIC population in reshuffling the diversity in the soybean genome among the RILs. The assessment of haplotype blocks indicated an uneven distribution of the parents’ genomes in RILs, suggesting cryptic influence against or in favor of certain parental genomes. The SoyMAGIC population is a recombined genetic material that will accelerate further genomic studies and the development of soybean cultivars with improved seed quality traits through the development and implementation of reliable molecular-based toolkits. Keywords: soybean (Glycine max L.), genetic linkage map, genotyping by sequencing, multi-parent advanced generation inter-crosses, Seed composition/quality Edited by: Kazuo N. Watanabe, University of Tsukuba, Japan Reviewed by: Giriraj Kumawat, ICAR Indian Institute of Soybean Research, India Milind B. Ratnaparkhe, ICAR Indian Institute of Soybean Research, India *Correspondence: Milad Eskandari meskanda@uoguelph.ca Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science Received: 16 May 2022 Accepted: 17 June 2022 Published: 07 July 2022 Received: 16 May 2022 Accepted: 17 June 2022 Published: 07 July 2022 ORIGINAL RESEARCH published: 07 July 2022 doi: 10.3389/fpls.2022.945471 INTRODUCTION The latter is facilitated by cost-effective, continuing, and reliable advances in high-throughput genotyping and phenotyping technologies that facilitated the establishment and evaluation of MAGIC populations with a large number of RILs along with well-developed phenotypic datasets. Since the 1920s, soybean [Glycine max (L.) Merr.] has been one of the major sources of protein and oil for human food and livestock feed in Canada (Singh and Hymowitz, 1999). Demand for this “king of beans” has been steadily increasing year-over-year due to its nutritional values for human and livestock, as well as industrial applications (Thrane et al., 2017). This growing demand has created a significant market for varieties with increased seed quality and yield, along with a range of improved agronomic traits. However, one of the main challenges for soybean breeders is the complexity associated with accumulating many of the desired quantitative traits in new cultivars. Many of these traits are regulated by multiple genes, located in different genomic regions, and tend to be dynamically regulated by a range of environmental, molecular, and biochemical factors (Whiting et al., 2020). A crucial step toward overcoming this challenge is deciphering the genetic structure of these quantitative traits, which can provide a prospect for plant breeders on how to select and develop cultivars with accumulated required traits. number of RILs along with well developed phenotypic datasets. The objective of this study was to develop and establish Soy MAGIC, an 8-founder soybean MAGIC population carrying various agronomic and seed composition traits, which can be used by researchers as an everlasting platform for deciphering and fine mapping of QTL associated with their target traits, and also to develop new value-added cultivars. Here, we present the process of SoyMAGIC development, high-density genetic linkage map construction as well as genetic features and validation of the population as a new genetic tool in soybean. The SoyMAGIC population with hundreds of RILs, each with a unique genetic combination of the eight parents and phenotypic performance, delivers a broad genetic resource for improving genetic gains of important traits in breeding programs as well as allowing for high precision QTL mapping of complex traits in soybean. Producing genetically recombinant crops through crossing two genetically diverse parents, so-called bi-parental crosses, has been one of the most important and common approaches for genetic studies and cultivar developments by plant geneticists and breeders. INTRODUCTION populations in self-pollinated crops includes crossing multiple genetically diverse inbred parental lines for several cycles, followed by single-seed descent selection process to produce recombinant inbred lines (RILs) carrying a mosaic of genome blocks from all parents (Scott et al., 2020). So far, the successful establishment of MAGIC population has been presented for several strategic crops such as maize (Jiménez-Galindo et al., 2019), barley (Novakazi et al., 2020), rice (Ponce et al., 2018), soybean (Shivakumar et al., 2018), and wheat (Stadlmeier et al., 2018). Scientific research publications in which MAGIC populations are used as the platform is showing a 250% increase in the last 10 years (Diouf and Pascual, 2021). The latter is facilitated by cost-effective, continuing, and reliable advances in high-throughput genotyping and phenotyping technologies that facilitated the establishment and evaluation of MAGIC populations with a large number of RILs along with well-developed phenotypic datasets. The objective of this study was to develop and establish Soy MAGIC, an 8-founder soybean MAGIC population carrying various agronomic and seed composition traits, which can be used by researchers as an everlasting platform for deciphering and fine mapping of QTL associated with their target traits, and also to develop new value-added cultivars. Here, we present the process of SoyMAGIC development, high-density genetic linkage map construction as well as genetic features and validation of the population as a new genetic tool in soybean. The SoyMAGIC population with hundreds of RILs, each with a unique genetic combination of the eight parents and phenotypic performance, delivers a broad genetic resource for improving genetic gains of important traits in breeding programs as well as allowing for high precision QTL mapping of complex traits in soybean. populations in self-pollinated crops includes crossing multiple genetically diverse inbred parental lines for several cycles, followed by single-seed descent selection process to produce recombinant inbred lines (RILs) carrying a mosaic of genome blocks from all parents (Scott et al., 2020). So far, the successful establishment of MAGIC population has been presented for several strategic crops such as maize (Jiménez-Galindo et al., 2019), barley (Novakazi et al., 2020), rice (Ponce et al., 2018), soybean (Shivakumar et al., 2018), and wheat (Stadlmeier et al., 2018). Scientific research publications in which MAGIC populations are used as the platform is showing a 250% increase in the last 10 years (Diouf and Pascual, 2021). INTRODUCTION Genetic variation of the parental lines provides the opportunity to decipher and map genomic regions, quantitative trait loci (QTL), which are associated with the trait of interest (Miles et al., 2008). A wide range of genetic studies have been conducted to date to identify QTL regions associated with soybean seed quality traits using bi-parental populations (Eskandari et al., 2013; Pei et al., 2018; Chen et al., 2021). Nevertheless, bi-parental populations despite having strong mapping power suffer from insufficiency of recombination events and genetic diversity for a given locus, which results from genetic segregation of loci coming from only two parents (Diouf and Pascual, 2021). In addition, in respect to soybean seed quality traits, as each QTL has a smaller effect on the trait (Diers et al., 1992; Hu et al., 2021), achieving higher mapping resolution, i.e., “fine mapping,” for developing more durable molecular markers, can be challenging using this type of populations. Citation: Hashemi SM, Perry G, Rajcan I and Eskandari M (2022) SoyMAGIC: An Unprecedented Platform for Genetic Studies and Breeding Activities in Soybean. Front. Plant Sci. 13:945471. doi: 10.3389/fpls.2022.945471 Front. Plant Sci. 13:945471. doi: 10.3389/fpls.2022.945471 July 2022 \| Volume 13 \| Article 945471 1 Frontiers in Plant Science \| www.frontiersin.org Soybean 8-Parent MAGIC Population Hashemi et al. Frontiers in Plant Science \| www.frontiersin.org DNA Extraction and High-Throughput Genotyping yp g Young leaves were collected from each individual RILs and parental lines and stored at −80° C after lyophilization. Afterward, DNA was extracted using the Macherey-Nagle NuceloSpin II DNA kit (MACHEREY-NAGEL, Germany) according to the manufacturer’s instructions. DNA quality and quantity were assessed through Nano-drop spectrophotometer ND-1000 (Nanodrop Technologies, Inc., Wilmington, DE, United States) along with a Qubit v2.0 Fluorometer (Thermo Fisher Scientific Inc., United States), respectively. DNA quality of parental lines was verified using 1% agarose gel (Voltage) and stained with ethidium bromide prior to imaging on a GelDoc system (Supplementary Figure S1). FIGURE 1 \| The conical cross used to establish the SoyMAGIC population. Capital words are representing eight elite parental cultivars, (A) OAC Prosper, (B) OAC 13-55C-HL, (C) OAC 07-78C-LL, (D) AC X790P, (E) RG 46, (F) RG 22, (G) RG 11, and (H) RG 23. Two-way crosses are represented by lower case letters (ab, bc, cd, de, ef, fg, gh, and ha). Four-way crosses are represented by four lowercase letters (abcd, bcde, bcde, fgde, ghef, hafg, ghab, and habc). Eight-way crosses are represented by eight lowercase letters (bcdeghab, fgdehabc, ghefabcd, hafgbcde, etc.). Black circles are showing the selfing generations, which ends up to the final RILs. FIGURE 1 \| The conical cross used to establish the SoyMAGIC population. Capital words are representing eight elite parental cultivars, (A) OAC Prosper, (B) OAC 13-55C-HL, (C) OAC 07-78C-LL, (D) AC X790P, (E) RG 46, (F) RG 22, (G) RG 11, and (H) RG 23. Two-way crosses are represented by lower case letters (ab, bc, cd, de, ef, fg, gh, and ha). Four-way crosses are represented by four lowercase letters (abcd, bcde, bcde, fgde, ghef, hafg, ghab, and habc). Eight-way crosses are represented by eight lowercase letters (bcdeghab, fgdehabc, ghefabcd, hafgbcde, etc.). Black circles are showing the selfing generations, which ends up to the final RILs. ( pp y g ) To genotype the RILs, sequencing libraries were prepared based on the genotyping by sequencing (GBS) protocol as explained by Elshire et al. (2011) except for the use of selective primers, which is described by Sonah et al. (2013) at the Plateforme d’analyses ge’nomiques (IBIS, Universite´ Laval). Normalized DNA concentrations of 10 ng/ml and restriction endonuclease of “ApeKI” were used in library preparation. Parental lines were genotyped by whole genome sequencing to obtain comprehensive genetic information as well as enough material for further investigations. Development of Soybean MAGIC Population p To develop the SoyMAGIC population, the following eight elite soybean lines were used as the founders: (A) OAC Prosper (Eskandari et al., 2017), (B) OAC 13-55C-HL, (C) OAC 07-78C- LL, (D) AC X790P (Poysa and Buzzell, 2001), (E) RG 46, (F) RG 22, (G) RG 11, and (H) RG 23 (Figure 1). These genetically diverse parental lines were selected based on their diverse phenotypic performance for important agronomic and seed quality traits (Table 1). Parental lines were inter-crossed in the form of conical crosses, consisting of eight parents and three cycles of crosses (Figure 1). In the first cycle, for each cross, the F1 seeds of eight 2-way mating combinations of the eight parents were generated in a way that each parent was used once as the female parent and once as the male parent. In the second cycle, F1 seeds of eight 4-way crosses, executed between the 2-way F1 plants, were generated such that each founding parent is present only once as the female and once as the male. Following the same pattern, the F1 seeds of eight 8-way crosses were generated by crossing the 4-way F1 plants. The plants resulting from the advanced inter-crossing stage were progressed four generations by single seed decent (SSD) to create 721 homozygous recombinant inbreed individuals. To address these limitations, various strategies have been proposed, including Advanced Intercrossed Lines (AILs), and Genome-Wide Association Studies (GWAS; Darvasi and Soller, 1995; Ozaki et al., 2002). However, AILs suffer from a low degree of genetic variation as a result of the presence of only two parents, and GWAS efficiency is also limited because of undetermined pedigree, missing parental information, and obtaining some false positive responses (Tam et al., 2019). A novel approach called “Multi-parent Advanced Generation Inter Crosses (MAGIC),” which was introduced by Kover et al. (2009), can to some extent address the above issues. In this approach, MAGIC populations resolve the issues associated with bi-parental analyses, and have a greater overall power in terms of genetic diversity, population structure, and mapping resolution (Huang et al., 2015; Diouf and Pascual, 2021). Developing MAGIC July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 2 Soybean 8-Parent MAGIC Population Hashemi et al. FIGURE 1 \| The conical cross used to establish the SoyMAGIC population. Development of Soybean MAGIC Population Capital words are representing eight elite parental cultivars, (A) OAC Prosper, (B) OAC 13-55C-HL, (C) OAC 07-78C-LL, (D) AC X790P, (E) RG 46, (F) RG 22, (G) RG 11, and (H) RG 23. Two-way crosses are represented by lower case letters (ab, bc, cd, de, ef, fg, gh, and ha). Four-way crosses are represented by four lowercase letters (abcd, bcde, bcde, fgde, ghef, hafg, ghab, and habc). Eight-way crosses are represented by eight lowercase letters (bcdeghab, fgdehabc, ghefabcd, hafgbcde, etc.). Black circles are showing the selfing generations, which ends up to the final RILs. pest, and weed management treatments. Plants in three middle rows were harvested after reaching full maturity. t The total chemical composition of soybean seed (30 g) was measured using Perten DA 7250 SD Near-Infrared Reflectance (NIR) spectrometer (Perten Instruments, Hägersten, Sweden). Seed samples were placed in a 9 mm diameter clear glass bottle at 4 mm height for the NIR spectrometer. Evaluation of seeds was performed for chemical components concentration as intact (without any treatment) using calibrations provided by Perten Instruments, as reported by Whiting et al. (2020). Three technical replications were applied for each measurement. Statistical analysis and visualization of the phenotype data were completed using R software packages including ggplot2, heatmaply, pastecs, and plotly. 1https://vcftools.github.io/ DNA Extraction and High-Throughput Genotyping Sequencing reads of parental lines were aligned to the reference genome, “William 82.” For the RILs, the variant call format (VCF) file was filtered out via VCFtools.1 After removing markers with more than 80% missing rate 183,482 SNPs remained out of 2,797,528 SNP markers. After individual level filtering, out of 760 individuals, 721 remained. Only bi-allelic SNPs remained. SNP imputation for the missing genotypes was carried out based on the haplotype structure of parental lines. TABLE 1 \| Descriptive characteristics of the parental lines for establishing the SoyMAGIC population. SoyMAGIC population. Parent ID Parent name Comment/characteristic A OAC Prosper High protein, high yield, large seed, semi- determinate B OAC 13-55C-HL High protein, high yield, and high linoleic C OAC 07-78C-LL High protein, high yield, and low linoleic D AC X790P Average yield, very high protein, large seed size E RG 46 High protein and oleic acid F RG 22 High oil, low stearic, and high linoleic acids G RG 11 High oil, high oleic, and low linolenic acids H RG 23 High protein, low stearic, and high oleic acids Parent ID Parent name Comment/characteristic mental Design and Phenotyping The RIL population was propagated in Ridgetown, Ontario, Canada (42°26′55.32″ N, 81°52′41.49″ W), during 2020 and 2021. The experiment was set up as a randomized complete block design (RCBD) with nearest neighbor adjustment and two replicates. Each plot consisted of five rows, 4.2 m long, with a row spacing of 43 cm. The rows were trimmed to 3.8 m in length after emergence, and the inside three rows were harvested. In each plot, 500 soybean seeds were planted to reach a plant density of 54 seeds per square meter (m−1). The plots were managed using conventional standard tillage, standard Physical map investigation and visualization were completed using rMVP and ggplot2 packages, R software (Wickham, 2017; Yin et al., 2021). Allelic contribution of parental lines in each chromosome was measured using “calc.genoprob” function with an error probability of 0.01 in the qtl2 package, R software (Broman et al., 2019). July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 3 Hashemi et al. Soybean 8-Parent MAGIC Population Genomic Features and Recombination Frequency of SoyMAGICt Genomic Features and Recombination Frequency of SoyMAGICt Phenotypic Variation in SoyMAGICh Phenotypic Variation in SoyMAGIC The normal distribution of phenotypic data was verified and confirmed by Shapiro Wilk test after removing outliers. As illustrated in Table 2, descriptive statistics of phenotypic data for RILs and parental lines were calculated. Almost all the selected seed composition traits showed lower minimums and higher maximums for RILs than parental lines. Moreover, the mean value of the protein and oil concentration was recorded higher in RILs than in parental lines. In terms of the fatty acids, the mean value of oleic, palmitic, and stearic acids decreased, whereas the mean value of linolenic and linoleic acids increased in RILs as compared to the parental lines. Amino acids such as histidine, alanine, tryptophan, phenylalanine, tyrosine, and proline had 2https://www.soybase.org/gb2/gbrowse/glyma.Wm82.gnm4/ Population Structure Principal component analysis (PCA) was carried out using TASSEL V5.2 to calculate the patterns of multi-locus variation (Bradbury et al., 2007). To illustrate the dispersion of the RILs in the population, the first two principal components (PCs) were used. According to the method of (Nei and Li, 1979), pairwise similarity coefficients were determined for all pairwise combinations of the RILs. To explore and visualized the familial relatedness among RILs, a Kinship matrix was also calculated using Genome Associated Prediction Integrated Tool (GAPIT) package in R (Lipka et al., 2012; Supplementary Figures S2 and S3). After discarding markers with a MAF ≤0.05 and heterozygous rate ≤0.13 from the 183,342 polymorphic SNPs and 721 individuals, 716 individuals with 122,747 SNPs remained, which were distributed across the whole soybean genome with an average spacing of 0.915 kb. Marker distribution varied among and within 20 chromosomes of soybean (Figure 3A). In the physical map, the largest and smallest numbers of markers were observed in Chromosomes 18 and 11 with 13,476 and 1,644 SNPs, respectively (Figure 3B). The mean genome-wide SNP number was recorded as 6,317 per chromosome (Figure 3B). Comparison of detected chromosome-wide markers with a gene density of G. max cultivar “William 82, genome assembly version 4” (Schmutz et al., 2010) demonstrated higher SNP frequency in the centromeric region of chromosome 2, 4, 18, and 20.2h Construction of Genetic Linkage Map Markers with heterozygosity of more than 12.5% were discarded. The distribution of average major allele frequency (AF), minor AF, and proportion of heterozygotes is illustrated in Figure 4. The average proportion of heterozygotes was 0.121 and 0.034 in the RILs and the parental lines, respectively. Average minor AF was 0.268 in parental lines and 0.188 among RILs, while the average major allele frequency was 0.732 and 0.812 in parental lines and RILs, respectively. The results indicated that the average MAF of the RILs was ranged from 0.101 on chromosome 19 to 0.337 on chromosome 14. This suggests that the SoyMAGIC RILs have higher average MAF and adequate polymorphism than the threshold (MAF < 0.05) for further genomic studies. Additionally, genome-wide and chromosome-wide assessment of parent’s allelic probability suggested that some parents contributed more to the SoyMAGIC RILs than others. Parents A and B with an average contribution of 19.3% and 14.2%, respectively, were more influential than the others (Figure 5). In contrast, parents D and E with an average contribution of 9.6% and 9.3%, respectively, were the least influential ones. Chromosomes 5 and 15 were recorded as the most unbalanced chromosomes with a maximum representation of parents A and G, respectively, and a minimum representation of parent F in both chromosomes. Construction of Genetic Linkage Map Construction of Genetic Linkage Map Genetic linkage map constriction of SoyMAGIC population was conducted using the inclusive composite interval mapping (ICIM- ADD) method in GAPL V1.2 software (Zhang et al., 2019). Before running the map construction, quality of the genotypic data was checked by the software. First, “SNP data conversion” function was used to convert the genetic dataset to the format of the software. Non-polymorphed markers either in parents or progenies and markers which were missing in one or more parents were filtered out. Afterward, identification and filtering of redundant markers was applied to remove the markers with a missing rate of ≥10%, while the markers with the minimum missing rate were set to present the co-localized markers. In a particular population, a set of co-localized markers was defined as one bin. Markers with heterozygosity of more than 12.5% were discarded. “Map construction in multi-parent derived pure-line populations” function was used to construct the genetic linkage map of SoyMAGIC population. Anchoring of markers with known chromosome ID on the physical map was the first step. Then, a grouping of markers was accomplished through anchored marker information and a threshold of marker recombination frequency (REC) of 0.3 for unanchored markers. For marker ordering, the two-optTSP and nearest-neighbor algorithms were used (Lin and Kernighen, 1973). Eventually, a window size of five-SNP was used as the rippling standard to measure the sum of adjacent recombination frequencies. Kosambi’s mapping function was used to convert the recombination frequency into map distance and the visualization of the genetic map was carried out using LinkageMapView package in R software (Ouellette et al., 2018). g p Genetic linkage map constriction of SoyMAGIC population was conducted using the inclusive composite interval mapping (ICIM- ADD) method in GAPL V1.2 software (Zhang et al., 2019). Before running the map construction, quality of the genotypic data was checked by the software. First, “SNP data conversion” function was used to convert the genetic dataset to the format of the software. Non-polymorphed markers either in parents or progenies and markers which were missing in one or more parents were filtered out. Afterward, identification and filtering of redundant markers was applied to remove the markers with a missing rate of ≥10%, while the markers with the minimum missing rate were set to present the co-localized markers. In a particular population, a set of co-localized markers was defined as one bin. RESULTS Population Development and Genotyping A set of 721 soybean MAGIC RILs was produced through three and four generations of advanced inter-crossing and self- pollination, respectively (Figure 1). GBS of RILs resulted in a total of 183,342 SNPs that were polymorphic between the eight parents and RILs. The RILs were on average 87.9% homozygous and appeared highly diverse and clustered uniformly relative to their eight parents, among which RG11, RG22, and RG23 were closer to each other than the other parent-to-parent relationships (Figure 2). July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 4 Hashemi et al. Soybean 8-Parent MAGIC Population higher mean values, whereas others had a lower mean for the RILs than the parental lines. Pearson’s correlation coefficient l f h d l l d b h parental lines and RILs. A positive correlation between all measured amino acids and seed protein concentration (r > 0.9) was observed. l b d b h FIGURE 2 \| PCA and phylogenetic relationships of the 716 SoyMAGIC RILs and eight parental lines (in red) based on 122747 SNP markers. A B FIGURE 3 \| SNP marker distribution on the genome of SoyMAGIC RILs. (A) Genome-wide distribution of SNP markers in the RILs of soybean MAGIC population. The number of SNPs is calculated and visualized in 1 Mb window size for each of the chromosomes (Chr). The number of markers per Mb is color-coded. (B) Number of SNP markers for each chromosome. The mean number of SNPs, 6317, across the whole genome was used as a baseline for intra-chromosome comparisons. Chromosomes 18 and 11 with highest and lowest number of SNPs are highlighted, respectively. FIGURE 2 \| PCA and phylogenetic relationships of the 716 SoyMAGIC RILs and eight parental lines (in red) based on 122747 SNP markers. FIGURE 2 \| PCA and phylogenetic relationships of the 716 SoyMAGIC RILs and eight parental lines (in red) based on 122747 SNP markers. A B FIGURE 3 \| SNP marker distribution on the genome of SoyMAGIC RILs. (A) Genome-wide distribution of SNP markers in the RILs of soybean MAGIC population. The number of SNPs is calculated and visualized in 1 Mb window size for each of the chromosomes (Chr). The number of markers per Mb is color-coded. (B) Number of SNP markers for each chromosome. RESULTS Summary statistic tables describe genome-wide proportion of heterozygosity and frequency of major and minor alleles of SoyMAGIC population in parental lines and RILs. FIGURE 5 \| Chromosome-wide and genome-wide allele contribution of parental lines. WG represents the contribution of parental lines in whole genome. A B FIGURE 4 \| Summary and pattern of genetic features in RILs and parental lines of SoyMAGIC population after filtering out of low-quality SNPs. (A) and (B) display chromosome-wide distribution of minor allele frequency and mean proportion of heterozygosity in the SoyMAGIC parental lines and RILs, respectively. Summary statistic tables describe genome-wide proportion of heterozygosity and frequency of major and minor alleles of SoyMAGIC population in parental lines and RILs. FIGURE 5 \| Chromosome-wide and genome-wide allele contribution of parental lines. WG represents the contribution of parental lines in whole genome. FIGURE 5 \| Chromosome-wide and genome-wide allele contribution of parental lines. WG represents the contribution of parental lines in whole genome. wide and genome-wide allele contribution of parental lines. WG represents the contribution of parental lines in whole genome. acids and fatty acids. In addition, as was expected, oleic acid showed a significant negative correlation with linoleic and linolenic acids (Figure 6). groups (LGs) with a total genome size of 3,770.75 centiMorgans (cM; Table 3). The highest and lowest map length was observed in LG18 and LG12 with 341.15 and 71.01 cM, respectively. The average length, across the LGs, was 188.54 cM. The number of markers for each linkage group ranged from 237 to 1,422 with an average of 600.35 marker. Additionally, the average marker interval was 0.37 cM. LG4 with an average distance RESULTS The mean number of SNPs, 6317, across the whole genome was used as a baseline for intra-chromosome comparisons. Chromosomes 18 and 11 with highest and lowest number of SNPs are highlighted, respectively. B A A B FIGURE 3 \| SNP marker distribution on the genome of SoyMAGIC RILs. (A) Genome-wide distribution of SNP markers in the RILs of soybean MAGIC population. The number of SNPs is calculated and visualized in 1 Mb window size for each of the chromosomes (Chr). The number of markers per Mb is color-coded. (B) Number of SNP markers for each chromosome. The mean number of SNPs, 6317, across the whole genome was used as a baseline for intra-chromosome comparisons. Chromosomes 18 and 11 with highest and lowest number of SNPs are highlighted, respectively. parental lines and RILs. A positive correlation between all measured amino acids and seed protein concentration (r > 0.9) was observed. However, negative correlation was observed between the amino higher mean values, whereas others had a lower mean for the RILs than the parental lines. Pearson’s correlation coefficient analysis of the seed quality traits was also measured among both July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 5 Soybean 8-Parent MAGIC Population Hashemi et al. A B FIGURE 4 \| Summary and pattern of genetic features in RILs and parental lines of SoyMAGIC population after filtering out of low-quality SNPs. (A) and (B) display chromosome-wide distribution of minor allele frequency and mean proportion of heterozygosity in the SoyMAGIC parental lines and RILs, respectively. Summary statistic tables describe genome-wide proportion of heterozygosity and frequency of major and minor alleles of SoyMAGIC population in parental lines and RILs. acids and fatty acids. In addition, as was expected, oleic acid showed a significant negative correlation with linoleic and linolenic acids (Figure 6). groups (LGs) with a total genome size of 3,770.75 centiMorgans (cM; Table 3). The highest and lowest map length was observed in LG18 and LG12 with 341.15 and 71.01 cM, respectively. The average length, across the LGs, was 188.54cM. The number A B FIGURE 4 \| Summary and pattern of genetic features in RILs and parental lines of SoyMAGIC population after filtering out of low-quality SNPs. (A) and (B) display chromosome-wide distribution of minor allele frequency and mean proportion of heterozygosity in the SoyMAGIC parental lines and RILs, respectively. Genetic Linkage Mapti After filtering out missing and low-quality markers using GAPL V1.2, 12,007 polymorphic SNPs were grouped into 20 linkage July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 6 Soybean 8-Parent MAGIC Population Hashemi et al. TABLE 2 \| Quantitative statistics for seed composition traits of parents and RILs in SoyMAGIC population. Traits RILs Parents Min Max Mean SD Min Max Mean SD Protein 38.35 49.49 44.16 1.424 41.27 48.39 43.91 1.998 Oil 18.45 24.47 21.55 0.958 18.20 23.27 21.24 1.453 Palmitic acid 9.96 14.02 12.14 0.491 11.54 12.89 12.33 0.443 Stearic acid 2.80 5.45 4.14 0.364 3.37 5.54 4.40 0.651 Oleic acid 15.10 40.34 26.72 3.543 17.75 39.61 27.77 6.053 Linoleic acid 39.18 57.56 48.56 2.862 37.31 53.98 47.63 5.331 Linolenic acid 3.42 10.49 6.79 0.942 3.83 9.06 6.54 1.484 Alanine 1.66 2.02 1.85 0.045 1.75 1.98 1.83 0.064 Arginine 2.78 3.81 3.33 0.138 2.96 3.61 3.27 0.183 Cysteine 0.44 0.63 0.53 0.033 0.52 0.61 0.56 0.034 Glycine 1.67 2.05 1.85 0.051 1.77 2.01 1.85 0.071 Histidine 1.02 1.27 1.14 0.035 1.06 1.25 1.13 0.054 Isoleucine 1.82 2.23 2.06 0.056 1.92 2.19 2.01 0.081 Leucine 2.98 3.76 3.41 0.101 3.18 3.66 3.37 0.139 Lysine 2.28 3.02 2.76 0.078 2.52 2.98 2.73 0.122 Methionine 0.47 0.60 0.54 0.019 0.52 0.59 0.56 0.021 Phenylalanine 1.99 2.54 2.29 0.071 2.12 2.49 2.25 0.107 Proline 1.87 2.33 2.12 0.067 1.96 2.28 2.08 0.101 Serine 1.63 2.10 1.88 0.071 1.75 2.08 1.87 0.091 Threonine 1.46 1.76 1.60 0.044 1.51 1.73 1.59 0.065 TABLE 2 \| Quantitative statistics for seed composition traits of parents and RILs in SoyMAGIC population. reciprocal conical design (Figure 1). To capture the maternal cytoplasmic genetic variance of parents (Morgan, 2013), the reciprocal conical crossing strategy was used during population development. of 0.15 cM was recorded as the densest LG, whereas LG7 had the largest average interval distance of 0.60 cM. The maximum and minimum interval distances were observed in LG19 and LG20 with 20.03 and 2.57 cM, respectively. p p p Soybean seed compositions, particularly oil and protein concentrations, are among the most studied traits in soybean due to their economic importance in the food and feed industries (Kumawat et al., 2016). Frontiers in Plant Science \| www.frontiersin.org Genetic Linkage Mapti Phenotypically, larger standard deviations, maximum and minimum values of the selected traits of RILs compared to parental lines (Table 2), confirmed the transgressive segregations and indicated the capability of SoyMAGIC population in reshuffling the genome in RILs. In fact, intensification of the genetic variation across the genome of RILs was because of the way that the population is developed. Similar results were reported for multi-parent populations of other plant crops such as rice, maize, cowpea, and eggplant, confirming the competence of multi-parent populations in reshuffling of genome and improving the recombination level (Dell’Acqua et al., 2015; Huynh et al., 2018; Ponce et al., 2020; Mangino et al., 2022). Since the eight parents were all completely inbred lines, the plants in each F1 set were homogeneously heterozygous. Theoretically, the F1s resulting from the four-way crosses, on the other hand, segregate and show substantial heterogeneity (Figure 1). This heterozygosity and heterogeneity generated individuals with recombined genotypes and phenotypes. Furthermore, using four generations of SSD selection, in which we did not apply any targeted selective pressure for any of the target traits, a genetically and phenotypically diverse RIL population consisting of 721 was generated and established as the SoyMAGIC population. DISCUSSION MAGIC populations are exceptional genetic resources for improving the recombination frequency of resultant RILs and discovering marker-trait relationships with high accuracy and resolution accordingly (Scott et al., 2020). Multiple parents with greater phenotypic and genetic variation, as well as multiple rounds of inter-crossing and selfing, enhance the number of recombination events and therefore maximize mapping accuracy (Huang et al., 2015). Through inter-crossing diverse parents for a particular trait, the genetic variability in the final RILs increases, which is a decisive advantage of developing these types of populations for genetic studies (Scott et al., 2020). Several studies have previously exploited MAGIC populations for investigating genetic control of important trait in strategic crops such as maize (Jiménez-Galindo et al., 2019), rice (Ponce et al., 2018) and wheat (Stadlmeier et al., 2018). Here, we report the establishment of a soybean MAGIC (SoyMAGIC) population developed by combining eight parental lines that were genetically and phenotypically diverse for several agronomic and seed quality traits (Table 1, Supplementary Table S1, and Figure 1). In plant breeding programs, a large population size is one of the necessary factors to maximize the mapping resolution (Beavis, 1998; Rosenthal and Borschbach, 2014). The SoyMAGIC population was maintained reasonably large at 721 RILs, to accumulate a wider range of recombination events, using a In plant breeding programs, a large population size is one of the necessary factors to maximize the mapping resolution (Beavis, 1998; Rosenthal and Borschbach, 2014). The SoyMAGIC population was maintained reasonably large at 721 RILs, to accumulate a wider range of recombination events, using a To discriminate genotypes for their genetic diversity in plant genetic and breeding activities, GBS has already been confirmed to be an exceptionally efficient and cost-effective approach for July 2022 \| Volume 13 \| Article 945471 7 Hashemi et al. Soybean 8-Parent MAGIC Population FIGURE 6 \| Pearson’s (r) correlation coefficient among seed quality traits in RILs of SoyMAGIC population. FIGURE 6 \| Pearson’s (r) correlation coefficient among seed quality traits in RILs of SoyMAGIC population. FIGURE 6 \| Pearson’s (r) correlation coefficient among seed quality traits in RILs of SoyMAGIC population TABLE 3 \| Information on linkage map of the SoyMAGIC population. Linkage group Map length No. of markers No. Frontiers in Plant Science \| www.frontiersin.org DISCUSSION of bins Interval distance Max Average 1 151.08 377 110 6.17 0.4 2 165.7 421 116 3.76 0.39 3 180.18 709 215 5.55 0.25 4 212.03 1,422 484 6.03 0.15 5 153.77 391 131 4.18 0.39 6 215.75 464 167 5.4 0.46 7 180.69 303 95 5.67 0.6 8 182.65 496 163 6.55 0.37 9 273.47 655 229 5.8 0.42 10 183.13 352 113 7.62 0.52 11 137.45 266 98 5.7 0.52 12 71.01 247 77 3.09 0.29 13 179.05 381 143 4.79 0.47 14 263.07 1,158 493 7.52 0.23 15 230.97 1,110 378 5.24 0.21 16 203.09 609 233 7.12 0.33 17 189.05 593 199 6.54 0.32 18 341.15 1,126 322 5.65 0.3 19 137.37 237 63 20.03 0.58 20 120.1 690 232 2.57 0.17 Total 3770.75 12,007 4,061 20.03 0.37 TABLE 3 \| Information on linkage map of the SoyMAGIC population. the genotyping of large multi and bi-parental populations (He et al., 2014; Kishor et al., 2021). WGS of parental lines has also been reported as a highly effective genotyping strategy in multiparent plant breeding programs, which can be employed in further genetic investigations such as QTL mapping and identification of candidate genes (Islam et al., 2016; Thyssen et al., 2019). Detection of 183,342 SNP markers across the genome, confirmed that GBS of RILs, imputed using WGS of the parental lines, could be a suitable method for generating a high-resolution map for soybean multiparent genotyping. In this study, higher number of SNP markers was observed around telomeric regions of most of the chromosomes, whereas chromosome 5, 7, 12 and 13 exhibited higher SNP density around centromeric area (Figure 3). These results reflect the strength of SoyMAGIC population in reshuffling alleles across the genome and providing a highly recombined genomic platform suitable for discovering QTL/candidate genes associated with complex traits. Theoretically, in an 8-parent MAGIC population, each of the parental lines should contribute 12.5%. However, certain paternal lines contributed more to the SoyMAGIC population than others (Figure 5). The observed variance in the contribution of founders might be caused by a variety of genotypic or environmental factors such as fertility reduction or male sterility due to environmental conditions (Brauner-Otto, 2014; Li et al., 2019). July 2022 \| Volume 13 \| Article 945471 8 Soybean 8-Parent MAGIC Population Hashemi et al. DISCUSSION Genotypic and phenotypic data generated for these studies will be stored and made available to breeders for improving their selection criteria and establishing efficient breeding strategies. DISCUSSION in order to maintain maternal genetic materials and high recombination rate in the RILs The population represents a FIGURE 7 \| Genetic linkage map constructed from SoyMAGIC. It has been shown that SNP discovery in soybeans is a challenging and time-consuming process (Wu et al., 2010). Limited sequence variation in currently cultivated varieties as well as the complicated nature of the soybean genome are two critical factors causing the complications (Choi et al., 2007). Considering these challenges, we have constructed a new and high-density genetic linkage map that contains 12,007 SNP markers with a genome length of 3,770.75 cM by employing an eight-parent RIL population. Compare to the previous studies on soybean genetic linkage maps of bi-parental populations (Hyten et al., 2010; Song et al., 2016), the current map demonstrated a greater number of distinct sites, comparable genome length, and shorter average bin size (Table 3, Figure 7). In comparison to bi-parental populations (Hyten et al., 2010), the SoyMAGIC population displayed a significantly higher number of marker alleles at each locus, which reflects the capacity of SoyMAGIC for enhancing genetic variation and recombination frequency in the population. Establishing genetic linkage map is an important step for the dissection of genome regions associated with important agronomic and quality traits through identifying the location of quantitative trait loci (QTL; Williams, 2018). Through improving genetic recombination in RILs, SoyMAGIC has provided a desired platform for discovering marker’s location across the genome and constructing a high-density genetic linkage map, which, in turn, provided a strong platform for further marker-trait association investigations. So far, several MAGIC population-derived RILs have been developed to dissect the genome of many crops using different mapping strategies (Scott et al., 2020). For instance, Huynh et al. (2018) used linkage map in an eight parent cowpea MAGIC population with 305 RILs, leading to the successful detection of four QTL underlying flowering time. Huang et al. (2021) using genome- wide association mapping in an 8-way upland cotton MAGIC population, discovered 177 SNPs strongly associated with nine agronomic traits in multiple environments. SoyMAGIC population will provide researchers with immortal diverse plant materials that can be tested across a wide range of environments with different types of biotic and abiotic stresses for discovering environment-specific effects of genomic regions associated with traits. Frontiers in Plant Science \| www.frontiersin.org REFERENCES Eskandari, M., Cober, E. R., and Rajcan, I. (2013). Genetic control of soybean seed oil: II. QTL and genes that increase oil concentration without decreasing protein or with increased seed yield. Theor. Appl. Genet. 126, 1677–1687. doi: 10.1007/s00122-013-2083-z Beavis, W. D. (1998). QTL Analyses: Power, Precision, and Accuracy. 1st Edn. Boca Raton, FL: CRC Press. He, J., Zhao, X., Laroche, A., Lu, Z. X., Liu, H. K., and Li, Z. (2014). Genotyping- by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding. Front. Plant Sci. 5, 1–8. doi: 10.3389/fpls.2014.00484 Bradbury, P. J., Zhang, Z., Kroon, D. E., Casstevens, T. M., Ramdoss, Y., and Buckler, E. S. (2007). TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635. doi: 10.1093/bioinformatics/btm308f Hu, Q., Zhang, Y., Ma, R., An, J., Huang, W., Wu, Y., et al. (2021). Genetic dissection of seed appearance quality using recombinant inbred lines in soybean. Mol. Breed. 41:72. doi: 10.1007/s11032-021-01262-9 Brauner-Otto, S. R. (2014). Environmental quality and fertility: the effects of plant density, species richness, and plant diversity on fertility limitation. Popul. Environ. 36, 1–31. doi: 10.1007/s11111-013-0199-3 Huang, C., Shen, C., Wen, T., Gao, B., Zhu, D., Li, D., et al. (2021). Genome- wide association mapping for agronomic traits in an 8-way upland cotton MAGIC population by SLAF-seq. Theor. Appl. Genet. 134, 2459–2468. doi: 10.1007/s00122-021-03835-w Broman, K. W., Gatti, D. M., Simecek, P., Furlotte, N. A., Prins, P., Sen, S., et al. (2019). R/qtl2: software for mapping quantitative trait loci with high- dimensional data and multiparent populations. Genetics 211, 495–502. doi: 10.1534/genetics.118.301595 Huang, B. E., Verbyla, K. L., Verbyla, A. P., Raghavan, C., Singh, V. K., Gaur, P., et al. (2015). MAGIC populations in crops: current status and future prospects. Theor. Appl. Genet. 128, 999–1017. doi: 10.1007/s00122-015-2506-0 Chen, H., Pan, X., Wang, F., Liu, C., Wang, X., Li, Y., et al. (2021). Novel QTL and meta-QTL mapping for major quality traits in soybean. Front. Plant Sci. 12, 1–22. doi: 10.3389/fpls.2021.774270 Huynh, B. L., Ehlers, J. D., Huang, B. E., Muñoz-Amatriaín, M., Lonardi, S., Santos, J. R. P., et al. (2018). A multi-parent advanced generation inter-cross (MAGIC) population for genetic analysis and improvement of cowpea (Vigna unguiculata L. Walp.). Plant J. 93, 1129–1142. doi: 10.1111/tpj.13827 Choi, I. Y., Hyten, D. L., Matukumalli, L. K., Song, Q., Chaky, J. M., Quigley, C. V., et al. (2007). FUNDING This project was funded in part through the Ontario Regional Priorities Partnership Program (ON-RP3), a collaborative initiative between the Agricultural Adaptation Council, Ontario Genomics, the Government of Canada through Genome Canada, and SeCan. ACKNOWLEDGMENTS The original contributions presented in the study are publicly available. This data can be found here: https://github.com/ SeyedMH/SoyMAGIC. The authors acknowledge Robert Brandt and Lin Liao for managing field trails and making crosses during the development of the SoyMAGIC population and Sepideh Torabi for sharing insights on soybean genomics experiments and research. SUPPLEMENTARY MATERIAL ME: conceptualization. SH: validation, data curation, visualization, and writing. SH and GP: formal analysis. SH, ME, IR, and GP: review and editing. ME and SH: project administration. All authors have read and agreed to the published version of the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.945471/ full#supplementary-material CONCLUSION In addition to serving as an immortal genetic resource for precise marker-traits association studies and precise QTL mapping, SoyMAGIC will support breeding programs in the long run by offering valuable pre-breeding resources. The preliminary phenotypic data collected on agronomic and seed quality traits along with the SNP data set showed large phenotypic and genetic diversity among the lines within the population, which indicate the potential benefits and advantages of using this diverse germplasm in genetic studies and breeding activities by the soybean community. SoyMAGIC has been established by inter-crossing eight founders using reciprocal conical crosses in order to maintain maternal genetic materials and high recombination rate in the RILs. The population represents a valuable plant germplasm resource, which consists of 721 highly July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 9 Soybean 8-Parent MAGIC Population Hashemi et al. recombined RILs with a large degree of phenotypic variation. 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Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Schmutz, J., Cannon, S. B., Schlueter, J., Ma, J., Mitros, T., Nelson, W., et al. (2010). Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183. doi: 10.1038/nature08670 Scott, M. F., Ladejobi, O., Amer, S., Bentley, A. R., Biernaskie, J., Boden, S. A., et al. (2020). Multi-parent populations in crops: a toolbox integrating genomics and genetic mapping with breeding. Heredity (Edinb). 125, 396–416. doi: 10.1038/s41437-020-0336-6 Copyright © 2022 Hashemi, Perry, Rajcan and Eskandari. 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. Shivakumar, M., Kumawat, G., Gireesh, C., Ramesh, S. V., and Husain, S. M. (2018). Soybean MAGIC population: a novel resource for genetics and plant breeding. Curr. Sci. 114, 906–908. doi: 10.18520/cs/v114/i04/906-908 July 2022 \| Volume 13 \| Article 945471 Frontiers in Plant Science \| www.frontiersin.org 11
https://openalex.org/W4311040520	https://www.researchsquare.com/article/rs-2314353/latest.pdf	English	null	Racial and Ethnic Differences in Self-reported Satisfaction With Fertility Clinics and Doctors	Research Square (Research Square)	2,022	cc-by	8,615	Racial and Ethnic Differences in Self-reported Satisfaction With Fertility Clinics and Doctors Ange Wang (  angewang31@gmail.com ) Ange Wang (  angewang31@gmail.com ) Racial and Ethnic Differences in Self-reported Satisfaction With Fertility Clinics and Doctors Ange Wang (  angewang31@gmail.com ) Stanford University School of Medicine https://orcid.org/0000-0002-7133-0950 Jake Anderson-Bialis Jerrine R. Morris Jamie Corley Deborah Anderson-Bialis Lauren Citro Muhammad Esa Seegulam Victor Fujimoto Research Article Keywords: assisted reproductive technologies, fertility, disparities, race/ethnicity, patient satisfaction, clinic satisfaction Posted Date: November 30th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2314353/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Ful License Version of Record: A version of this preprint was published at Archives of Gynecology and Obstetrics on April 18th, 2023. See the published version at https://doi.org/10.1007/s00404-023-07043-3. Racial and Ethnic Differences in Self-reported Satisfaction With Fertility Clinics and Doctors Ange Wang (  angewang31@gmail.com ) Stanford University School of Medicine https://orcid.org/0000-0002-7133-0950 Jake Anderson-Bialis Jerrine R. Morris Jamie Corley Deborah Anderson-Bialis Lauren Citro Muhammad Esa Seegulam Victor Fujimoto Research Article Keywords: assisted reproductive technologies, fertility, disparities, race/ethnicity, patient satisfaction, clinic satisfaction Posted Date: November 30th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2314353/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Ful License Version of Record: A version of this preprint was published at Archives of Gynecology and Obstetrics on April 18th, 2023. See the published version at https://doi.org/10.1007/s00404-023-07043-3. Research Article DOI: https://doi.org/10.21203/rs.3.rs-2314353/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Archives of Gynecology and Obstetrics on April 18th, 2023. See the published version at https://doi.org/10.1007/s00404-023-07043-3. Page 1/25 Page 1/25 Abstract Objective: To investigate if differences in self-reported satisfaction with fertility clinics and doctors differ by race/ethnicity. Study Design: We used cross-sectional survey data from FertilityIQ online questionnaires completed by patients receiving U.S. fertility care from July 2015 to December 2020. Univariate and multivariate logistic and linear regression analyses performed to assess association of race/ethnicity on patient reported clinic and physician satisfaction. Results: Our total sample size included 21,472 patients (15,986 Caucasian, 1,856 Black, 1,780 LatinX, 771 East Asian, 619 South Asian, 273 Middle Eastern, 187 Native American self-reported). When adjusting for all confounders (demographic and patient satisfaction), we found that Black patients rated their doctors more highly (OR 1.30, 95% CI 1.04-1.62 p=0.022 logistic and Coef 0.082, 95% CI 0.013-0.15 p=0.02 linear), while other ethnic groups did not show significant differences compared to Caucasian patients. East Asians had borderline lower satisfaction with clinic satisfaction in logistic regression (OR 0.74 95% CI 0.55-1.00 p=0.05), while significant differences were not found for other ethnic groups for clinic satisfaction. Conclusions: In summary, some but not all minority groups differed in their self-reported perception of satisfaction with fertility clinic and doctors compared to Caucasian patients. Cultural differences towards surveys may contribute to some of these findings, and satisfaction by racial/ethnic group may also be modified by results of care. Introduction Prior literature has demonstrated disparities in assisted reproductive technology outcomes by racial/ethnic group. Minorities and particularly Black patients have been found to have worse outcomes when compared to Caucasian patients in multiple studies.(1, 2) An analysis of 38,309 cycles using Society for Assisted Reproductive Technology Clinic Outcome Reporting System (SART CORS) data found increased miscarriage rate and decreased live birth rate compared to Black women compared to white women, even after controlling for uterine and tubal factor disease.(3) Studies have also reported that even in cycles with preimplantation genetic testing (PGT), Black race was associated with lower live birth rate in frozen embryo transfer cycles (adjusted risk ratio, 0.82; 95% confidence interval [CI], 0.73–0.92) and a higher risk of clinical loss (adjusted risk ratio, 1.59; 95% CI, 1.28–1.99).(4) Another study reported that Black recipients with donor embryo transfer cycles had a lower probability of live birth than white donors and recipients (adjusted risk ratio, 0.84; 95% confidence interval, 0.71– 0.99).(5) Disparities in assisted reproductive technology (ART) outcomes for Asian and Hispanic groups have also been reported, with a large SART study finding that adjusted odds of pregnancy were reduced for Asians (0.86), and that live birth odds were reduced for multiple minority groups: Asians (0.90), Black (0.62), and Hispanic (0.87).(6) Studies on South Asian populations have reported inconsistent information on disparities, including some studies reporting worse outcomes in this population, while other have reported equivalent outcomes.(7, 8) Studies on Hispanic patients have been limited by sample size, though some studies have found that live birth rates from ART may be decreased for this population.(6) Literature on patient perspective of self-reported fertility care satisfaction (in general and also by race/ethnicity) is extremely limited. Introduction A study of 1,460 patients at an academic fertility center in Illinois found that Black women Page 2/25 (14.7%) were more likely to report race as a barrier to fertility treatment compared with other races, though satisfaction with fertility care was not assessed.(9) A prior FertilityIQ analysis of 7,456 women from 2015–2018 reported a number of factors associated with positive patient-reported experience.(10) Other literature has investigated patient satisfaction in fertility care, with studies generally reporting high general patient satisfaction (94%).(11–14) In other fields of medicine, several studies have suggested that minority groups (including East Asians and Black patients) may report lower satisfaction that White groups (15, 16), though not all studies have found these effects.(17) In general, the literature on patient perspective and satisfaction by race/ethnicity for fertility care and other medical fields is very limited, and this is an important are for future research. (14.7%) Given the known disparities in ART outcomes and the lack of literature on patient satisfaction in fertility care by race/ethnicity, we aimed to use the large, multi-racial FertilityIQ database to investigate if differences in self- reported satisfaction with fertility clinics and doctors also differ by race/ethnicity, using seven distinct categories categorized by FertilityIQ (Caucasian, Black, East Asian, South Asian, LatinX, Middle Eastern, Native American). This is one of the first studies of this subject matter of racial/ethnic satisfaction with fertility care, and the largest to date. Materials And Methods This is a cross-sectional survey study using online-based FertiliyIQ questionnaires, which were voluntarily completed by English-speaking patients seeking care at U.S. fertility centers. FertilityIQ (www.fertilityiq.com) is an online website that contains extensive fertility-related resources (with both free and paid resources), including clinic and physician satisfaction questionnaires, courses on fertility and assisted reproductive technology topics, and information from fertility experts. All surveys were conducted in English from July 2015 to December 2020. FertilityIQ has voluntary surveys available on most United States fertility clinics and doctors. This study was determined to be exempt from Institutional Review Board by the University of California, San Francisco, as this did not involve human subjects and only deidentified survey data was used for analysis. Study and participants The FertilityIQ questionnaire has been described in prior literature.(10) To summarize, the online survey contains 115 questions on a variety of fertility-related topics including patient demographics, fertility diagnoses and treatment, and clinic and doctor information. The full survey is available at www.fertilityiq.com. The survey includes both mandatory and optional questions (with the majority being optional), as well as skip logic where applicable. The survey contains a variety of question formats, including multiple choice, free text, and Likert scale, depending on the question of interest. All surveys were voluntarily filled out by patients with experience at U.S. fertility clinics. Patient responses and clinic experiences were not verified. Racial/ethnic and outcomes classification We classified patients into the following self-reported racial/ethnic groups based on the FertilityIQ questionnaire: Caucasian, Black, East Asian (EA), South Asian (SA), LatinX, Middle Eastern (ME), and Native American (NA), according to the ethnicities surveyed in the questionnaire. Multi-racial or unknown race were excluded from the analysis. Page 3/25 Page 3/25 In terms of classification of doctor and clinic satisfaction outcomes, we used methodology similar to a prior analysis on FertilityIQ general patient satisfaction.(10) Patient and clinic satisfaction was classified according to the answer to the question ‘‘Would you recommend this fertility clinic/physician to a best friend?” which was answered on a 1–10 scale (1 = no, 10 = very likely to recommend). This is a required question on the FertilityIQ questionnaire. We evaluated doctor and clinic satisfaction as both categorical and continuous outcomes. For categorical outcomes, a score of 9 or 10 was categorized as a positive patient satisfaction outcome. Per prior literature, this positive patient satisfaction outcome was chosen based on busines literature on net promotor score (NPS) in customer surveys, which is designed to ‘‘limit the promoter designation to only those who are the most enthusiastic consumers to avoid ‘grade inflation’ that occurs with traditional customer-satisfaction assessments.’’(10, 18) For continuous outcomes, the exact numerical score (1–10) was used in analysis. Statistical analysis We investigated the outcomes of clinic and doctor satisfaction as both categorical and continuous outcomes, using logistic and linear regression models respectively. We studied the outcomes using univariate and multivariate models. In our multivariate models, we adjusted for both demographic confounders, as well as other confounders which may affect satisfaction with fertility clinics/doctors based on prior literature. Demographic confounders included age (continuous), insurance status (Yes/No), gender (Male, Female, Other, Decline to state), LGBTQ status (Yes/No), income ($0-49K, $50-99K, $100-199K, $200-499K, $500K+), education (less than high school, HS/GED, some college, bachelor’s degree, master’s degree, professional degree, doctorate), state, and profession. Patient satisfaction confounders included questionnaire elements such as results of treatment, treated as number/human, good communicator, trustworthy, billing, wait time, convenience, understand, adjustment of plan, presence of primary RN, error made, frequency of seeing doctor (patient satisfaction questions were either a Likert scale or Yes/No). These patient satisfaction confounders were chosen based on factors found to be significantly associated with a positive fertility clinic experience using previous FertilityIQ data.(10) All tests were 2-sided with significance at the alpha = 0.05 level. Statistical analyses were performed using Stata Version 15 (Stata Corporation). Results Our total sample size included 21,472 patients (15,986 Caucasian, 1,856 Black, 1,780 LatinX, 771 EA, 619 SA, 273 Middle Eastern, 187 Native American) (Table 1). In terms of baseline characteristics, the groups were overall similar on age (p = 0.61, ranging from mean of 35.72 years for Native American patients to 36.76 years for Middle Eastern patients). The groups significantly differed on all other demographic and patient satisfaction characteristics (p < 0.05), though not all differences were clinically significant. Over 90% of participants identified as female and non-LGBT. Caucasian, EA, SA, and Middle Eastern groups reported higher income with a larger percentage of respondents over $200K per year, and a higher percentage of respondents with college degree or higher. Unexplained infertility was the most common diagnosis across all races, followed by male factor and PCOS. Racial/ethnic groups also differed significantly on all patient satisfaction confounders (p < 0.01), though not all differences appeared to be clinically significant and may have been driven by the large sample size. Results Page 4/25 Table 1 Baseline characteristics (see next page) Caucasian Black East Asian South Asian LatinX Middle Eastern Native American p- value N 15986 1856 771 619 1780 273 187 Percentage 74.5% 8.6% 3.6% 2.9% 8.3% 1.3% 0.9% Demographic characteristics: Age at survey, mean (SD) 36.30 (9.43) 35.94 (8.36) 36.44 (8.01) 36.48 (10.89) 36.33 (8.73) 36.76 (11.35) 35.72 (10.15) 0.61 Gender < 0.001 Female 15416 (96.5%) 1802 (97.2%) 732 (95.3%) 570 (92.8%) 1722 (96.9%) 253 (92.7%) 181 (97.3%) Male 535 (3.3%) 47 (2.5%) 32 (4.2%) 43 (7.0%) 54 (3.0%) 20 (7.3%) 5 (2.7%) Other 10 (0.1%) 1 (0.1%) 0 (0.0%) 0 (0.0%) 1 (0.1%) 0 (0.0%) 0 (0.0%) Decline to state 14 (0.1%) 3 (0.2%) 4 (0.5%) 1 (0.2%) 0 (0.0%) 0 (0.0%) 0 (0.0%) LGBT (self- classified) < 0.001 Yes 719 (4.5%) 140 (7.6%) 33 (4.3%) 21 (3.4%) 104 (5.9%) 11 (4.1%) 17 (9.3%) No 15100 (94.9%) 1697 (92.0%) 723 (94.4%) 581 (94.9%) 1642 (92.6%) 254 (94.4%) 166 (90.7%) Decline to state 93 (0.6%) 8 (0.4%) 10 (1.3%) 10 (1.6%) 27 (1.5%) 4 (1.5%) 0 (0.0%) Income < 0.001 $0-49K 1061 (6.6%) 482 (26.1%) 29 (3.8%) 37 (6.0%) 327 (18.4%) 38 (14.0%) 41 (21.9%) $50-99K 5459 (34.2%) 775 (41.9%) 142 (18.6%) 138 (22.4%) 728 (41.0%) 61 (22.4%) 95 (50.8%) $100-199K 6000 (37.6%) 387 (20.9%) 269 (35.2%) 237 (38.5%) 497 (28.0%) 81 (29.8%) 37 (19.8%) $200-499K 2235 (14.0%) 110 (5.9%) 211 (27.6%) 122 (19.8%) 133 (7.5%) 51 (18.8%) 9 (4.8%) $500K + 366 (2.3%) 16 (0.8%) 44 (5.7%) 26 (4.2%) 12 (0.7%) 14 (5.1%) 1 (0.5%) Decline to state Table 1 Baseline characteristics (see next page) Page 5/25 Page 5/25 Caucasian Black East Asian South Asian LatinX Middle Eastern Native American p- value Education < 0.001 Less than HS 4 (< 1%) 4 (0.2%) 1 (0.1%) 1 (0.2%) 5 (0.3%) 0 (0.0%) 0 (0.0%) HS/GED 281 (1.8%) 149 (8.0%) 10 (1.3%) 12 (1.9%) 135 (7.6%) 11 (4.1%) 24 (12.9%) Some college 2126 (13.3%) 518 (27.9%) 45 (5.9%) 54 (8.8%) 526 (29.6%) 41 (15.1%) 75 (40.3%) Bachelor's degree 6382 (40.0%) 507 (27.3%) 279 (36.4%) 205 (33.2%) 589 (33.1%) 68 (25.1%) 51 (27.4%) Master's degree 4755 (29.8%) 466 (25.1%) 221 (28.8%) 198 (32.1%) 343 (19.3%) 73 (26.9%) 24 (12.9%) Professional degree 1692 (10.6%) 146 (7.9%) 161 (21.0%) 101 (16.4%) 138 (7.8%) 54 (19.9%) 11 (5.9%) Doctorate 727 (4.6%) 64 (3.5%) 50 (6.5%) 46 (7.5%) 43 (2.4%) 24 (8.9%) 1 (0.5%) Insurance coverage (for infertility) 11037 (69.0%) 1303 (70.2%) 502 (65.1%) 429 (69.3%) 1155 (64.9%) 155 (56.8%) 106 (56.7%) < 0.001 Diagnosis Ovulatory 1490 (9.3%) 100 (5.4%) 38 (4.9%) 37 (6.0%) 151 (8.5%) 12 (4.4%) 21 (11.2%) <0.001 DOR 3019 (18.9%) 271 (14.6%) 163 (21.1%) 103 (16.6%) 302 (17.0%) 58 (21.2%) 23 (12.3%) <0.001 Endometriosis 1855 (11.6%) 177 (9.5%) 70 (9.1%) 73 (11.8%) 242 (13.6%) 29 (10.6%) 29 (15.5%) <0.001 Male factor 3612 (22.6%) 333 (17.9%) 113 (14.7%) 91 (14.7%) 354 (19.9%) 62 (22.7%) 41 (21.9%) <0.001 PCOS 3066 (19.2%) 343 (18.5%) 95 (12.3%) 131 (21.2%) 371 (20.8%) 42 (15.4%) 50 (26.7%) <0.001 Eqq quality 1861 (11.6%) 190 (10.2%) 110 (14.3%) 83 (13.4%) 203 (11.4%) 44 (16.1%) 23 (12.3%) 0.01 Tubal 1118 (7.0%) 406 (21.9%) 39 (5.1%) 51 (8.2%) 189 (10.6%) 18 (6.6%) 16 (8.6%) <0.001 Uterine 828 (5.2%) 358 (19.3%) 53 (6.9%) 43 (6.9%) 125 (7.0%) 12 (4.4%) 7 (3.7%) <0.001 Immunological 266 (1.7%) 22 (1.2%) 10 (1.3%) 11 (1.8%) 23 (1.3%) 9 (3.3%) 2 (1.1%) 0.15 RPL 1346 (8.4%) 117 (6.3%) 50 (6.5%) 44 (7.1%) 101 (5.7%) 18 (6.6%) 14 (7.5%) <0.001 Page 6/25 Caucasian Black East Asian South Asian LatinX Middle Eastern Native American p- value Unexplained 5107 (31.9%) 373 (20.1%) 214 (27.8%) 195 (31.5%) 451 (25.3%) 71 (26.0%) 59 (31.6%) <0.001 Other 1570 (9.8%) 184 (9.9%) 67 (8.7%) 52 (8.4%) 167 (9.4%) 17 (6.2%) 22 (11.8%) 0.29 Patient satisfaction characteristics: Obtained desired result* 2.72 (1.26) 2.53 (1.24) 2.75 (1.26) 2.73 (1.28) 2.66 (1.27) 2.79 (1.26) 2.51 (1.32) <0.001 Treated as a human instead of number* 4.29 (1.18) 4.42 (1.10) 4.21 (1.25) 4.22 (1.25) 4.34 (1.17) 4.36 (1.14) 4.38 (1.16) <0.001 MD communication* 4.30 (1.07) 4.46 (0.98) 4.21 (1.15) 4.28 (1.10) 4.43 (1.05) 4.42 (1.02) 4.32 (1.14) <0.001 MD trustworthiness* 4.49 (0.97) 4.53 (0.93) 4.31 (1.10) 4.43 (1.00) 4.52 (0.97) 4.44 (0.99) 4.57 (0.93) <0.001 Billing department* 3.96 (1.15) 3.97 (1.15) 3.70 (1.22) 3.76 (1.21) 4.00 (1.17) 3.98 (1.18) 3.94 (1.14) <0.001 Wait time <0.001 No wait 867 (5.5%) 193 (10.4%) 53 (6.9%) 46 (7.6%) 152 (8.6%) 24 (9.1%) 20 (10.7%) Within an hour 3603 (22.7%) 407 (22.0%) 173 (22.7%) 122 (20.1%) 423 (24.0%) 75 (28.4%) 50 (26.7%) End of day 8144 (51.3%) 753 (40.7%) 315 (41.3%) 295 (48.6%) 7 762 (43.2%) 99 (37.5%) 72 (38.5%) Within 24 hours 2040 (12.9%) 324 (17.5%) 140 (18.3%) 97 (16.0%) 262 (14.9%) 42 (15.9%) 25 (13.4%) More than 24 hours 729 (4.6%) 85 (4.6%) 46 (6.0%) 33 (5.4%) 94 (5.3%) 12 (4.5%) 12 (6.4%) Not sure 492 (3.1%) 87 (4.7%) 36 (4.7%) 14 (2.3%) 71 (4.0%) 12 (4.5%) 8 (4.3%) Appointment and scheduling convenience 4.37 (0.96) 4.55 (0.84) 4.22 (1.03) 4.33 (0.95) 4.44 (0.94) 4.43 (0.90) 4.58 (0.76) < 0.001 Understanding on next steps* 4.22 (1.03) 4.36 (0.99) 4.13 (1.06) 4.16 (1.09) 4.33 (1.03) 4.24 (1.09) 4.26 (1.18) < 0.001 Adjustment after setbacks* 4.18 (1.16) 4.17 (1.18) 4.09 (1.20) 4.03 (1.25) 4.25 (1.17) 4.25 (1.06) 4.26 (1.18) 0.00 Page 7/25 Caucasian Black East Asian South Asian LatinX Middle Eastern Native American p- value Frequency of seeing doctor* 3.27 (1.35) 3.32 (1.43) 3.29 (1.40) 3.49 (1.38) 3.41 (1.40) 3.40 (1.36) 3.31 (1.41) < 0.001 Univariate analyses On univariate analyses for doctor satisfaction as a categorical outcome (Table 2A), we found that when compared to the reference group of Caucasian patients, Black (OR 1.17, 95% CI 1.06–1.30, p = 0.002) and LatinX patients (OR 1.18, 95% CI 1.06–1.31, p = 0.002) rated their doctors more favorably. EA patients rated their doctors significantly lower (OR 0.78, 95% CI 0.68–0.91, p = 0.001), while all other racial groups did not significantly differ from Caucasian patients. The same trends were found for doctor satisfaction as a linear outcome (Table 2B), with Black (Coeff 0.28, 95% CI 0.15 to 0.42, p < 0.001) and LatinX patients (Coeff 0.22 95% CI 0.08 to 0.36, p = 0.003) rating doctors significantly higher than Caucasian patients, and EA significantly lower (Coeff − 0.37 95% CI -0.60 to -0.16, p < 0.001). For clinic satisfaction, univariate regression (both logistic and linear regression, Tables 2C-2D) found similar trends, with Black and LatinX patients rating clinics more favorably, while EA patients rated clinics significantly lower. In addition, for clinic satisfaction, SA patients also rated clinics significantly lower? as both a categorical (OR 0.77 95% CI 0.66–0.91, p = 0.002) and continuous outcome (Coeff − 0.29 95% CI -0.51 to -0.06, p = 0.014). There were no significant differences for ME or NA patients on clinic satisfaction, when compared to Caucasian patients. Table 2A-D. Satisfaction scores by ethnicity -univariate Table 2A-D. Satisfaction scores by ethnicity -univariate Page 8/25 Univariate logistic regression - doctor satisfaction OR (95% CI) p-value Race/ethnicity Caucasian Reference Black 1.17 (1.06-1.30) 0.002 East Asian 0.78 (0.68-0.91) 0.001 South Asian 0.88 (0.75-1.04) 0.131 LatinX 1.18 (1.06-1.31) 0.002 Middle Eastern 1.01 (0.79-1.30) 0.912 Native American 1.35 (0.99-1.86) 0.061 Univariate linear regression - doctor satisfaction Coeff (95% CI) p-value Race/ethnicity Caucasian Reference Black 0.28 (0.15 to 0.42) <0.001 East Asian -0.37 (-0.60 to -0.16) <0.001 South Asian -0.21 (00.45 to 0.02) 0.07 LatinX 0.22 (0.08 to 0.36) 0.003 Middle Eastern -0.14 (-0.21 to 0.48) 0.442 Native American -0.18 (-0.24 to 0.59) 0.401 Page 9/25 Page 9/25 Page 9/25 Univariate logistic regression clinic satisfaction OR (95% CI) p-value Race/ethnicity Caucasian Reference Black 1.21 (1.09-1.34) <0.001 East Asian 0.69 (0.60 - 0.80) <0.001 South Asian 0.77 (0.65 - 0.91) 0.002 LatinX 1.18 (1.06-1.31) 0.002 Middle Eastern 0.80 (0.62-1.02) 0.071 Native American 1.32 (0.96-1.82) 0.088 Univariate linear regression clinic satisfaction Coeff (95% CI) p-value Race/ethnicity Caucasian Reference Black 0.28 (0.14 to 0.42) <0.001 East Asian -0.50 (-0.71 to -0.30) <0.001 South Asian -0.29 (-0.51 to -0.06) 0.014 LatinX 0.21 (0.07 to 0.34) 0.004 Middle Eastern -0.05 (-0.29 to 0.39) 0.76 Native American 0.39 (-0.02 to 0.79) 0.06 Multivariate analysis For doctor satisfaction, when adjusting for all confounders (demographic and patient satisfaction), we found that Black patients still rated their doctors more highly (OR 1.30, 95% CI 1.04–1.62 p = 0.022 logistic and Coef 0.082, 95% CI 0.013–0.15 p = 0.02 linear) when compared to Caucasian patients. However, for doctor satisfaction, other ethnic groups did not show significant differences on multivariate logistic or linear regression when compared to Caucasian patients (Tables 3A-B). In the multivariate adjusted analysis for clinic satisfaction (Tables 3C-D), we found that EA had borderline lower satisfaction with clinic in logistic regression (OR 0.74, 95% CI 0.55-1.00 p = 0.05), while significant differences were not found for other ethnic groups. For clinic satisfaction as a linear outcome, no group significantly differed from Caucasian patients. In the multivariate adjusted analysis, most patient satisfaction confounders were highly significant, while most demographic confounders were not significant with the exception of insurance and certain income groups in terms of association with doctor and clinic satisfaction outcomes. Page 10/25 Page 10/25 Page 10/25 In a separate analysis adjusting for demographic confounders only, we found that EA rated both their physician and clinics lower, though other groups were not significantly different (with the exception of SA rating their clinics lower on categorical satisfaction only) (Supplementary Material). Table 3. Multivariate satisfaction scores by ethnicity Discussion In summary, in this large cohort of self-reported fertility care satisfaction, we found that after adjustment of confounders, Black patients reported higher doctor satisfaction and East Asians reported borderline lower clinic satisfaction. Despite some differences in unadjusted satisfaction among multiple racial/ethnic groups, most differences did not persist after adjustment for demographic and patient satisfaction confounders. These patterns are particularly interesting given the known disparities in ART outcomes, with Black patients consistently being found in literature to have worse outcomes than other racial groups.(1) Overall, our data are reassuring in that minority groups do not appear to report worse satisfaction scores with fertility care, though more investigation is needed on both fertility patient satisfaction as well as outcome disparities in outcomes by race/ethnicity. In summary, in this large cohort of self-reported fertility care satisfactio confounders, Black patients reported higher doctor satisfaction and Eas satisfaction. Despite some differences in unadjusted satisfaction amon differences did not persist after adjustment for demographic and patien patterns are particularly interesting given the known disparities in ART o being found in literature to have worse outcomes than other racial grou that minority groups do not appear to report worse satisfaction scores w investigation is needed on both fertility patient satisfaction as well as o race/ethnicity. Table 3. Multivariate satisfaction scores by ethnicity Page 11/25 Multivariate logistic regression - doctor satisfaction OR (95% CI) p-value Race/ethnicity Caucasian Reference Black 1.30 (1.04-1.62) 0.022 East Asian 0.77 (0.55 - 1.08) 0.14 South Asian 1.21 (0.81 - 1.81) 0.34 LatinX 0.93 (0.74 - 1.16) 0.50 Middle Eastern 1.22 (0.66 - 2.25) 0.53 Native American 1.69 (0.83 - 3.42) 0.15 Demographic characteristics: Age at survey 1.00 (0.99 - 1.00) 0.60 Insurance No Reference Yes 0.88 (0.77-1.00) 0.06 Gender Decline Reference Female 1.21 (0.81 - 1.81) 0.80 Male 0.39 (0.12-16.2) 0.86 Other 0.13 (0.01-3.30) 0.22 LGBT Decline to state Reference No 0.99 (0.45-2.18) 0.45 Yes 1.12 (0.49-2.57) 0.49 Income $0-49K Reference $50-99K 0.97 (0.77-1.23) 0.81 $100-199K 0.91 (0.71-1.17) 0.46 $200-499K 0.81 (0.60-1.09) 0.16 $500K + 0.48 (0.12-1.82) 0.28 Decline to state 0.84 (0.59-1.20) 0.34 Education Less than HS Reference HS/GED 2.28 (0.34-15.13) 0.39 Multivariate logistic regression - doctor satisfaction Some college 3.27 (0.51-21.09) 0.21 Bachelor's degree 2.80 (0.43-18.09) 0.28 Master's degree 2.19 (0.34-14.14) 0.41 Professional degree 2.72 (0.42-17.79) 0.30 Doctorate 2.11 (0.32-13.92) 0.44 Patient satisfaction characteristics: Obtained desired result* 1.69 (1.61-1.77) <0.001 Treated as a human instead of number* 2.09 (1.92-2.28) <0.001 MD communication* 2.18 (1.97-2.41) <0.001 MD trustworthiness* 3.50 (3.07-4.00) <0.001 Billing department* 1.17 (1.10-1.24) <0.001 Wait time No wait Reference Within an hour 0.62 (0.44-0.87) <0.01 End of day 0.57 (0.41-0.80) 0.001 Within 24 hours 0.46 (0.32-0.65) <0.001 More than 24 hours 0.49 (0.31-0.78) <0.01 Not sure 0.49 (0.31-0.79) <0.01 Appointment and scheduling convenience 1.15 (1.07-1.24) <0.001 Understanding on next steps* 1.47 (1.35-1.60) <0.001 Adjustments after setbacks 1.79 (1.67-1.93) <0.001 Primary RN No Reference Not sure 1.02 (0.86-1.22) 0.80 Yes 1.12 (0.98-1.28) 0.09 Clinical error made (%)* 0.94 (0.82-1.07) 0.34 Frequency of seeing doctor* 1.34 (1.28-1.41) <0.001 State and profession included in multivariate analysis, not displayed due to space constraints Page 13/25 Page 13/25 Multivariate logistic regression - clinic satisfaction Multivariate logistic regression - clinic satisfaction OR (95% CI) p-value Race/ethnicity Caucasian Reference Black 1.04 (0.85 - 1.27) 0.73 East Asian 0.74 (0.55 - 1.00) 0.05 South Asian 0.99 (0.70 - 1.40) 0.95 LatinX 1.02 (0.82 - 1.26) 0.87 Middle Eastern 0.66 (0.39 - 1.10) 0.11 Native American 1.54 (0.79 - 3.01) 0.20 Demographic characteristics: Age at survey 1.00 (0.99-1.01) 0.91 Insurance No Reference Yes 1.05 (0.93-1.18) 0.46 Gender Decline Reference Female 1.97 (0.24-16.2) 0.53 Male 1.60 (0.19-13.4) 0.67 Other 8.55 (0.22-3326.1) 0.48 LGBT Decline to state Reference No 0.62 (0.29-1.31) 0.21 Yes 0.74 (0.34-1.64) 0.46 Income $0-49K Reference $50-99K 0.94 (0.75-1.17) 0.56 $100-199K 0.86 (0.68-1.08) 0.20 $200-499K 0.75 (0.57-0.99) 0.041 $500K + 0.52 (0.16-1.63) 0.16 Decline to state 0.82 (0.59-1.14) 0.59 Education HS/GED 2.84 (0.29-27.6) 0.29 Some college 3.09 (0.33-29.3) 0.33 Bachelor's degree 1.94 (0.25-22.24) 0.25 Master's degree 1.94 (0.20-18.39) 0.20 Professional degree 2.03 (0.21-19.35) 0.21 Doctorate 2.09 (0.22-20.15) 0.22 Patient satisfaction characteristics: Obtained desired result 1.52 (1.45-1.59) <0.001 Treated as a human instead of number* 1.56 (1.45-1.68) <0.001 MD communication* 1.29 (1.18-1.42) <0.001 MD trustworthiness* 2.06 (1.84-2.31) <0.001 Billing department* 1.73 (1.64-1.82) <0.001 Wait time No wait Reference Within an hour 1.02 (0.75-1.38) 0.91 End of day 0.74 (0.55-0.99) 0.041 Within 24 hours 0.50 (0.36-0.68) <0.001 More than 24 hours 0.29 (0.19-0.45) <0.001 Not sure 0.43 (0.29-0.65) <0.001 Appointment and scheduling convenience 1.57 (1.47-1.68) <0.001 Understanding on next steps* 1.39 (1.29-1.51) <0.001 Adjustments after setbacks 1.48 (1.38-1.60) <0.001 Primary RN No Reference Not sure 1.14 (0.97-1.35) 0.11 Yes 1.14 (1.22-1.56) <0.001 Clinical error made (%)* 0.52 (0.46-0.58) <0.001 Frequency of seeing doctor* 1.03 (0.98-1.07) 0.25 State and profession included in multivariate analysis not displayed due to space constraints Multivariate linear regression - doctor satisfaction Multivariate linear regression - doctor satisfaction Coeff (95% CI) p- value Race/ethnicity Caucasian Reference Black 0.082 (0.013-0.15) 0.02 East Asian 0.066 (-0.039 to 0.17) 0.22 South Asian 0.35 (-0.82 to 0.15) 0.55 LatinX -0.0018 (-0.72 to 0.68) 0.96 Middle Eastern 0.11 (-0.66 to 0.29) 0.22 Native American -0.041 (-0.24 to 0.16) 0.69 Demographic characteristics: Age at survey -0.0020 (-0.0040 to 0.000023) 0.053 Insurance No Reference Yes -0.014 (-0.055 to 0.027) 0.49 Gender Decline Reference Female -0.47 (-1.12 to 0.19) 0.16 Male -0.49 (-1.15 to 0.17) 0.14 Other -0.58 (-1.65 to 0.49) 0.29 LGBT Decline to state Reference No -.081 (-0.31 to 0.15) 0.49 Yes -0.077 (-0.32 to 0.17) 0.53 Income $0-49K Reference $50-99K -0.033 (-0.11 to 0.040) 0.38 $100-199K -0.076 (-0.15 to 0.0016) 0.055 $200-499K -0.22 (-0.31 to -0.13) <0.001 $500K + -0.19 (0.52 to 0.15) 0.28 Less than HS Reference HS/GED -0.21 (-0.97 to 0.55) 0.58 Some college -0.17 (-0.92 to 0.59) 0.67 Bachelor's degree -0.25 (-1.00 to 0.50) 0.52 Master's degree -0.33 (-1.08 to 0.42) 0.39 Professional degree -0.33 (-1.08 to 0.42) 0.39 Doctorate -0.35 (-1.10 to 0.41) 0.37 Patient satisfaction characteristics: Obtained desired result 0.24 (0.22 to 0.26) <0.001 Treated as a human instead of number* 0.46 (0.44 to 0.49) <0.001 MD communication* 0.43 (0.39 to 0.46) <0.001 MD trustworthiness* 1.02 (0.98 to 1.05) <0.001 Billing department* 0.06 (0.043 to 0.081) <0.001 Wait time No wait Reference Within an hour 0.12 (-0.71 to 0.096) 0.76 End of day 0.036 (-0.044 to 0.12) 0.38 Within 24 hours -0.065 (-0.16 to 0.28) 0.17 More than 24 hours -0.21 (-0.33 to -0.91) 0.001 Not sure -0.032 (-0.17 to 0.10) 0.64 Appointment and scheduling convenience 0.058 (0.35 to 0.82) <0.001 Understanding on next steps* 0.17 (0.14 to 0.20) <0.001 Adjustments after setbacks 0.38 (0.35 to 0.40) <0.001 Primary RN No Reference Not sure -0.018 (-0.074 to 0.038) 0.53 Yes 0.05 (0.0043 to 0.088) 0.031 Frequency of seeing doctor* 0.058 (0.042 to 0.723) <0.001 Frequency of seeing doctor 0.058 (0.042 to 0.723) 0.001 State and profession included in multivariate analysis, not displayed due to space constraints Multivariate linear regression - clinic satisfaction Coeff (95% CI) p-value Race/ethnicity Caucasian Reference Black 0.0030 (-0.080 to 0.086) 0.94 East Asian 0.046 (-0.79 to 0.17) 0.47 South Asian 0.10 (-0.041 to 0.24) 0.16 LatinX -0.0081 (-0.092 to 0.076) 0.85 Middle Eastern 0.15 (-0.063 to 0.36) 0.17 Native American 0.069 (-0.17 to 0.31) 0.57 Demographic characteristics: Age at survey -0.00055 (-0.0030 to 0.0019) 0.66 Insurance No Reference Yes 0.091 (0.042 to 0.14) <0.001 Gender Decline Reference Female -0.15 (-0.94 to 0.64) 0.71 Male -0.20 (-0.99 to 0.60) 0.63 Other -0.20 (-1.49 to 1.08) 0.76 LGBT Decline to state Reference No -0.21 (-0.49 to 0.062) 0.13 Yes -0.25 (-0.54 to 0.039) 0.089 Income $0-49K Reference $50-99K -0.041 (-013 to 0.47) 0.36 $100-199K -0.12 (-0.21 to -0.26) 0.012 $200-499K -0.33 (-0.44 to -0.22) <0.001 Decline to state -0.17 (-0.30 to -0.38) 0.012 Decline to state -0.17 (-0.30 to -0.38) 0.012 Decline to state Education Less than HS Reference HS/GED -02.9 (-1.21 to 0.62) 0.53 Some college -0.25 (-1.16 to 0.65) 0.58 Bachelor's degree -0.34 (-1.24 to 0.57) 0.47 Master's degree -0.39 (-1.30 to 0.52) 0.40 Professional degree -0.45 (-1.36 to 0.45) 0.33 Doctorate -0.49 (-1.40 to 0.42) 0.29 Patient satisfaction characteristics: Obtained desired result 0.23 (0.20 to 0.25) <0.001 Treated as a human instead of number* 0.39 (0.36 to 0.43) <0.001 MD communication* 0.17 (0.13 to 0.21) <0.001 MD trustworthiness* 0.89 (0.85 to 0.93) <0.001 Billing department* 0.31 (0.29 to 0.34) <0.001 Wait time No wait Reference Within an hour 0.13 (0.025 to 0.23) 0.015 End of day 0.091 (-0.0056 to 0.19) 0.065 Within 24 hours -0.15 (-0.26 to -0.38) 0.008 More than 24 hours -0.69 (-0.83 to -0.54) <0.001 Not sure -0.27 (-0.43 to -0.11) 0.001 Appointment and scheduling convenience 0.30 (0.27 to 0.33) <0.001 Understanding on next steps* 0.13 (0.90 to 0.16) <0.001 Adjustments after setbacks 0.29 (0.26 to 0.32) <0.001 Primary RN Page 19/25 No Reference Not sure 0.026 (-0.042 to 0.093) 0.45 Yes 0.12 (0.070 to 0.17) <0.001 Clinical error made (%)* -0.36 (-0.41 to -0.30) <0.001 Frequency of seeing doctor* -0.036 (-0.054 to -0.18) <0.001 *State and profession included in multivariate analysis, not displayed due to space constraints Comparison with existing literature Prior literature has consistently reported disparities in ART outcomes, with minorities and particularly Black patients having poorer outcomes when compared to Caucasian patients.(1, 2) An analysis of 38,309 cycles using SART CORS data found increased miscarriage rate and decreased live birth rate compared to Black women compared to white women, even after controlling for uterine and tubal factor disease.(3) Another study of 138,927 SART ART cycles found increased fetal loss rate in Black women compared to other racial groups.(6) Several studies have reported significantly lower implantation, clinical pregnancy, and live birth rates in Black ART patients (3, 19), though not all studies have found these conclusions.(20, 21) It has been hypothesized that an increased prevalence of leiomyomas and possible difference in baseline diagnoses of BMI may contribute to these dipartites, though some studies controlling for these factors have continued to find disparities. Several more recent studies have continued to report disparities in Black ART outcomes; a recent analysis of 7,002 SART CORS cycles found that Black race was associated with lower live birth rate in frozen embryo transfer cycles (aRR 0.82; 95% CI 0.73–0.92) and a higher clinical loss rate (aRR 1.59; 95% CI, 1.28–1.99) compared with White women. Additionally, Black women were found to have a lower rate of implantation compared to other racial groups even in PGT cycles.(4) Another study of 1,601 donor embryo transfer cycles from 2008–2015 found that Black recipients had a lower probability of live birth than white donors and recipients (aRR 0.84; 95% confidence interval, 0.71–0.99), while live birth disparities were not found in Hispanic, Asian, and other races compared with white recipients.(5) Page 20/25 Disparities in ART outcomes for Asian and Hispanic groups have also been reported. Comparison with existing literature The prior referenced study of over 130,000 ART cycles from SART found that odds of pregnancy were reduced for Asians (0.86) and live birth odds were reduced for Asians (0.90), Black (0.62), and Hispanics (0.87).(6) Another SART study of 25,843 white and 1,429 Asian women found that Asian women had significantly decreased cumulative pregnancy rate and live birth rate when compared to white women, and that Asian ethnicity was an independent predictor of poor ART outcomes after adjustment for confounders.(22) Multiple other studies have also reported worse ART outcomes in Asian patients, including both large database and smaller independent center studies.(23–25) Studies on SA populations specifically have reported inconsistent information on disparities, including some studies reporting worse outcomes in this population, while other have reported equivalent outcomes.(7, 8) One study of 196 Caucasian and 117 South Asian women reported equivalent outcomes in frozen embryo transfers, but significantly lower outcomes in fresh transfers, suggesting that the relationship between race/ethnicity and ART outcomes may depend on ART procedure type.(26) Studies on Hispanic patients have been limited by sample size; in the SART Writing Group 2010 paper, live birth rates from ART were 13% lower for Hispanic than for white women.(6) However, a study of the military health care system found no significant differences in IVF outcomes for Hispanic and white patients.(27) More investigation is warranted in this population. It is also important to note that the data on this subject of racial/ethnic disparities in ART outcomes may be limited by incomplete report of race/ethnicity in datasets used for analysis on this subject (28), leading to possible bias in the results. Additionally, studies have typically reported minority groups of Black, Asian, and Hispanic, even though these groups may contain substantial heterogeneity and additional subgroups. Additionally, data suggests that access to fertility care is also adversely affected by race/ethnicity, as infertility care is accessed at a higher rate by non-Hispanic white women with higher socioeconomic parameters, and at a lower rate for Hispanic women.(29, 30) It is also important to note that the data on this subject of racial/ethnic disparities in ART outcomes may be limited by incomplete report of race/ethnicity in datasets used for analysis on this subject (28), leading to possible bias in the results. Additionally, studies have typically reported minority groups of Black, Asian, and Hispanic, even though these groups may contain substantial heterogeneity and additional subgroups. Comparison with existing literature Additionally, data suggests that access to fertility care is also adversely affected by race/ethnicity, as infertility care is accessed at a higher rate by non-Hispanic white women with higher socioeconomic parameters, and at a lower rate for Hispanic women.(29, 30) Literature on patient perspective on self-reported fertility care satisfaction by race/ethnicity is extremely limited, despite the more extensive literature on disparities in outcomes as above. A study of 1,460 patients at an academic fertility center in Illinois found that Black women (14.7%) were more likely to report that race was a barrier to getting fertility treatment compared with White (0.0%), Hispanic (5.1%), and Asian (5.4%) women, though satisfaction with fertility care was not assessed.(9) A prior FertilityIQ analysis of 7,456 women from 2015–2018 reported a number of factors associated with positive patient-reported experience (including positive results, being treated like a human instead of number, good communication and expectation setting by doctor, shorter wait times, scheduling, and billing satisfaction).(10) However, stratifications by race/ethnicity group were not available in this analysis, and we have updated this data with these stratifications as well as a substantially large sample size. A few other studies have investigated patient satisfaction in fertility care, with an older study of 1,499 patients in the Netherlands reporting high general patient satisfaction (94%), with waiting times, information provision, and emotional support the least positive aspects of care.(11) Prior literature includes smaller studies that have investigated multiple aspects of satisfaction with fertility care, including general patient satisfaction and provider perception (12–14), though study on fertility satisfaction by ethnicity is very limited. In other fields of medicine, a large study of around 250,000 Press-Ganey surveys found that Asian, younger and female patients provided less favorable ratings than other race/ethnicity older and male patients.(15) This is in line with our findings of EA reporting lower clinic satisfaction than other groups. Other large studies have suggested that minority groups may report lower satisfaction than Caucasian groups with medical care, including a European study of 138,878 cancer patients (16) and a study of 7,795 patients on satisfaction with Page 21/25 Page 21/25 physician, nursing, and overall care during hospitalization.(17) In contrast, a smaller study of 527 surveys found no differences in outpatient pediatric surgical care by race/ethnicity or socioeconomic are. Comparison with existing literature These findings from other studies may not be completely generalizable to fertility care given the differences in patient population and higher percentage of self-pay patients in fertility. In general, given the limited research, more research in patient satisfaction by race/ethnicity in medicine (not just fertility care) is warranted. Our analysis is the largest and one of the first to investigate patient found that despite disparities in ART outcomes, Black patients were satisfaction significantly higher than other groups. EA were the only significantly lower than other groups, while other disparities were no expected, there were baseline differences between the groups in term characteristics. Though some disparities were found on univariate a most differences in unadjusted satisfaction scores no longer persis Black patients did not report lower satisfaction despite literature sug population. Overall, data is reassuring in that minority groups do no fertility care, with the exception of EA for clinic satisfaction. Howeve underlying disparities in fertility care for minority populations. Our analysis is the largest and one of the first to investigate patient satisfaction in fertility care by ethnicity. We found that despite disparities in ART outcomes, Black patients were the only group to rate their physician satisfaction significantly higher than other groups. EA were the only group to rate their clinic satisfaction as significantly lower than other groups, while other disparities were not seen after adjustment for confounders. As expected, there were baseline differences between the groups in terms of demographic and clinic satisfaction characteristics. Though some disparities were found on univariate analysis, after adjustment of all confounders, most differences in unadjusted satisfaction scores no longer persisted with the exceptions above. Interestingly, Black patients did not report lower satisfaction despite literature suggesting worse ART outcomes in this population. Overall, data is reassuring in that minority groups do not appear to have worse satisfaction with fertility care, with the exception of EA for clinic satisfaction. However, more investigation is needed into the underlying disparities in fertility care for minority populations. Conclusion In summary, some but not all minority groups differed in their self-reported perception of satisfaction with fertility clinic and doctors compared to Caucasian patients. On fully adjusted analyses, some of these differences were no longer apparent, though Black patients continued to rate doctors more highly, while EA rated clinics lower. Cultural differences towards surveys may contribute to some of these findings, and satisfaction by racial/ethnic group may also be modified by results of care. Author contributions Page 23/25 Author contributions A Wang: Protocol/project development, Data collection or management, Data analysis, Manuscript writing/editing J Anderson-Bialis: Protocol/project development, Data collection or management, Manuscript writing/editing JR Morris: Protocol/project development, Data collection or management, Data analysis, Manuscript writing/editing J Corley: Data analysis, Manuscript writing/editing D Anderson-Bialis: Protocol/project development, Manuscript writing/editing L Citro: Protocol/project development, Manuscript writing/editing M Esa Seegulam: Manuscript writing/editing V Fujimoto: Protocol/project development, Data analysis, Manuscript writing/editing References A Wang: Protocol/project development, Data collection or management, Data analysis, Manuscript writing/editing J Anderson-Bialis: Protocol/project development, Data collection or management, Manuscript writing/editing JR Morris: Protocol/project development, Data collection or management, Data analysis, Manuscript writing/editing J Corley: Data analysis, Manuscript writing/editing D Anderson-Bialis: Protocol/project development, Manuscript writing/editing L Citro: Protocol/project development, Manuscript writing/editing M Esa Seegulam: Manuscript writing/editing V Fujimoto: Protocol/project development, Data analysis, Manuscript writing/editing Strengths and Limitations The strengths of this analysis include the large sample size, detailed information available on potential confounders, and the novelty of the subject matter. FertilityIQ is the largest database of patient satisfaction on fertility care in the United States, and our large sample size is an asset in allowing seven categories of racial/ethnic stratification. Additionally, the prior literature on patient satisfaction by race/ethnicity in fertility care is extremely limited, and ours is by far the largest study in this area. We were also able to investigate racial/ethnic groups including Middle Eastern and Native American which have traditionally not been as frequently studied (though sample sizes were limited for these groups), and were also able to study East Asian and South Asians separately (as this is a heterogeneous group which has often been studied as one racial category). FertilityIQ also collects extensive demographic information which as used in multivariate analysis, as some of these factors may be confounders for patient satisfaction. The limitations of the study include its cross-sectional nature, self-reported voluntary survey data which may be subject to bias in terms of recall bias or who chooses to fill out an online survey, and small but statistically significant differences that may not be clinically significant (as a result of large sample size). The survey was available online but not sent out to patients, so patients had to choose to go to the website to voluntarily, which may lead to response bias. Additionally, there may be cultural differences in terms of who chooses to fill out a survey and how different groups respond to survey questions, or cultural differences in expectations of care. It is important to note that given known disparities in access to care, this survey can only be interpreted in the context of those who were able to obtain fertility care. The survey was also limited to English speaking patients, which is a potential bias in the analysis by not capturing non-English speaking patients. We also had to exclude multi- racial respondents from the analysis due to difficulty analyzing multi-racial data; however, as the population of multi-racial individuals will continue to increase, is it important to develop ways to adequately analyze this population. Though the data must be interpreted with caution, our large sample size gives us confidence in our results for the different racial/ethnic groups studied. Page 22/25 Page 22/25 Disclosures/Conflicts of Interest: None. IRB: An Institutional Review Board (IRB) through UCSF has determined that this research study does not involve human subjects and is therefore exempt. IRB: An Institutional Review Board (IRB) through UCSF has determined that this research study does not involve human subjects and is therefore exempt. SYNOPSIS: Minority groups differed in self-reported fertility care satisfaction compared to Caucasian patients, though most of these differences were no longer present on fully adjusted analyses. ACKNOWLEDGEMENTS The authors wish to acknowledge the fertility patients who have provided valuable information by filling out surveys on FertilityIQ. Author contributions: AW, JAB, JRM, and VF were involved in study conception and design. AW performed the data analysis. AW, JAB, and JRM performed data interpretation. AW wrote the initial draft of the manuscript. All authors contributed to additional data interpretation and final approval of the manuscript. Presentations: These data were presented in part as a poster presentation at American Society for Reproductive Medicine 2021 in Baltimore, MD. Funding sources: There are no funding sources to report for this study. Funding sources: There are no funding sources to report for this study. Funding sources: There are no funding sources to report for this study. 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Schmidt L, Holstein BE, Boivin J, Tjornhoj-Thomsen T, Blaabjerg J, Hald F et al (2003) High ratings of satisfaction with fertility treatment are common: findings from the Copenhagen Multi-centre Psychosocial Infertility (COMPI) Research Programme. Hum Reprod 18:2638–2646 14. Gonen LD (2016) Satisfaction with in vitro fertilization treatment: patients' experiences and professionals' perceptions. Fertil Res Pract 2:6 14. Gonen LD (2016) Satisfaction with in vitro fertilization treatment: patients' experiences and professionals' perceptions. Fertil Res Pract 2:6 15. Liao L, Chung S, Altamirano J, Garcia L, Fassiotto M, Maldonado B et al (2020) The association between Asian patient race/ethnicity and lower satisfaction scores. BMC Health Serv Res 20:678 15. References 1. Quinn M, Fujimoto V (2016) Racial and ethnic disparities in assisted reproductive technology access and outcomes. Fertil Steril 105:1119–1123 2. Wellons MF, Fujimoto VY, Baker VL, Barrington DS, Broomfield D, Catherino WH et al (2012) Race matters: a systematic review of racial/ethnic disparity in Society for Assisted Reproductive Technology reported outcomes. Fertil Steril 98:406–409 3. Seifer DB, Frazier LM, Grainger DA (2008) Disparity in assisted reproductive technologies outcomes in black women compared with white women. Fertil Steril 90:1701–1710 3. Seifer DB, Frazier LM, Grainger DA (2008) Disparity in assisted reproductive technologies outcomes in black women compared with white women. Fertil Steril 90:1701–1710 4. Makhijani R, Godiwala P, Grady J, Christy A, Thornton K, Grow D et al (2022) Black race associated with lower live birth rate in frozen-thawed blastocyst transfer cycles: an analysis of 7,002 Society for Assisted Reproductive Technology frozen-thawed blastocyst transfer cycles. Fertil Steril 117:360–367 4. Makhijani R, Godiwala P, Grady J, Christy A, Thornton K, Grow D et al (2022) Black race associated with lower live birth rate in frozen-thawed blastocyst transfer cycles: an analysis of 7,002 Society for Assisted Reproductive Technology frozen-thawed blastocyst transfer cycles. Fertil Steril 117:360–367 5. Liu Y, Hipp HS, Nagy ZP, Capelouto SM, Shapiro DB, Spencer JB et al (2021) The effect of donor and recipient race on outcomes of assisted reproduction. Am J Obstet Gynecol 224:374 e1- e12 5. Liu Y, Hipp HS, Nagy ZP, Capelouto SM, Shapiro DB, Spencer JB et al (2021) The effect of donor and recipient race on outcomes of assisted reproduction. Am J Obstet Gynecol 224:374 e1- e12 6. Fujimoto VY, Luke B, Brown MB, Jain T, Armstrong A, Grainger DA et al (2010) Racial and ethnic disparities in assisted reproductive technology outcomes in the United States. Fertil Steril 93:382–390 6. Fujimoto VY, Luke B, Brown MB, Jain T, Armstrong A, Grainger DA et al (2010) Racial and ethnic disparities in assisted reproductive technology outcomes in the United States. Fertil Steril 93:382–390 7. Palep-Singh M, Picton HM, Vrotsou K, Maruthini D, Balen AH (2007) South Asian women with polycystic ovary syndrome exhibit greater sensitivity to gonadotropin stimulation with reduced fertilization and ongoing pregnancy rates than their Caucasian counterparts. Eur J Obstet Gynecol Reprod Biol 134:202–207 7. References Liao L, Chung S, Altamirano J, Garcia L, Fassiotto M, Maldonado B et al (2020) The association between Asian patient race/ethnicity and lower satisfaction scores. BMC Health Serv Res 20:678 16. Pinder RJ, Ferguson J, Moller H (2016) Minority ethnicity patient satisfaction and experience: results of the National Cancer Patient Experience Survey in England. BMJ Open 6:e011938 16. Pinder RJ, Ferguson J, Moller H (2016) Minority ethnicity patient satisfaction and experience: results of the National Cancer Patient Experience Survey in England. BMJ Open 6:e011938 17. Woods SE, Bivins R, Oteng K, Engel A (2005) The influence of ethnicity on patient satisfaction. Ethn Health 10:235–242 17. Woods SE, Bivins R, Oteng K, Engel A (2005) The influence of ethnicity on patient satisfaction. Ethn Health 10:235–242 Page 24/25 Page 24/25 18. Reichheld FF (2003) The one number you need to grow. Harv Bus Rev 81:46–54 18. Reichheld FF (2003) The one number you need to grow. Harv Bus Rev 81:46–5 19. Feinberg EC, Larsen FW, Catherino WH, Zhang J, Armstrong AY (2006) Comparison of assisted reproductive technology utilization and outcomes between Caucasian and African American patients in an equal-access- to-care setting. Fertil Steril 85:888–894 20. Dayal MB, Gindoff P, Dubey A, Spitzer TL, Bergin A, Peak D et al (2009) Does ethnicity influence in vitro fertilization (IVF) birth outcomes? Fertil Steril 91:2414–2418 21. Bendikson K, Cramer DW, Vitonis A, Hornstein MD (2005) Ethnic background and in vitro fertilization outcomes. Int J Gynaecol Obstet 88:342–346 22. Purcell K, Schembri M, Frazier LM, Rall MJ, Shen S, Croughan M et al (2007) Asian ethnicity is associated with reduced pregnancy outcomes after assisted reproductive technology. Fertil Steril 87:297–302 23. Baker VL, Luke B, Brown MB, Alvero R, Frattarelli JL, Usadi R et al (2010) Multivariate analysis of factors affecting probability of pregnancy and live birth with in vitro fertilization: an analysis of the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System. Fertil Steril 94:1410–1416 24. Shapiro AJ, Darmon SK, Barad DH, Albertini DF, Gleicher N, Kushnir VA (2017) Effect of race and ethnicity on utilization and outcomes of assisted reproductive technology in the USA. Reprod Biol Endocrinol 15:44 25. Langen ES, Shahine LK, Lamb JD, Lathi RB, Milki AA, Fujimoto VY et al (2010) Asian ethnicity and poor outcomes after in vitro fertilization blastocyst transfer. Obstet Gynecol 115:591–596 26. References Shah MS, Caballes M, Lathi RB, Baker VL, Westphal LM, Milki AA (2016) In vitro fertilization outcomes after fresh and frozen blastocyst transfer in South Asian compared with Caucasian women. Fertil Steril 105:1484–1487 27. Feinberg EC, Larsen FW, Wah RM, Alvero RJ, Armstrong AY (2007) Economics may not explain Hispanic underutilization of assisted reproductive technology services. Fertil Steril 88:1439–1441 27. Feinberg EC, Larsen FW, Wah RM, Alvero RJ, Armstrong AY (2007) Economics may not explain Hispanic underutilization of assisted reproductive technology services. Fertil Steril 88:1439–1441 28. Seifer DB, Zackula R, Grainger DA, Society for Assisted Reproductive Technology Writing Group R (2010) Trends of racial disparities in assisted reproductive technology outcomes in black women compared with white women: Society for Assisted Reproductive Technology 1999 and 2000 vs. Fertil Steril 93:2004–2006 29. Komorowski AS, Jain T (2022) A review of disparities in access to infertility care and treatment outcomes among Hispanic women. Reprod Biol Endocrinol 20:1 28. Seifer DB, Zackula R, Grainger DA, Society for Assisted Reproductive Technology Writing Group R (2010) Trends of racial disparities in assisted reproductive technology outcomes in black women compared with white women: Society for Assisted Reproductive Technology 1999 and 2000 vs. Fertil Steril 93:2004–2006 28. Seifer DB, Zackula R, Grainger DA, Society for Assisted Reproductive Technology Writing Group R (2010) Trends of racial disparities in assisted reproductive technology outcomes in black women compared with white women: Society for Assisted Reproductive Technology 1999 and 2000 vs. Fertil Steril 93:2004–2006 29. Komorowski AS, Jain T (2022) A review of disparities in access to infertility care and treatment outcomes among Hispanic women. Reprod Biol Endocrinol 20:1 29. Komorowski AS, Jain T (2022) A review of disparities in access to infertility care and treatment outcomes among Hispanic women. Reprod Biol Endocrinol 20:1 30. Jain T, Hornstein MD (2005) Disparities in access to infertility services in a state with mandated insurance coverage. Fertil Steril 84:221–223 30. Jain T, Hornstein MD (2005) Disparities in access to infertility services in a state with mandated insurance coverage. Fertil Steril 84:221–223 Page 25/25 Page 25/25
https://openalex.org/W4253697928	https://www.qeios.com/read/INIJD9/pdf	English	null	ACKR2 wt Allele	Definitions	2,020	cc-by	114	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios ACKR2 wt Allele National Cancer Institute National Cancer Institute Qeios ID: INIJD9 · https://doi.org/10.32388/INIJD9 Source National Cancer Institute. ACKR2 wt Allele. NCI Thesaurus. Code C51317. Human ACKR2 wild-type allele is located in the vicinity of 3p21.3 and is approximately 58 kb in length. This allele, which encodes atypical chemokine receptor 2 protein, is involved in the recruitment of effector immune cells to sites of inflammation. The allele also plays a role in chemokine-driven leukocytes recirculation. Variant alleles have been implicated in chemokine effects relating to the development and growth of vascular tumors. Qeios ID: INIJD9 · https://doi.org/10.32388/INIJD9 1/1
W3133742434.txt	https://www.mdpi.com/2071-1050/13/5/2715/pdf	en		Eco-Economic Coordination Analysis of the Yellow River Basin in China: Insights from Major Function-Oriented Zoning	Sustainability	2,021	cc-by	12,261	sustainability Article Eco-Economic Coordination Analysis of the Yellow River Basin in China: Insights from Major Function-Oriented Zoning Zhongwu Zhang 1 , Tianying Chang 1 , Xuning Qiao 2,3, , Yongju Yang 2,3 , Jing Guo 2 and Han Zhang 2 1 2 3 Citation: Zhang, Z.; Chang, T.; Qiao, X.; Yang, Y.; Guo, J.; Zhang, H. Eco-Economic Coordination Analysis of the Yellow River Basin in China: School of Geography, Shanxi Normal University, Linfen 041000, China; zhangzw@sxnu.edu.cn (Z.Z.); changtianying0@hotmail.com (T.C.) School of Surveying and Land Information Engineering, Henan Polytechnic University, Jiaozuo 454003, China; yjuz@hpu.edu.cn (Y.Y.); gguojing99@hotmail.com (J.G.); zhang_hann@hotmail.com (H.Z.) Research Centre of Arable Land Protection and Urban-Rural High-Quality Development of Yellow River Basin, Henan Polytechnic University, Jiaozuo 454003, China Correspondence: qiaoxuning@hpu.edu.cn Abstract: The ecological-economic coordination degree model is widely used to analyze eco-economic coordination relationships, but methods for determining the relative weights of the ecological and economic systems lack a scientific basis. Examining the Yellow River Basin based on Major FunctionOriented Zoning (MFOZ) in China, the study surveyed 42 experts and used the analytic hierarchy process (AHP)to calculate the ecological and economic weights of the different main function zones. It also improved the model and evaluated the coordination degree of the ecological economic system in 642counties of eight provinces in the Yellow River Basin from 1991 to 2015. The results indicate that (1) the ecological value of the basin increased from 823 billion Yuan in 2001 to 1142 billion Yuan in 2015; (2) the GDP shows a linear growth trend: high- and medium–high-value areas of per capita GDP are clustered around nine metropolitan areas, while cold spots are distributed in ecological protection and agricultural development zones; (3) the ecological and economic coordination of the river basin first rose and then declined; and (4) the coordinated development areas are concentrated in five urban agglomerations that are highly consistent with the per capita GDP hotspots. Insights from Major Function-Oriented Zoning. Sustainability 2021, 13, 2715. Keywords: ecology and economy; coordination; sustainable development; Major Function-Oriented Zoning (MFOZ); analytic hierarchy process; Yellow River Basin https://doi.org/10.3390/su13052715 Academic Editor: Antonio Boggia 1. Introduction Received: 18 January 2021 Accepted: 25 February 2021 Published: 3 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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/). The eco-environmental system is the basis for maintaining material circulation and biological survival, and economic development is the result of human use of natural and developed resources. With the rapid development of the economy and society, the contradictions between regional economic development, resource carrying capacity, and the ecological environment have become more prominent. The coordinated development of ecological environmental and economic systems has become an important basis for whether or not the region can achieve sustainable development [1]. In the 1660s, Petty linked the creation of economic value with the ecological environment for the first time. He believed that environmental factors, such as labor and land, can create value together. Since then, there have been similar studies on the coordinated development of the ecological environment and social economy, such as Malthus’s population theory and Ricardo’s theory of land rent. At present, the evaluation of the coordination relationship between the ecological environment and economic development is a hot topic in academic circles. Scholars have achieved diverse findings through large-scale research on the global [2,3], country [4,5], and province and city [6,7] trends. Research at the small scale for a county, to comprehensively study its watershed, is insufficient. As a composite ecosystem, watersheds have received increasing attention due to the sensitivity of their ecological environment and diversity of scales [8]. Sustainability 2021, 13, 2715. https://doi.org/10.3390/su13052715 https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 2715 2 of 18 In terms of evaluation methods, the first approach is to construct an ecologicaleconomic index system [9,10]; however, itis difficult to obtain data on the evaluation of county and smaller-scale units, the selection of indicators is very different, and the comparison of the measurement results is weak. The second is to construct a coupled evaluation model, including the Driving force–State–Response (DSR) model proposed by the United Nations Commission on Sustainable Development [11], the analytic hierarchy process (AHP) evaluation model [12], the ecological footprint evaluation model [13], and the coordinated development model [14,15]. The coordinated development degree model of the economy and environment has the widest application range;it avoids the difficulty of reflecting whether the overall regional ecological economic system is in high-level coordination or low-level coordination, and it can more objectively and clearly reflect the degree of interaction between the ecological environment system and the socio-economic system. The determination of the parameters in this model is subjective, and it is generally assumed that the ecological and economic systems are equally important. However, in reality, the effects of the ecological and economic systems in the different regions are not the same. To reflect this difference, to alleviate the contradiction between the ecological environment and economic development caused by urbanization and industrialization in China, and to promote the coordination and sustainability of economic development and ecological protection, the State Council of China—based on the regional resource and environmental carrying capacity, the existing development density, and development potential—approved and implemented the “National Plan for MFOZ” in 2010. The plan divides the land into optimized, key, restricted, and prohibited development areas according to development methods [16]. Major Function-Oriented Zoning (MFOZ) is a strategic, basic, and restrictive plan for China’s land and space development;it is of great strategic significance to promote the formation of a land and space development pattern that is coordinated with the population, economy, resources, and environment, and to promote coordinated economic, social, and environmental development [17,18]. At the same time, it provides a new idea for the determination of model parameters; that is, based on the positioning of the main functions of different regions, the analytic hierarchy process is used to determine the weight of the economic and ecological systems. The Yellow River Basin is an important economic zone and ecological barrier in China, and it plays a particularly important role in China’s economic and social development and ecological security [19]. Due to the particular natural environment and geographic location, the economic and ecological environment of the Yellow River Basin is undergoing complex changes and is always facing severe challenges. Jin [20] and Xu et al. [21] put forward the idea of using regional classification guidance, coordinating and handling the “four major relationships”, and promoting the ecological protection and high-quality development of the Yellow River Basin with the basic development pattern of “three regions and seven groups”. However, they did not indicate how to coordinate the ecological-economic relationship between the different main function areas and how to achieve sustainable development. Related research has shown that GDP and GDP per capita can objectively reflect the levels of socio-economic development of a region, and are important indicators to measure the living standards of its people [22]. Among the ecological indicators, net primary productivity (NPP) is positively correlated with the supply and regulation functions of the ecosystem; its temporal and spatial changes track the changes in ecosystem functions [23,24], reflecting not only the ecosystem itself, but also the material, energy cycle, and change process [25], which can have important impacts on the natural environment. Therefore, the research is based on MFOZ, combined with coordination degree theory, to build an ecological-economic coordination degree model, with GDP, per capita GDP, and NPP as the economic and ecosystem indicators. The study quantitatively analyzes the ecological-economic system coordination degree of MFOZ for 642 counties in eight provinces of the Yellow River Basin from 1991 to 2015, and provides decision support for the ecological and economic coordination and sustainable development of MFOZ in the Yellow River Basin. Sustainability 2021, 13, 2715 Mongolia, Shaanxi, Shanxi, Henan, and Shandong, and finally into the Bohai Sea in Kenli County, Dongying City, Shandong Province (Figure 1). The drainage area is about 750,000 km2, with a total length of 5464 km. It is the second-longest river in China and the fifthlongest river in the world. There are many mountains in the basin, and the height differ3 of from 18 ence between east and west is huge, forming a three-level ladder from west to east, high to low, which makes the natural conditions, such as geomorphology, climate, precipitation, and evaporation, of the different regions vary greatly. This region has an important position in China’s economic development, with great growth potential and de2. Research Area, Materials, and Methods velopment space. As of 2018, the total population of the Yellow River Basin is 420 million, 2.1. Research Area accounting for about 30.3% of the country, and the regional GDP is 23.9 trillion Yuan [26], ◦ E–119◦ E and 32◦ N–42◦ N;it is about 1900 km river basin between orThe about 26.5% of is thelocated total. The basin96 is currently the area most strongly affected by the long from east between to west and 1100and km land. wide In from north to south, originating from relationship humans recent decades, rapid urbanization in the some northern foot of the Bayan Har Mountains on the Qinghai–Tibet Plateau in China. areas of the Yellow River Basin has led to an excessive load on the total amount ofThe water basin is vast, through nine provinces, Qinghai,The Sichuan, Gansu, Ningxia, used, and flowing the water carrying capacity has including been overloaded. ecological environment Inner Mongolia, Shaanxi, Shanxi, Henan, and Shandong, and finally into the Bohai Sea inand has continued to deteriorate, restricting the sustainable development of the economy Kenli County, Dongying City, Shandong Province (Figure 1). The drainage area is about society [27]. 2 , with a total length of 5464 km. It is the second-longest river in China and the 750,000 In kmSeptember 2019, at the Symposium on Ecological Protection and High-Quality Defifth-longest the world. in the the height velopmentriver of theinYellow RiverThere Basin,are thismany issue mountains was upgraded to abasin, majorand national strategy. difference between east and west is huge, forming a three-level ladder from west to east, Most of Sichuan Province is located in the Yangtze River Basin. Only Aba Tibetan and from high to low, which makes the natural conditions, such as geomorphology, climate, Qiang Autonomous Prefecture and Ganzi Tibetan Autonomous Prefecture flow through precipitation, and evaporation, of the different regions vary greatly. This region has an the Yellow River Basin; their population and economic total account for 0.7% and 0.3% of important position in China’s economic development, with great growth potential and the Yellow River Basin, which has a weak influence on the economic and social developdevelopment space. As of 2018, the total population of the Yellow River Basin is 420 million, ment pattern of the Yellow River Basin. The eastern Inner Mongolia region has been inaccounting for about 30.3% of the country, and the regional GDP is 23.9 trillion Yuan [26], cluded in the spatial scope of the “Northeast Region Revitalization Plan”, and its ecoor about 26.5% of the total. The basin is currently the area most strongly affected by the nomic and social development is closely related to the Northeast region.As the Yellow relationship between humans and land. In recent decades, rapid urbanization in some River Basin covers eight provinces, the study area in this article encompasses over areas of the Yellow River Basin has led to an excessive load on the total amount of water 642counties (cities, districts), including the upper reaches of Gansu, Ningxia, and Qinghai; used, and the water carrying capacity has been overloaded. The ecological environment the middle reaches of Inner Mongolia (excluding eastern Mongolia), Shaanxi, and Shanxi; has continued to deteriorate, restricting the sustainable development of the economy and and the lower reaches of Henan and Shandong. society [27]. Figure 1. Location of the Yellow River Basin. In September 2019, at the Symposium on Ecological Protection and High-Quality Development of the Yellow River Basin, this issue was upgraded to a major national strategy. Most of Sichuan Province is located in the Yangtze River Basin. Only Aba Tibetan and Qiang Autonomous Prefecture and Ganzi Tibetan Autonomous Prefecture flow through the Yellow River Basin; their population and economic total account for 0.7% and 0.3% of the Yellow River Basin, which has a weak influence on the economic and social development pattern of the Yellow River Basin. The eastern Inner Mongolia region has been included in the spatial scope of the “Northeast Region Revitalization Plan”, and its economic and social development is closely related to the Northeast region.As the Yellow River Basin covers eight provinces, the study area in this article encompasses over 642 counties (cities, districts), including the upper reaches of Gansu, Ningxia, and Qinghai; the middle reaches of Inner Mongolia (excluding eastern Mongolia), Shaanxi, and Shanxi; and the lower reaches of Henan and Shandong. Sustainability 2021, 13, 2715 4 of 18 2.2. Data Source and Preprocessing The 1991–2015 NPP data come from the monthly NPP of China’s terrestrial ecosystems north of 18◦ N latitude 1 km raster dataset (1985–2015) (http://www.geodoi.ac.cn/WebCn/ doi.aspx?ID=1212&paperID=82fbe955-6d1a-42c9-afd3-af5c36b5863f&tdsourcetag=s_pctim_ aiomsg). The spatial resolution is 1 km, and the unit is gC/(m2 ·a). The image data for the NPP China region for 25 years (from 1991 to 2015) were obtained, which are based on the monthly meteorological data for China’s land from 1985 to 2015, the soil texture data for China, and the land cover and vegetation index data products based on MODIS and AVHRR remote sensing images. The data were entered into the Carnegie–Ames–Stanford Approach (CASA) model and used to develop a1 km raster dataset of the monthly net primary productivity of China’s terrestrial ecosystem north of the 18◦ N latitude for 31 years (1985–2015) [28,29]. In ArcGIS 10.2, the Yellow River Basin vector file was used to mask the abovementioned processed data to obtain the NPP data for 642 counties (cities, districts) in the eightprovinces ofthe Yellow River Basin from 1991 to 2015. The GDP and GDP per capita of the counties (cities) in the Yellow River Basin are derived from the Statistical Yearbooks of the Provinces of the Yellow River Basin (1992–2016), the Yellow River Basin Yearbooks of the Provinces (1992–2016), the Statistics of Shandong Province and Qinghai Province Yearbook (1992–2016), and the Yearbook of Shandong Province and Qinghai Province (1992–2016). Among them, the GDP and per capita GDP indicators of some counties and cities are missing. Based on the data for adjacent years, interpolation and gray correlation forecasting methods were used to supplement them. 2.3. Research Methods 2.3.1. NPP Change Trend Analysis Method The unary linear regression analysis method can be used to simulate the change trend of each raster pixel from 1991 to 2015, thereby reflecting the temporal and spatial evolution characteristics of the regional NPP. It is calculated as follows: θi = n × ∑in=1 i × NPPi − ∑in=1 i ∑in=1 NPPi n × ∑in=1 i2 − (∑in=1 i )2 (1) where θi is the slope of NPP change over the years in the Yellow River Basin; n is the number of years; NPPi is the average value of NPP in the ith year; and θi > 0 indicates that NPP is increasing, and the opposite shows a decreasing trend. The absolute value of θi reflects the rate at which NPP increases or decreases. 2.3.2. Ecological Value Evaluation The photosynthesis of vegetation is accompanied by the ecological process of the synthesis of organic matter essential for human survival and development, such as energy fixation, carbon sequestration, and oxygen production. The oxidation ratio of vegetation NPP will affect terrestrial carbon sinks [30]. To effectively and quantitatively evaluate the ecological effects produced in the process of NPP synthesis, using the estimation results of NPP, this paper expresses the ecological value in three parts: the value of organic matter production, the value of fixed CO2 , and the value of O2 released. According to the photosynthesis equation, the mass ratio between primary productivity and CO2 and O2 is 100:163:120, and the mass of the three can be calculated. At the same time, the shadow price method is used to unify the three through price forms to obtain the ecological value calculation results [24]. 1. The Value of Organic Matter j j Nv = NPPi ×c 0.45 (2) Sustainability 2021, 13, 2715 5 of 18 j j In the formula, Nv is the value of organic matter produced in the jth year; NPPi is the average annual organic matter produced in the jth year in the region; and 0.45 is the carbon content of NPP. Considering that coal prices have been declining from 2012 to 2016, the closing price of Qinhuangdao 5500 kcal thermal coal has dropped by about 100 Yuan/t per year, and the ecological value of coal prices during this period has a large deviation. Prices have stabilized since January 2017. In 2017, the annual average price of 5500 kcal thermal coal was 611.7 Yuan/t (https://www.cctd.com.cn/uploadfile/2018/0328/20180328093649264. pdf). The unit price converted into standard coal is 480.6 Yuan/t, slightly higher than the 2010 standard coal unit price of 453.31 Yuan/t. Therefore, the study uses 480.6 Yuan/t as the unit price of standard coal to calculate the ecological value, and c is 480.6 Yuan/t. 2. Fixed CO2 Value and Release O2 Value According to the photosynthesis equation, CO2 absorption and O2 emissions can be measured, and then the carbon tax method and the oxygen production cost method are used to measure their value [31]. The carbon tax conversion cost is 702.95 Yuan/t, and the oxygen production cost is 400 Yuan/t [25]. 3. Ecological Value The value of organic matter produced, the value of fixed CO2 , and the value of O2 j released are integrated to obtain the ecological value of vegetation, Ev . c j j Ev = NPPi × + 702.95 + 400 (3) 0.45 2.3.3. Analytic Hierarchy Process The analytic hierarchy process was proposed by Saaty of the United States; it treats complex decision-making issues as a system and decomposes it into multiple index levels to calculate the weight of each index level. The steps are as follows: The matrix A is established according to the corresponding evaluation criteria and expert judgment, and the importance of Ai and Aj is determined according to the scale of Saaty1~9. According to Formula (4), one calculates the weight, W A , of each indicator, where λmax is the maximum eigenvalue of matrix A. AW A = λmax W A (4) Moreover, it is necessary to use CR to judge whether matrix A passes the consistency test. When CR ≤ 0.1, it can be judged that the matrix passes the consistency test. The specific calculation is as in Formula (5), where RI is the random consistency index. CR = (λmax − n)/(n − 1) RI (5) 2.3.4. Eco-Economic Coordination Evaluation As the two most important subsystems, the ecological economic system is tightly related to the natural environment and human living environment. Eco-economic coordination can measure the degree of harmony between the ecological and economic subsystems or the internal elements of the system [5]; it can represent the relationship between the natural environment and the human environment and measure the degree of harmony and synchronization between the two systems [22]. If the evaluation results of the two systems are synchronized, the region is in a coordinated state, and vice versa. The greater the dispersion is, the higher the degree of disorder is [25]. This study is based on MFOZ, and we selected GDP, per capita GDP, and NPP as the economic system indicators and ecosystem indicators to construct a model of ecologicaleconomy coordinated development. The MFOZ of the eight provinces in the Yellow River Basin divides the territorial space into four categories: optimized development zones, key Sustainability 2021, 13, 2715 6 of 18 development zones, restricted development zones, and prohibited development zones. Different types of territorial spaces have their own emphasis on ecological protection and economic development. The optimized development zone is an urbanized area where the economy is relatively developed, the population is dense, the development intensity is high, and resource and environmental problems are prominent. Industrialization and urbanization development should be optimized. Key development zones should improve their innovation capabilities, take a new path of industrialization and urbanization, and promote accelerated economic development to become an important growth pole supporting the future economic development of the Yellow River Basin and even the entire country. Restricted development zones are agricultural areas whose main function is to provide agricultural products and undertake important tasks in the construction of the national grain production core area, or areas with fragile ecosystems and low resource and environmental carrying capacity. The area should enhance comprehensive agricultural production capacity, and ecological products production capacity restricts the development of large-scale, high-intensity industrialization and urbanization. Prohibited development zones include representative natural ecosystems, natural concentrated distribution areas of rare animal and plant species, natural and cultural relics with special value, and other key ecological function areas. Compulsory protection shall be implemented, and human-made activities shall be strictly controlled. Various factors interfere with the natural ecology and improve environmental quality. In the evaluation of regional coordination relationships and model construction, different ecological and economic system weight values were set according to the main function positioning of the different regions in the Yellow River Basin. According to the comprehensive evaluation results of the economic development level and ecological environment level of each region in the Yellow River Basin, and utilizing the concept of the dispersion coefficient, the ecological-economic coordination degree model was constructed to quantitatively evaluate the coordination of the economic development and ecological environment of the counties (cities) in the study area.  γ   d×r   C= h i2   d +r   (6) 2 In the formula, C is the degree of ecological-economic coordination; d is the level of regional economic development—that is, GDP per capita; r is the level of ecological environment—that is, the ecological value per unit area; and γ is 2, which is the adjustment coefficient [32]. The value range of C is [0, 1]; the larger the value of C, the smaller the dispersion between d and r, and the more coordinated the regional economy and ecology, and vice versa. To more fully reflect the degree of coordination of the ecological economy of the river basin, C, d, and r are used to construct a coordinated development degree function: T = αd + βr √ D = C×T (7) (8) In the formula, T is the comprehensive evaluation index of ecological economy, reflecting the overall level of ecology and economy in the basin; D is the coordinated development index of ecological economy, which is a comprehensive index for examining the degree of coordination and overall development of the basin’s ecological economy; and α and β are undetermined weights. The value range of T is (0, 1); the larger the T, the higher the overall level of regional ecological environment and economic development. The value range of D is (0, 1); the larger the D, the higher the coordinated development of the regional ecological economy. When the value range of D is (0, 0.5), the regional ecological economy is in a stage of disordered development; when the value range of D is (0.5, 0.6), it is in a Sustainability 2021, 13, 2715 7 of 18 weak coordination stage; when the value range of D is (0.6, 1), it is in a stage of coordinated development. According to the requirements of the MFOZ of the eight provinces in the Yellow River Basin, there are differences in the requirements for economic development and ecological protection in each type of development zone. Therefore, when calculating the comprehensive evaluation index of ecological economy and the coordinated development index of the Yellow River Basin, the values of α and β mainly consider the requirements of different types of areas for economic development and ecological protection. By issuing questionnaires to the 42 experts (these 42 experts came from universities and research institutes related to the basin, including the Chinese Academy of Sciences, East China Normal University, Henan Polytechnic University, Henan Institute of Geography, Henan University, Central China Normal University, Shanxi Normal University, Qinghai Normal University, Northwest Normal University, Sichuan Agricultural University, and so on. The male to female ratio was 2:1, with doctors accounting for 86%, professors accounting for 36%, and associate professors accounting for 42%) participating in the MFOZ of the Yellow River Basin, the ecological and economic relative importance of the different major function zones in the upper, middle, and lower reaches of the Yellow River Basin were judged, the consistency was tested, and the geometric average was calculated. For the correlation weight matrix, the α and β weight values were obtained by solving the eigenvector corresponding to the largest eigen root (Table 1). Since the prohibited development zones are mostly distributed in the form of points in the optimized, key, and restricted development zones (mainly distributed in the restricted development zones), the first three types of areas were considered in the study. Table 1. Economic and ecological weights of the Major Function-Oriented Zoning (MFOZ) in the Yellow River Basin. ZoneType α β Upstream key development zone Upstream restricted development zone Midstream key development zone Middle restricted development zone Downstream key development zone Downstream restricted development zone Downstream optimized development zone 0.59 0.29 0.77 0.41 0.67 0.37 0.51 0.41 0.71 0.23 0.59 0.33 0.63 0.49 Based on the main function types of different regions, d and r were calculated, and the regions were divided into three types: economic lag, synchronization, and ecological lag. 3. The Spatio-Temporal Pattern of NPP in the Yellow River Basin 3.1. Temporal and Spatial Distribution Characteristics of Annual Average NPP of Vegetation From 1991 to 2015, the annual average NPP of the Yellow River Basin changed from 196 to264 g·m−2 ·a−1 (Figure 2), showing an increasing trend in fluctuations. The average value in 25 years was 224 g·m−2 ·a−1 . The annual average NPP was mainly concentrated between 100 and 300 g·m−2 ·a−1 , accounting for 55–83% of the study area, with an average of 70%. The second is an NPP < 100 g·m−2 ·a−1 , accounting for the highest proportion of the total basin area in 2001, reaching 27% with an average of 18%. Sustainability 2021, 13, 2715 nability 2021, 13, x FOR PEER Sustainability 2021, REVIEW 13, x FOR PEER REVIEW 8 of 18 8 of 18 8 of 18 Figure 2. The inter-annual variation ofvariation thevariation Yellow River 1991–2015. Figure 2. The inter-annual of Yellow theBasin, Yellow River Basin, 1991–2015. Figure 2. The inter-annual of the River Basin, 1991–2015. From 1991 toFrom 2015, 1991 the annual NPP average of the Yellow Basin showed an showed an River Basin to 2015,average the annual NPP ofRiver the Yellow overall high in the south and low (Figure The overall spatialoverall characteristic of high in theofsouth and low in the (Figure 3).The spatial characteristic in the south andnorth lowin inthe thenorth north (Figure3).3).The in study has the lowest NPP, most of lowhich is located vegetation in vegetation the northwest ofnorthwest the study of area has thearea lowest NPP, most ofNPP, which is in the the northwest ofthe the study area has the lowest most of which is loinMongolia; Inner Mongolia; this isthis because the northwest is a hilly gully theon Loess cated in Innercated this is because theisnorthwest is anorthwest hilly and area onarea the on in Inner Mongolia; because the isgully aand hilly and gully area the Plateau, with serious soil erosion, sparse vegetation, and poor ecological conditions. The Loess Plateau,Loess withPlateau, serious with soil erosion, vegetation, poor ecological condiserious sparse soil erosion, sparseand vegetation, and poor ecological condihighest NPP of thevegetation vegetation located in in the south; this isisdue forest tions. The highest NPP of the isisvegetation located thisthe duetotothe thehigh high tions. The highest NPP of the is located in south; this is due to coverage the high in in this area, and it this is main production area of of agricultural forest coverage this area, and it the is area, the main production area agricultural forest coverage in and it is the main production areaproducts. ofproducts. agricultural products. Figure 3. Distribution and slope of theand netslope primary productivity (NPP) in the Yellow 1991–2015. Figure 3. Distribution of the net primary productivity (NPP)River in theBasin, Yellow River Basin, Basin, 1991–2015. 1991–2015. Distribution and Yellow River 3.2. Analysis of3.2. theAnalysis Change Trend inVegetation of the Change TrendNPP inVegetation NPP 3.2. Analysis of the Change Trend inVegetation NPP Based on the Based pixel scale, inter-annual in vegetation NPP in the Yellow on thethe pixel scale, the change inter-annual change in vegetation NPP in the Yellow Based thewas pixel the inter-annual change in vegetation NPP the change Yellow River Basin from 1991 to on 2015 analyzed by unary linear the NPP change River Basin from 1991 toscale, 2015 was analyzed by regression, unary linear regression, theinNPP River Basin from 1991 to 2015 was analyzed by unary linear regression, the NPP change trend of eachtrend grid in obtained, andobtained, the results were of the eachstudy grid area in thewas study area was and the reclassified results werebyreclassified by trend of each grid in the study area was obtained, and the results were reclassified by the natural discontinuity method (Figure 3). This can be roughly divided into catthe natural discontinuity method (Figure 3). This can be roughly seven divided into seven catthe natural discontinuity method (Figure 3). This can be roughly divided into seven egories: serious reduction, moderate reduction, slight reduction, basically unchanged, egories: serious reduction, moderate reduction, slight reduction, basically unchanged, categories: serious reduction, moderate slightinreduction, unchanged, slight increase, moderate increase, andincrease, severe increase. Theincrease. trend vegetation slight increase, moderate andreduction, severe The trendbasically inNPPvegetation NPPslight increase, moderate increase, and severe increase. The trend in vegetation change in thechange Yellow in River Basin shows regional spatial differences. with NPPchange the Yellow River obvious Basin shows obvious regional spatialAreas differences. Areas with in the Yellow River in Basin obvious regional spatial differences. Areas with slight and slight and moderate reductions NPPshows are mainly distributed Ningxia, central Inner slight and moderate reductions in NPP are mainlyindistributed in Ningxia, central Inner Mongolia, northern Shaanxi, and most municipal districts. This area is close to the Mu Mongolia, northern Shaanxi, and most municipal districts. This area is close to the Mu Sustainability 2021, 13, 2715 ainability 2021, 13, x FOR PEER REVIEW 9 of 18 9 of 18 moderate reductions in NPP are mainly distributed in Ningxia, central Inner Mongolia, Us Desert, the Luliang Mountains, the coal resource-rich areas, northern Shaanxi, and most municipal districts. Thisand areavarious is close urban to the agMu Us Desert, glomerations the watershed. Thethe ecological environment is and extremely theinLuliang Mountains, coal resource-rich areas, variousfragile, urban with agglomerations low crop yields, levels ofThe industrialization and urbanization, and afragile, harsh ecological in thehigh watershed. ecological environment is extremely with low crop yields, environment. Areas with serious reductionsin are mainly distributed around the high levels of industrialization andNPP urbanization, and a harsh ecological environment. areas of slight and moderate reduction, which are distributed in dots and lines. The avAreas with serious reductions in NPP are mainly distributed around the areas of slight erage annual NPP changereduction, slope is low, andare thedistributed ecologicalinenvironment is deteriorating. and moderate which dots and lines. The average annual NPP changeincreases slope is low, andare themainly ecological environment is deteriorating. Areas Areas with severe in NPP located in the “Two Rivers and One Wa-with severe increases NPPas areBailong mainly located in the “Two Rivers One Water” areas, ter” ecological areas,insuch River, Baishui River, Westand Hanshui River,ecological Sisuch as Bailong River,Hanzhong Baishui River, WestFenhe Hanshui River,Valley Sichuan-Dianyun chuan-Dianyun Forest Area, Basin, River Basin, andForest Area, Hanzhong Basin, Fenhe River City, ValleyHanzhong Basin, andCity, Huanghuaihai Plain, including Longnan Huanghuaihai Plain, including Longnan Linfen City, Zhumadian City, Hanzhong City, Linfen City, Zhumadian City, Heze City, and other cities under the City, Heze and other cities under the jurisdiction of the counties. The ecological thebeen counties. The ecological area has been effectively environmentjurisdiction in this areaofhas effectively protected,environment and the NPPinisthis increasing annuprotected, and the is increasing annually. The areas with slight ally. The areas with slight andNPP moderate increases in NPP have a wide range,and andmoderate those increases in aNPP have a wide range, andexpanded those withtoclose to a significant increase have aexpanded to with close to significant increase have the surrounding area, showing the surrounding area, showing a trend and in concentrated andthe contiguous and trend in concentrated and contiguous distribution indicating that ecologicaldistribution enindicating that the ecological environment of the Yellow River Basin is improving each vironment of the Yellow River Basin is improving each year. year. 4. Evaluation of the Ecological and Economic Coordination Relationship in the Yellow 4. Evaluation of the Ecological and Economic Coordination Relationship in the Yellow River Basin River Basin 4.1. The Dynamic Characteristics of Ecological of Value and Economic Value in the Yellow 4.1. The Dynamic Characteristics Ecological Value and Economic Value inRiver the Yellow River Basin Basin To facilitateTo thefacilitate analysisthe of analysis the time-series characteristics of the ecological economy economy of of the time-series characteristics of the ecological of the Yellow Basin, theBasin, valuethe of value NPP in Yellow 1991 to 1991 2015to 2015 was theRiver Yellow River of the NPP in the River YellowBasin Riverfrom Basin from calculated the standard coal price of 480.6 Yuan/t. studyused was calculated based based on theon standard coal price of 480.6 Yuan/t. ThisThis studyused the the general retail price index of the Yellow River Basin, and theGDP GDPofofthe theYellow YellowRiver River Basin was general retail price index of the Yellow River Basin, and the revisedtotoobtain obtainthe theGDP GDPdata datafor forthe theYellow Yellow River River Basin Basin based based on on comparacomparable prices in Basin was revised 2015.From 1991 to 2015, the ecological valuevalue of the of Yellow River Basin ble prices in 2015.From 1991 to 2015, the ecological the Yellow Rivershowed Basin an upward 2−14.991x (y = 0.586x 14.991x + 1047.9, R2 = 0.178); the lowest valuevalue was 823 showed an trend upward trend (y2=−0.586x + 1047.9, R2 =0.178);the lowest wasbillion Yuan in 2001, and the highest was 1142 billion Yuan in 2015 (Figure 4). The GDP of the Yellow 823 billion Yuan in 2001, and the highest was 1142 billion Yuan in 2015 (Figure 4).The 2 = 0.8606), from 350 billion River Basin increased linearly (y = 62.757x − 308.36, R Yuan in GDP of the Yellow River Basin increased linearly (y = 62.757x−308.36, R2 1991 to 15,160Yuan billion Yuanto15,160billion in 2015—a 43-fold increase. =0.8606),from350 billion in 1991 Yuan in 2015—a 43-fold increase. Figure 4. 4. Dynamic of the the NPP NPP value value and and GDP GDP of of the the Yellow Yellow River River Basin, Basin, 1991–2015. 1991–2015. Figure Dynamic changes changes of 4.2. The Spatial Distribution Characteristics of Ecological Value in the Yellow River Basin Ecological value and ecological value per unit area are signs used to judge the quality of the regional ecological environment.The ecological value of the Yellow River Basin Sustainability 2021, 13, 2715 10 of 18 4.2. The Spatial Distribution Characteristics of Ecological Value in the Yellow River Basin Ecological value and ecological value per unit area are signs used to judge the quality of the regional ecological environment. The ecological value of the Yellow River Basin in 2015 was 1103 billion Yuan, accounting for approximately 7.28% of the GDP in 2015.As can be seen from Table 2, Golmud City, Zhiduo County, Dulan County, Haixi Mongolian, the Tibetan Autonomous Prefecture, Maduo County, and five other cities and counties have the highest ecological value, with an average value of 33.94 billion Yuan; the ecological value of urban areas, such as Xuchang City District, Yima City, Jincheng City District, Linxia City, and Wulanchabu City District, is the lowest. The average ecological value per unit area of the Yellow River Basin in 2015 was 573,900 Yuan/km2 . The ecological value per unit area of five counties, including Pingdingshan City District, Mian County, Tanghe County, FangchengCounty, and JuCounty, ranks among the top five, with an average value of 983,200 Yuan/km2 . The average ecological value of five counties and cities, including Suzhou District, Ejina Banner, Yijinhuoluo Banner, Guyang County, and Yuanping City, is 61,900 Yuan/km2 , which is only one-tenth of the average for the basin. Combining the above two indicators, most counties have higher ecological values, while urban areas generally have lower ecological values. Table 2. Ecological value of the eightprovinces in the Yellow River Basin in 2015. Highest Values Ecological value (100 million Yuan) Ecological value per unit area (10,000 Yuan/km2 ) Lowest Values Golmud 624.15 Xuchang City District 0.94 Zhiduo County 378.13 Yima City 0.88 Dulan County 289.41 Jincheng City District 0.87 Haixi Mongolian and Tibetan Autonomous Prefecture 205.86 Linxia City 0.70 Maduo County 199.21 Ulanqab City District 0.36 Pingdingshan City District 99.08 Suzhou District 7.01 Mian County 98.75 Ejina Banner 6.34 Tanghe County 98.74 Yijinhuoluo Banner 6.30 Fangcheng County 97.74 Guyang County 6.23 Ju County 97.26 Yuanping City 5.07 4.3. The Spatial Characteristics of the Economic Development Level of the Yellow River Basin We used ARCGIS and natural clustering to divide the economic development of the Yellow River Basin in 2015 into five levels: high value, medium–high value, medium value, medium–low value, and low value (Figure 5). As a whole, values are low in the south and high in the north, and the lower reaches of the Yellow River Basin are higher than the upper and middle reaches. In the upper reaches of the basin, contrary to NPP, areas with high and medium–high per capita GDP mainly appear in the Golmud and Jiuquan cities in the northwest. This area is a key development zone in Qinghai Province and Gansu Province, and a carrier of new industrialization and urbanization in the upper reaches of the Yellow River Basin. The low–medium and medium per capita GDP present a situation of overall dispersion and partial concentration. Areas with low per capita GDP are contiguously distributed, encompassing 95 counties and districts. These account for 32% of the entire low valuearea. This is mainly distributed in agricultural development areas, such as the Ningxia Plain Agricultural Area and the Liupan Mountain Forest Ecological Barrier Area, the Qinba Mountains, the Daliangshan Mountains, and other ecologically restricted development zones. Agriculture in this region is relatively developed and the ecological environment is Sustainability 2021, 13, 2715 11 of 18 Sustainability 2021, 13, x FOR PEER REVIEW good, but of 18 the industrial foundation is weak, the level of urbanization is low, and the 11 urban economy is underdeveloped. Figure 5. 5. The The spatial spatial distribution distributionof ofper percapita capitaGDP GDPand andthe theanalysis analysisofofcold coldand and hotspots per capita GDP in the YelFigure hotspots of of per capita GDP in the Yellow low River Basin in 2015. River Basin in 2015. In the middle reaches, the high-value and medium–high-value areas of per capita GDP account for 58% of the entire study area. area. They are mainly distributed in restricted development zones, including Sunite Left development zones, including Sunite Left Banner, Banner, Shenmu Shenmu County, County, and and Yulin Yulin City, and key development development zones, zones,including includingZhungeer ZhungeerBanner, Banner, Tuoketuo County, Baotou Tuoketuo County, Baotou City,City, and and Wushen Banner. The low–medium medium per capita are dispersed Wushen Banner. The low–medium and and medium per capita GDPGDP areasareas are dispersed but but partially concentrated in Huanglong County, Fu County, Xiangning partially concentrated in Huanglong County, Fu County, ZhidanZhidan County,County, Xiangning County, Pu County, Liangcheng County, among with low with per capita GDP are County, Pu and County, and Liangcheng County,others. amongAreas others. Areas low per capita located 123 counties, includingincluding Ningwu County, County, County,Xiand Fangshan GDP areinlocated in 123counties, NingwuLin County, LinXiCounty, County, and County. They are They mainly in mountainous areas inareas Shanxi, south of Helan Fangshan County. aredistributed mainly distributed in mountainous in Shanxi, south of Mountain and Qinling Mountain. This area is dominated by mountains, with complex Helan Mountain and Qinling Mountain. This area is dominated by mountains, with terrain and inconvenient transportation, which is not conducive to high-quality economic complex terrain and inconvenient transportation, which is not conducive to high-quality development. economic development. In the lower reaches, areas with high and medium–high per capita GDP are mainly located counties, including Dongying City,City, ZiboZibo City, City, KenliKenli County, Qinglocated in in 12 12cities citiesand and counties, including Dongying County, dao City, Weihai City, Zhengzhou City District, and others. This area is located in Qingdao City, Weihai City, Zhengzhou City District, and others. This area is locatedthe in optimized development zonezone and key zone of the of Yellow River Basin, the optimized development anddevelopment key development zone the Yellow Rivermainly Basin, distributed on bothon sides of sides the Beijing–Guangzhou and Longhai railway lines. The difmainly distributed both of the Beijing–Guangzhou and Longhai railway lines. fusion and spillover effectseffects of talent, capital, technology, and policies are obvious, and The diffusion and spillover of talent, capital, technology, and policies are obvious, the transportation location advantage is prominent. The median GDP per capita area is and the transportation location advantage is prominent. The median GDP per capita armainly distributed within the boundary of the river basin, such as at the junction of Henan ea is mainly distributed within the boundary of the river basin, such as at the junction of Province, Shanxi Shanxi Province, and Shandong Province. This area is an ecological or agriculHenan Province, Province, and Shandong Province. This area is an ecological or tural area with relatively developed agriculture and a good ecological environment, but agricultural area with relatively developed agriculture and a good ecological environthe urban vitality is insufficient. are a large number low– ment, but economic the urbandevelopment economic development vitality isThere insufficient. There are of a large medium and low-value areas that are distributed contiguously, mainly in the agricultural number of low–medium and low-value areas that are distributed contiguously, mainly areas the Huanghuai of the Beijing–Guangzhou railway line. in theof agricultural areasPlain of theeast Huanghuai Plain east of the Beijing–Guangzhou railway The spatial agglomeration characteristics of the Yellow River Basin economy are line. analyzed using the cold- and hotspot analysis method [25]. The high-value areas and The spatial agglomeration characteristics of the Yellow River Basin economy are medium–high-value areas of per capita GDP are mainly concentrated in the nine metropolianalyzed using the cold- and hotspot analysis method [25]. The high-value areas and tan areas, with Jinan, Qingdao, and Zhengzhou in the lower Yellow River Basin and Xining, medium–high-value areas of per capita GDP are mainly concentrated in the nine metLanzhou, Hohhot, Yinchuan, Xixian, and Taiyuan as the core (Figure 5). The high level of ropolitan areas, with Jinan, Qingdao, and Zhengzhou in the lower Yellow River Basin economic development has formed a hot area in the Basin, which has a significant role in and Xining, Lanzhou, Hohhot, Yinchuan, Xixian, and Taiyuan as the core(Figure 5). The driving the economic growth of the region. The cold-spot areas mainly have low per capita high level of economic development has formed a hot area in the Basin, which has a sigGDP, andare distributed in ecologically restricted development zones, such as Dingxi City nificant role in driving the economic growth of the region. The cold-spot areas mainly and Baiyin City of Gansu Province along the Yellow River Agricultural Industrial Zone; have low per capita GDP, andare distributed in ecologically restricted development zones, such as Dingxi City and Baiyin City of Gansu Province along the Yellow River Agricultural Industrial Zone; Linfen City and Changzhi City of Shanxi Province City and other Fenwei plain agricultural areas;the TongbaiDabie Mountain ecological area is Sustainability 2021, 13, 2715 12 of 18 Sustainability 2021, 13, x FOR PEER REVIEW 12 of 18 Linfen City and Changzhi City of Shanxi Province City and other Fenwei plain agricultural areas; the TongbaiDabie Mountain ecological area is dominated by Gushi County and County in Henan and the Huanghuai Plainofarea is dominated by isShangcheng dominated by Taikang CountyProvince; and Huaiyang County. The level economic developTaikang County and Huaiyang County. The level of economic development in this region ment in this region is low and agglomeration occurs, which has a negative effect on suris low and agglomeration occurs, which hasarea a negative effect on surrounding counties and rounding counties and cities. The transition is located between the cold and hot areas, cities. The transition area is located the cold and hot areas, and the economic and the economic agglomeration effectbetween is not significant. agglomeration effect is not significant. 4.4. Degreeof Coordinated Development of an Ecological Economy in the Yellow River Basin 4.4. Degreeof Coordinated Development of an Ecological Economy in the Yellow River Basin 4.4.1. Time-Series Analysis 4.4.1. Time-Series Analysis The degree of ecological and economic coordination of the Yellow River Basin from The degree of ecological and economic coordination of the Yellow River Basin from 1991 to 2015 was calculated using Equations (6)–(8). The maximum value was 0.40 in 1997, 1991 to 2015 was calculated using Equations (6)–(8). The maximum value was 0.40 in and theand minimum value was 0.22was in 2000 and 2009 (Figure 6).From 6). 1991 to 2000, 1997, the minimum value 0.22 in 2000 and 2009 (Figure From 1991coordito 2000, nation rose and then fell, showing an “inverted U-shaped” trend. This indicates that the coordination rose and then fell, showing an “inverted U-shaped” trend. This indicates that relationship between ecology and economy in theinbasin improved before 1997.The the relationship between ecology and economy the basin improved before 1997.ecoThe nomic foundation in theinbasin is weak and development is slow, nothas had a large economic foundation the basin is weak and development is butit slow,has butit not had a negative impact on the environment. From 1997 to 2000, the relationship between ecology large negative impact on the environment. From 1997 to 2000, the relationship between and economy in the basin became and the degree economic coordination in the ecology and economy in the basincloser, became closer, and theof degree of economic coordination basin dropped to the lowest level. From 2001 to 2009, coordination increased first and in the basin dropped to the lowest level. From 2001 to 2009, coordination increasedthen first declined. relationship between ecology economy in the basin is tense antagand thenThe declined. The relationship betweenand ecology and economy in the basinand is tense and onistic. After 2009, the relationship between the the economy andand environment tended antagonistic. After 2009, the relationship between economy environment tendedtoto ease, ease,and andthe thedegree degreeofofcoordination coordinationbetween betweenthe thetwo twoincreased increasedgradually. gradually. Figure6.6.Coordinated Coordinateddevelopment developmentofofthe theecological ecologicaleconomy economyin inthe theYellow Yellow River River Basin, Basin, 1991– 1991–2015. Figure 2015. 4.4.2. Analysis of Spatio-Temporal Changes In 1991, 12 regions were in aChanges state of coordinated development, 9were in a state of 4.4.2. Analysis of Spatio-Temporal weakly coordinated development, and 621were in a state of disorder. In 2015, 41 regions In 1991, 12 regions were in a state of coordinated development, 9were in a state of were in a state of coordinated development, 50were in weakly coordinated development, weakly coordinated development, and 621were in a state of disorder. In 2015, 41regions and 551were in disorder (Figure 7). From 1991 to 2015, the coordination status of 96counties were a state of coordinated 50were weakly coordinated development, and in cities changed. Of these,development, 84counties and cities in improved, while 12declined. Among and 551were in disorder (Figure 7). From 1991 to 2015, the coordination status of 96counthem, the coordinated development of ecological economy in the upper reaches of the ties andRiver citiesBasin changed. these, much, 84counties and citiesand improved, whilewere 12declined. Yellow has notOfchanged and the middle lower reaches generally Among them, the coordinated development of ecological economy in the upper reaches on the rise (Table 3). of the Yellow River Basin has not changed much, and the middle and lower reaches were generally on the rise (Table 3). Sustainability Sustainability 2021, 2021, 13, 13, 2715 x FOR PEER REVIEW 13 13 of of 18 18 Figure 7. The Yellow River River Basin, Basin, 1991–2015. 1991–2015. The distribution distribution of coordinated development of the ecological economy in the Yellow further analyzedevelopment thesespatio-temporal characteristics, this paper comTableTo 3. The coordinated and changes evolution in the Yellow River Basin from 1991 to 2015. bines the coordination and synchronization of the regional ecological economy to divide State Upstream Midstream Downstream Totallag, the coordinatedChange development of the ecological economy into disorder–economic Disorder–Weak coordination and disorder–ecological 3 17 48 disorder–synchronous development, lag; 28weak coordinaDisorder–Coordination 6 15 13 weak coordina34 Rise tion–economic lag, weak coordination–synchronous development, and Weaklag; coordination–Coordination 0 2 2develtion–ecological and coordination–economic0 lag, coordination–synchronous Coordination–Disorder lag. We calculated 1 1 coordinated 5 7 opment, and coordination–ecological the development Coordination–Weak coordination 0 0 0 0 Fall degree of 642 counties and cities in the Yellow River Basin in 1991 and 2015, and judged Weak coordination–Disorder 2 1 2 5 the coordinated development degree type (Figure 7).The number of counties, including 96 0, 4, the above types, in Total 1991 and 2015 were612, 2, 8,12 8, 1, 0, 8, 0,34and 3, and50 522, 4, 30, 42, 24, 1, and 15, respectively. From 1991 to 2015, the coordinated development of the ecological of thethese Yellow River Basin evolution was dominated by disorder–economic lag, To economy further analyze spatio-temporal characteristics, this paper combines accounting for 81–95%, and coordinatedofdevelopment relatively low. to divide the the coordination and synchronization the regional was ecological economy coordinated development of the ecological economy into disorder–economic lag, disorder– Table 3. The coordinated development and changes in thelag; Yellow River Basin from 1991 to 2015. synchronous development, and disorder–ecological weak coordination–economic lag, weak coordination–synchronous development, and weak coordination–ecological and Change State Upstream Midstream Downstream lag; Total coordination–economic lag, coordination–synchronous development, and coordination– Disorder–Weak coordination 3 17 28 48 ecological lag. We calculated the coordinated development degree of 642 counties and Rise Disorder–Coordination 6 15 13 34 cities in the Yellow River Basin in 1991 and 2015, and judged the coordinated development Weak coordination–Coordination 0 0 2 2 degree type (Figure 7). The number of counties, including the above types, in 1991 and Coordination–Disorder 1 1 5 7 2015 were612, 2, 8, 8, 1, 0, 8, 0, and 3, and 522, 4, 30, 42, 0, 4, 24, 1, and 15, respectively. From FalltoCoordination–Weak coordination 0 economy of the 0 Yellow River 0 1991 2015, the coordinated development of 0the ecological Weak coordination–Disorder 2 1 2 5 Basin was dominated by disorder–economic lag, accounting for 81–95%, and coordinated 12 34 50 96 development was Total relatively low. The coordinated development areas are mainly concentrated in the Shandong Peninsula urban agglomeration, with Jinanareas and are Qingdao as concentrated the center; theinCentral Plains urban The coordinated development mainly the Shandong Penagglomeration, with Zhengzhou as the center; the Guanzhong urban insula urban agglomeration, with Jinan and Qingdao as the Plain center; the agglomeration, Central Plains with Xi’an as the center; the HubaoEyu with Hohhot as the center; urban agglomeration, with Zhengzhouurban as theagglomeration, center; the Guanzhong Plain urban agandLanxi City Group, with Lanzhou City and Xining City as the center. The level of glomeration, with Xi’an as the center; the HubaoEyu urban agglomeration, with Hohhot economic development become thewith mainLanzhou factor affecting theXining coordinated development as the center; andLanxihas City Group, City and City as the center. of the ecological economy. Weak coordination areas scattered and closethe to the coordiThe level of economic development has become the are main factor affecting coordinatnation areas, including developed regions, such as Zouping Abaga ed development of the economically ecological economy. Weak coordination areas areCounty, scattered and Banner, City, Haixiareas, Mongolian, and economically Tibetan Autonomous Prefecture; Duolun close to Hohhot the coordination including developed regions,such as County, and Wuchuan County; Huachi County, Yumen County; and other economically Zouping County, Abaga Banner, Hohhot City,Haixi Mongolian, and Tibetan Autonounderdeveloped areas. The economically developed regions have low NPP andCounpoor mous Prefecture;Duolun County, and Wuchuan County;Huachi County, Yumen ecological environment quality. The economic level of economically underdeveloped area ty; and other economically underdeveloped areas. The economically developed regions is relatively low, the ecological value per unit area is high, and the ecological and economic have low NPP and poor ecological environment quality. The economic levelof economi- Sustainability 2021, 13, 2715 14 of 18 systems are in weak coordination with a low degree of coordination. The disordered areas are vast, accounting for 86% of the Yellow River Basin. Most of the areas are located in the upper reaches of the Yellow River Ecological Restricted Development Zones and key energy-rich development zones in the middle reaches. 5. Discussion 5.1. Comparison of NPP Data Accuracy in the Yellow River Basin Among the research objects, the NPP of vegetation in counties and districts under the jurisdiction of cities, such as Jiayuguan City, Delingha City, Xining City, and Haidong, Xi’an-Taiyuan Metropolitan Area, Dezhou, Binzhou, Dongying, and Weihai, is low. This is consistent with the fact that the urbanization level of the “one along nine tripods” metropolitan area and surrounding counties and cities in the nine provinces of the Yellow River Basin is relatively high and the vegetation coverage rate is low. The average NPP of the Yellow River Basin from 1991 to 2015 was 224 g·m−2 ·a−1 , and the variation range is 196–264 g·m−2 ·a−1 . This is consistent with the result that Tian et al. [33] calculated using MODIS17A3 data. The authors found that the average annual NPP of vegetation in the Yellow River Basin from 2000 to 2015 was228.20 g·m−2 ·a−1 , and the range of variation was179.60–258.10 g·m−2 ·a−1 . Zhu [34] used the CASA model and the BP neural network model to dynamically simulate the vegetation NPP of the Yellow River Basin from 1992 to 2015 and found that the average annual NPP was249.69 g·m−2 ·a−1 , which was close to the resultspresented in this paper:224 g·m−2 ·a−1 . 5.2. Reliability Verification of Ecological-Economic Coordination Model This paper improves the ecological and economic coordinated development model of the Yellow River Basin based on MFOZ. To verify the findings, the following discussion is offered. First, the ecological and economic coordination degree of the Yellow River Basin, calculated here, shows the following trend: before 1997, the relationship between ecology and economy improved, and the coordination degree of the ecological economy gradually increased; from 1997 to 2009, the relationship between ecology and economy became closer, showing an antagonistic state; after 2009, the relationship between ecology and economy tended to ease, and the degree of ecological economy coordination gradually increased. Sun et al. [35] used the concept of inclusive green growth to build an economic and ecological coordinated development model to evaluate the green economic growth level of 285 cities in China from 2003 to 2015. They found that China’s green economic growth level was low and relatively unstable before 2010; however, after 2010, the trend has been on the rise, which is consistent with the trend of ecological and economic coordination in the Yellow River Basin estimated in this study. Second, in this study, the degree of economic and ecological coordination is between 0 and 0.6, and the coordination degree is mainly disorder–economic lag. The coordination areas are mostly concentrated in key development zones with urban agglomeration as the core, while the disorder areas are mostly concentrated in agricultural development zones and ecologically restricted development zones in the middle and upper reaches of the Yellow River. From a regional perspective, Zameer et al. [36] established an eco-economic index system and a coupling coordination degree model and calculated that the economic and ecological coupling coordination degree of eastern, central, and western China from 2013 to 2018 was between 0.3 and 0.5, which is still low. This demonstrates the pattern that the west is lower than the middle and the east, which is consistent with this article’s findings. Third, compared with traditional model, the improved model solved the problem of lacking scientific basis, but its precision will inevitably be affected by the subjectivity of the expert scoring and the number of experts. Sustainability 2021, 13, x FOR PEER REVIEW Sustainability 2021, 13, 2715 15 of 18 15 of 18 5.3. Comparison of Research Results and Related Results 5.3. Comparison of Research Results and Related Results On the whole, the coordination of ecological economy in the Yellow River Basin is On the is whole, the coordination of ecological in the Yellow River Basin is low, low, which consistent with the result that Xu eteconomy al. [26] found when they evaluated the which is consistent with the result that Xu et al. [26] found when they evaluated nine nine provinces and regions in the Yellow River Basin from the two dimensions the of highprovinces and regions in the Yellow River Basin from the two dimensions of high-quality quality economic development and ecological security. economic development and ecological security. From the perspective of the upper, middle, and lower reaches of the basin, the ecoFrom perspective of the upper, middle, and lower reaches of thethe basin, the ecologilogical andthe economic coordination of the Yellow River Basin presents following patcal and economic coordination of the Yellow River Basin presents the following pattern: tern: downstream is higher than the middle reaches, and the middle reaches higher than downstream is higher than the middle reaches, and the middle reaches higher than the the upper reaches (Figure 8). Shi [19] came to a similar conclusion when studying the couupper reaches (Figure 8). Shi [19] came to a similar conclusion when studying the coupling and coordination of ecological protection and high-quality economic development pling and coordination of ecological protection and high-quality economic development in the Yellow River Basin. Zhang [37] studied the high-quality development level of cenin the Yellow River Basin. Zhang [37] studied the high-quality development level of tral cities in the Yellow River Basin from the five dimensions of economic structure, innocentral cities in the Yellow River Basin from the five dimensions of economic structure, vation-driven development, resource allocation, appropriate ecological livability, and innovation-driven development, resource allocation, appropriate ecological livability, and sharing of public services. He concluded that the economic and social development of the sharing of public services. He concluded that the economic and social development of the Yellow River Basin showed a pattern of“upstream backwardness, midstream rising, and Yellow River Basin showed a pattern of“upstream backwardness, midstream rising, and downstream development”, which is roughly the same as the results of this paper. downstream development”, which is roughly the same as the results of this paper. Figure 8. Degree of coordinated nine provinces (upper, coordinated development developmentofofthe theecological ecologicaleconomy economyininthe the nine provinces (upper, middle, and lower reaches) of the Yellow River Basin, 1991–2015. middle, and lower reaches) of the Yellow River Basin, 1991–2015. 5.4. Recommendationsfor Recommendationsfor Sustainable Sustainable Development Development Policy Policy of of the the Yellow Yellow River 5.4. River Basin Basin Region Region Ecological protection and high-quality development of the Yellow River Basin,asasa Ecological protection and high-quality development of the Yellow River Basin, a major national strategy, are important parts of regional coordinated and sustainable major national strategy, are important parts of regional coordinated and sustainable dedevelopment. The economic and ecologicalcoordination coordinationfluctuations fluctuationsofofthe the Yellow Yellow River River velopment. The economic and ecological Basin are related to China’s development strategies and policies in various periods. Basin are related to China’s development strategies and policies in various periods. Since Since 2010, the Resources Comprehensive PlanPlan (2010–2030)”, the “Yellow River 2010, the “National “NationalWater Water Resources Comprehensive (2010–2030)”, the “Yellow Basin Comprehensive Planning (2012–2030)”, the “National Development Planning” isRiver Basin Comprehensive Planning (2012–2030)”, the “National Development Plansued by the State Council, the National Development and Reform Commission, and other ning” issued by the State Council, the National Development and Reform Commission, government departments have all regarded the Yellow River Basin as a key area of ecoand other government departments have all regarded the Yellow River Basin as a key area nomic ecology in China, thereby promoting the steady improvement of the economic and of economic ecology in China, thereby promoting the steady improvement of the ecoecological coordination of the Yellow River Basin. nomic and ecological coordination of the Yellow River Basin. The degree of coordination of the upper and middle lower reaches of the Yellow River The degree of coordination of the upper and middle lower reaches of the Yellow Basin was the closest in 1991, but the coordination degree of the lower reaches was higher River Basin was the closest in 1991, but the coordination degree of the lower reaches was than that of the upper and middle reaches later, which has a strongrelationship with the higher than that of the upper and middle reaches later, which has a strongrelationship main function positioning of the upper and middle lower reaches. After 2004, the middle with the main function positioning of the upper and middle lower reaches. After 2004, the reaches were significantly higher than the lower reaches, which is closely related to the middle reaches were significantly higher than the lower reaches, which is closely related implementation of the policy of returning farmland to forest (grass) in the middle reaches to the implementation of the policy of returning farmland to forest (grass) in the middle of the Loess Plateau and the economic boost from energy development in the middle reaches reaches of of the the Loess Loess Plateau Plateau.and the economic boost from energy development in the middle reaches of the Loess Plateau. The territory upstream of the Yellow River Basin belongs to the national key limit development zone, and the inability to drive at the center of the city and urban agglom- Sustainability 2021, 13, 2715 16 of 18 eration has caused economic development to lag. Thus, policymakers should focus on thinking about river basin ecological compensation mechanisms, deepening the upgrades to western development policy, establishing a precise strategy for poverty alleviation in the west, improving the upstream area residents’ income and welfare, and solving the problem of the up-stream’s backward economy. The middle reaches of the Yellow River Basin are mostly located in the Loess Plateau, where soil erosion is serious and the ecological environment is poor. The middle three provinces are also rich in coal resources. In the process of economic development, coal resources have been developed on a large scale. In addition, the unreasonable industrial structure and backward technical equipment of the three provinces have led to various ecological problems and accelerated the deterioration of the regional ecological environment. The ecological lag in the middle reaches of the Yellow River Basin requires thinking about continuing to deepen the policy of returning farmland to forest (grass), and using the new national strategy of ecological protection and high-quality development in the Yellow River Basin to properly resolve the contradiction between energy development and ecological protection in provinces such as Shanxi, Shaanxi, and Inner Mongolia. Compared with the upper and middle reaches of the Yellow River Basin, the economic development level of the lower reaches of the Yellow River is higher, the ecological environment is better, and the degree of ecological and economic coordination is higher, but it is still lower than the national level. With the development of industrialization and urbanization, the contradiction between the attenuation of water resources and the rigid growth of water use in the lower reaches of the Yellow River Basin has become prominent, and the difficulty of ensuring agricultural water use has increased [20]. We should focus on improving the methods of ecological compensation, combining the ecological compensation mechanism of water resources with the compensation mechanism of cultivated land protection, and solving the problems of the unbalanced allocation of downstream water resources, food security, and farmers’ income. 6. Conclusions This paper improves the coordinated development model of ecological economy in the Yellow River Basin on the basis of main function areas, and considers the influence of different main function positions on the coordinated development of the ecological economy. It can be widely used in the analysis of the coordinated relationship between ecosystems and economic systems in different types of areas. The ecological and economic coordination of the Yellow River Basin has undergone three stages of change. From 1991 to 2000, coordination in the basin rose and then fell, presenting an “inverted U-shaped” trend. Before 1997, the relationship between ecology and economy in the basin tended to be better, and the economic foundation in the basin was weak and developed slowly, without significant negative impacts on the environment. From 1997 to 2000, the relationship between ecology and economy became increasingly close, and the coordination degree of economy in the basin decreased to the lowest level. From 2001 to 2009, ecological and economic coordination increased and then decreased. The coordination degree was very low, and the relationship between ecology and economy became closer. After 2009, coordination fluctuated upward. In the past 25 years, the coordinated development of ecological economy in the Yellow River Basin has been dominated by economic lagging imbalances, and the coordination of ecological economy presents the following pattern: downstream higher than midstream, and midstream higher than upstream. Generally speaking, the low level of economic development is the main factor for the imbalance of the ecological economy. The implementation of the “Develop the West” campaign plays an important role in improving western economic development and promoting the coordinated development of ecological economy. At the same time, the “Grain for Green” policy in the middle reaches has improved the service function of the ecosystem and promoted the coordinated development of the ecological economy. Sustainability 2021, 13, 2715 17 of 18 Research plays an important role in policy formulation for the Yellow River Basin. Based on the different main functions of the upper, middle, and lower reaches, corresponding countermeasures have been taken to solve the problem of coordinated development. The upstream areas should focus on solving the problem of upstream economic backwardness. The middle reaches should properly solve energy development and ecological development concerns. The downstream areas should mainly solve the problems of the unbalanced allocation of downstream water resources and low food security. Author Contributions: Conceptualization, X.Q.; methodology, X.Q. and T.C.; software, T.C., Z.Z., J.G. and H.Z.; validation, J.G. and H.Z.; formal analysis, T.C., J.G., H.Z. and Z.Z.; investigation, T.C.; resources, Z.Z.; data curation, Z.Z., J.G. and T.C.; writing—original draft preparation, Z.Z., T.C. and X.Q.; writing—review and editing, Y.Y.; visualization, Y.Y., T.C.; supervision, Z.Z., X.Q.; project administration, Z.Z.; funding acquisition, X.Q. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China, grant number 41971274; the Philosophy and Social Science Scholar’s Funds of Universities of Henan Province, grant number 2018-YXXZ-07; the Innovation research team of Henan Provincial University, grant number 2021-CXTD-08; theInnovation research team of Henan Polytechnic University, grant number CXTD2020-1. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not Applicable. Data Availability Statement: Not Applicable. Acknowledgments: We express our gratitude to anonymous reviewers and editors for their professional comments and suggestions. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Harte, M.J. Ecology, sustainability, and environment as capital. Ecol. Econ. 1995, 15, 157–164. 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https://openalex.org/W3049242285	https://europepmc.org/articles/pmc8549361?pdf=render	English	null	Post-weaning shifts in microbiome composition and metabolism revealed by over 25,000 pig gut metagenome assembled genomes	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	10,878	Received 12 December 2020; Accepted 12 April 2021; Published 09 August 2021 Author affiliations: 1iThree Institute, University of Technology Sydney, Sydney, New South Wales, Australia; 2NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales, Australia. Correspondence: Daniela Gaio, d.gaio@outlook.com Keywords: carbohydrate active enzymes; co-assembly; gut microbiome; post-weaning; shotgun metagenomics; time series. Abbreviations: AA, auxiliary activity; ANI, average nucleotide identity; CAZy, carbohydrate active enzyme; CBM, carbohydrate binding module; CE, carbohydrate esterase; fc, fold change; GH, glycoside hydrolase; GT, glycosyl transferase; GTDB, Genome Taxonomy Database; MAG, metagenome- assembled genome; NMDS, non-metric multidimensional scaling; PC, principal component; PCA, principal component analysis; PL, polysaccharide lyase; SLH, S-layer homology domain. Data statement: All supporting data, code and protocols have been provided within the article or through supplementary data files. One supplementary table and fourteen supplementary figures are available with the online version of this article. 000501 © 2021 The Authors Abstract Using a previously described metagenomics dataset of 27 billion reads, we reconstructed over 50 000 metagenome-assembled genomes (MAGs) of organisms resident in the porcine gut, 46.5 % of which were classified as >70 % complete with a <10 % contamination rate, and 24.4 % were nearly complete genomes. Here, we describe the generation and analysis of those MAGs using time-series samples. The gut microbial communities of piglets appear to follow a highly structured developmental pro- gramme in the weeks following weaning, and this development is robust to treatments including an intramuscular antibiotic treatment and two probiotic treatments. The high resolution we obtained allowed us to identify specific taxonomic ‘signatures’ that characterize the gut microbial development immediately after weaning. Additionally, we characterized the carbohydrate repertoire of the organisms resident in the porcine gut. We tracked the abundance shifts of 294 carbohydrate active enzymes, and identified the species and higher-level taxonomic groups carrying each of these enzymes in their MAGs. This knowledge can contribute to the design of probiotics and prebiotic interventions as a means to modify the piglet gut microbiome. DATA SUMMARYh Only a tiny fraction of known bacterial species are repre- sented in genome sequence databases and, in particular, difficult-to-culture and non-pathogenic organisms remain greatly underrepresented [1–3]. On the contrary, taxa of interest for their pathogenicity or potential in the biotech sector are more often sequenced and, therefore, tend to be better represented in public databases. Although an increasing diversity of reference genomes is becoming available, reference-based methods are not ideal for the discovery of new genomes, as they depend, directly or indirectly, on existing databases [4]. Moreover, proof of the nature of functional redundancy in the microbiome [5, 6], the early documented within-species genomic diver- sity [7], coupled with its striking functional implications [6, 8, 9], should make us ever more aware that important compositional and functional features may be masked completely when the view of the microbiome is restricted to a small conserved gene fragment and the assumptions Only a tiny fraction of known bacterial species are repre- sented in genome sequence databases and, in particular, difficult-to-culture and non-pathogenic organisms remain greatly underrepresented [1–3]. On the contrary, taxa of interest for their pathogenicity or potential in the biotech sector are more often sequenced and, therefore, tend to be better represented in public databases. Although an increasing diversity of reference genomes is becoming available, reference-based methods are not ideal for the discovery of new genomes, as they depend, directly or indirectly, on existing databases [4]. Moreover, proof of the nature of functional redundancy in the microbiome [5, 6], the early documented within-species genomic diver- sity [7], coupled with its striking functional implications [6, 8, 9], should make us ever more aware that important compositional and functional features may be masked completely when the view of the microbiome is restricted to a small conserved gene fragment and the assumptions The data were deposited in the National Center for Biotech- nology Information Sequence Read Archive under project accession number PRJNA526405. Workflows and code used for the preliminary data processing are available in the GitHub repository (https://github.com/koadman/metapigs). Code used for the data analysis and visualization are available in the GitHub repository (https://github.com/GaioTransposon/ metapigs_dry). OPEN DATA OPEN ACCESS OPEN DATA OPEN ACCESS OPEN DATA OPEN ACCESS OPEN DATA OPEN ACCESS RESEARCH ARTICLE Gaio et al., Microbial Genomics 2021;7:000501 DOI 10.1099/mgen.0.000501 RESEARCH ARTICLE DOI 10.1099/mgen.0.000501 Post-weaning shifts in microbiome composition and metabolism revealed by over 25 000 pig gut metagenome-assembled Daniela Gaio1,, Matthew Z. DeMaere1, Kay Anantanawat1, Toni A. Chapman2, Steven P. Djordje This is an open-access article distributed under the terms of the Creative Commons Attribution License. Impact Statement Shotgun metagenomic sequencing, however, allows a much more complete view of the microbial community. The tech- nique uses assembly and binning for the reconstruction of genomes from metagenomic reads, but it comes with many technical challenges. Among the main challenges are the detection of less prevalent taxa within the sample [10], and the high sequence similarity among strains of the same species, making assembly a particularly challenging task [11, 12]. However, various techniques have been developed to overcome these obstacles. Previous work in computational metagenomics has established that repeated sampling from an environment or subject can facilitate the reconstruction of genomes from species [13, 14] and strains [15] in the microbial community. Two major steps are typically involved in the data analysis: assembly and binning. The joint assembly of samples from an individual host is called co-assembly, while binning consists in the grouping of co-assemblies into metagenome- assembled genomes (MAGs) in the process of differential coverage binning [16]. The abundance per sample is then inferred from coverage depth and/or k-mer frequencies [17]. The rationale behind this type of binning method is based on the observation that the abundance of genetic material from the same organism changes in one subject or environ- ment over time in a highly correlated manner [13, 14, 16, 18]. There are two reasons that time-series binning facilitates the resolution of genomes: (i) strains are known to persist for long periods of time (i.e. decades) [19], and as such the abundance of the same organism in a subject or environment changing over time in a highly correlated manner improves the infer- ence of MAGs; (ii) at least in humans, over half of all species in the gut are represented by single strains [20]; therefore, co-assembly of time points boosts the per-genome coverage. The aim of this work was to assemble a large amount of metagenomic data into metagenome-assembled genomes (MAGs), and to use the MAGs to describe the developing gut microbial composition of post-weaning piglets, from a genomic and a functional perspective. A large number of shared species were found to undergo the same shifts in distinct hosts during the first 5 weeks after weaning. This type of fine, species-level tuning of the gut in piglets was not measured before and can be used to choose (or avoid) a time window for treatment with specific probiotics. We also confirmed confounding factors (e.g. Metagenomic samplesl Below, we briefly summarize the origin of the samples, but we refer to our previous report [21] for a thorough description of the animal trial and the sample processing workflow. Subjects were post-weaning piglets (n=126) from which faecal samples were collected between 1 and 10 times INTRODUCTION Advances in sequencing technology and computational anal- ysis enable the study of whole microbial communities from a given environment in a high-throughput manner, bypassing the need to cultivate individual organisms. High-throughput microbial community profiling is generally approached in a targeted (e.g. 16S rRNA) or untargeted (shotgun) manner. 1 Gaio et al., Microbial Genomics 2021;7:000501 on its conservation, as in the 16S rRNA amplicon profiling technique. on its conservation, as in the 16S rRNA amplicon profiling technique. Impact Statement breed, small differences in age) to signifi- cantly correlate with microbial composition, and we report the duration of their detection. This can guide researchers to make the proper adjustments to their animal trials, so as to minimize noise in the signal. Ultimately, we obtained a description of the carbohy- drate enzyme repertoire of these post-weaning piglets, by tracking temporal abundance shifts of nearly 300 carbohydrate active enzymes and their mapping, hence, specificity, to taxonomic groups, knowledge which can be applied to manufacture feed with the aim of tweaking the abundance of specific (e.g. health-promoting, low carbon emission) taxa. during a 40 day period. Piglets were aged 22.5±2.5 days at the start of the trial. The piglets were distributed over six treatment cohorts: a placebo group (control n=29); two probiotic groups (D-Scour n=18; ColiGuard n=18); one antibiotic group (neomycin n=24); and two antibiotic- then-probiotic treatment groups (neomycin+D-Scour n=18; neomycin+ColiGuard n=18). Additionally, 42 faecal samples derived from the piglets’ mothers, 18 samples derived from three distinct positive controls and 20 nega- tive controls were included. All piglets were sampled weekly, while a subset of the piglets (8 per cohort; n=48) was sampled twice weekly. In total, mothers were sampled once, while piglets were sampled between 1 and 10 times (median=6.0; mean=6.11). As previously described [21], samples were homogenized immediately after collection and stored at −80 °C until thawed for further processing. Extraction of DNA was performed with the PowerMi- crobiome DNA/RNA EP kit (Qiagen) and libraries were prepared from genomic DNA using the Hackflex method [22]. Libraries were normalized, pooled and sequenced on three Illumina NovaSeq S4 flow cells. The data were depos- ited in the National Center for Biotechnology Information Sequence Read Archive under project accession number PRJNA526405. The present work builds upon a large collection of post- weaning porcine gut samples for which we previously described the microbial community composition using an assembly-free phylogenetic profiling technique [21]. We reconstruct genomes from this large sample collection and analyse how those genomes change in abundance across time and treatment cohorts. To do so, we created co-assemblies for each individual host, pooling all the time point samples available from each subject, thereby increasing the power to reconstruct genomes from low-abundance microbes. In this study, we describe the associations we found between specific genomes and the post-weaning aging process in piglets, as well as the carbohydrate metabolism during the post-weaning period. Pooled assembly and binning Adapter-trimmed and quality-filtered paired-end reads from all samples were grouped by subject and assem- bled using megahit [26] (v1.1.3) (parameters: --min- contig-len=5 --prune-level=3 --max-tip-len=280), yielding a combined total of 8 389 418 contigs. Reads were mapped back to assembly contigs using bwa-mem [27] (v0.7.17- r1188). Format conversion to bam format was performed with SAMtools [28] (v1.9). Metagenome binning of the co-assemblies was performed for each subject (i.e. each pig, positive control and all negative controls were separately co-assembled) with MetaBAT2 [29] (v2.12.1) (parameters: --minContig 2500). MAG quality assignment Quality analysis of MAGs was performed with CheckM [30] (v1.0.13) following the lineage-specific workflow (lineage_set). Microbial diversity assessment and microbial abundance heatmaps were generated using GTDB clustered MAGs. After the exclusion of samples with low read counts (approxi- mately <10 000) (prune_samples function), samples were rarefied (rarefy_even_depth function) (PhyloSeq v1.28.0). To compare microbial diversity scores of samples from different time points, t-tests were applied, and significance values were adjusted with the Bonferroni method. To generate the heat- maps, taxa were filtered based on abundance, prevalence and within-samples variation, as indicated in the figure legends. Data analysis Analyses of MAGs were performed for the following sets of MAGs: (i) nearly complete genomes (≥90 % completeness and ≤5 % contamination) as estimated and taxonomically catego- rized by CheckM analysis (n=12.4×103); (ii) length filtered and ANI clustered MAGs from dRep analysis (n=22.4×103); (iii) all (unfiltered) MAGs taxonomically categorized by GTDB (n=51.2×103). Non-metric multidimensional scaling (NMDS) and network analysis were performed with PhyloSeq [35] using the median sequencing depth to normalize samples. Bray–Curtis value was used as a distance measure. Separately from the ordination analysis performed with PhyloSeq, we ran principal component analysis (PCA) with data normalized by proportions and transformed with the centred log-ratio. In the latter case, the data underwent the following transforma- tions: (i) library size normalization by proportions; (ii) sum of counts from MAGs falling under the same MAG (taxonomic or ANI) group assignment; (iii) mean by sampling time point and cohort; (iv) centred log-ratio transformation. MAG taxonomic assignment Taxonomic clustering of MAGs was performed with Kraken2 [31, 32] (v2.0.8) using, in parallel: a prebuilt 8 Gb database (MiniKraken DB_8 GB) constructed from bacterial, viral and archaeal genomes from RefSeq version Oct. 18 2017, and the Genome Taxonomy Database (GTDB) [33] (release 89.0). All MAGs were assigned a taxon at the species level. Higher taxonomic levels of the GTDB were retrieved from the latest release (release 89.0) of the bacterial and archaeal database publicly available at the GTDB website (https:// gtdb.ecogenomic.org/). Phyla composition profiles were obtained from presence/absence counts of MAGs, ignoring their relative abundances in each sample, using MAGs that were taxonomically assigned with either GTDB or CheckM. We refer to these as GTDB clustered MAGs and CheckM clustered MAGs. Age, breed, co-housing and piglet weight were tested for corre- lation with microbiome composition. Samples were pruned and rarefied as described above and PCA was performed with PhyloSeq (v1.28.0). As breed and age were confounded factors, we additionally performed PCA on subsets of equal age or breed. The resulting eigenvectors were tested to assess the significance of the correlations. For the categorical vari- ables age, breed and co-housing, Dunn tests were performed. For the continuous variable weight, the Spearman’s rank test was performed. Significance values were adjusted by Bonfer- roni correction for multiple testing. Sequence data processing A total of 911 samples were sequenced, generating a mean of 7 581 656 (median=3 936 038) paired-end reads per sample 2 Gaio et al., Microbial Genomics 2021;7:000501 We refer to these as 95 % average nucleotide identity (ANI) MAG clusters and 99 % ANI MAG clusters. for a total of 27.2 billion paired-end reads. BBDuk [23] (v38.22) was used for adapter trimming (parameters: k=23 hdist=1 tpe tbo mink=11), PhiX DNA removal (parameters: k=31 hdist=1) and quality filtering (parameters: ftm=0 qtrim=r trimq=20). Quality assessment was performed with fastqc [24] (v0.11.18) and multiqc [25] (v1.8). Completeness and contamination of MAGs According to quality analysis with CheckM [30], 46.5 % (n=23 798) of the total MAGs (n=51 170) were classified as ≥70 % complete with a ≤10 % contamination rate, while 24.4 % (n=12 486) were classified as nearly complete genomes as by the Bowers et al. [10] definition of MAGs with ≥90 % completeness and ≤5 % contamination (Fig. 1). Three hundred and thirty MAGs were ≥99 % complete and had a ≤0.1 % contamination rate. The MAGs with ≥60 % completeness and ≤10 % contamination rate ranged in size from 442 kb to 6.4 Mb (median=1.9 Mb), and their N50 values ranged from 4 to 728 kb (median=16 kb). Gene function analysis Protein prediction from MAGs was performed with Prodigal [38] (v2.6.3). The predicted proteins were clustered with cd- hit [39] (v4.6) using 90 and 100 % identity. Predicted proteins were mapped against a custom database of the UniRef90 database (release June 2020) using diamond [40] (v0.9.31). Before mapping, the database was filtered to contain only error-corrected sequences matching the uniref90_ec_filtered list from HUMAnN2 [41] (/utility_mapping) (v2.8.1). To run the filtering, SeqTK [42] (v1.3-r106) was used. MAG ANI clusters and taxonomic clusters Additionally, predicted proteins were mapped against the carbohydrate active enzyme (CAZy) database [43] with dbCAN2 [44] (v2.0.11), which utilizes diamond [40] and hmmer [45]. The proportion of species per enzyme ID was derived as follows: MAGs were grouped by subject, enzyme ID and GTDB-assigned species; distinct groups were selected and a count was obtained; the sum of distinct species falling within one enzyme ID was obtained and the proportion was derived. As a reference-free technique of MAG generation was adopted, we grouped the MAGs into clusters of similar genomes using two methods: (i) 95 and 99 % sequence similarity using the genome dereplication tool dRep [34]; and (ii) taxonomic clustering with Kraken2 [32] against the GTDB [33, 81]. Using the first method, nearly half of the MAGs (43.8 %) passed the established length-filtering threshold (500 000 nt) and were assigned a cluster ID based on ANI. A total of 1267 unique primary clusters (95 % ANI) and 4480 unique secondary clusters (99 % ANI) were obtained. Primary or secondary clusters that were found in only one subject were defined as unique, whereas clusters that were found in more than one subject were defined as common. Primary clusters (95 % ANI) were common among subjects at a higher rate than secondary clusters (99 % ANI): 98.6 and 86.2 % among piglets, and 91.4 and 72.1 % among the mothers, respectively, delineating strain specificity within hosts (Fig. S3). Differential abundance analysis Because MAGs were constructed independently for each host, highly similar MAGs are expected to be present in the data, representing the same species or strain recovered from different hosts. To group the MAGs into collections that represent MAGs of the same species or strain we carried out bin dereplication using dRep (v2.3.2) [34]. All MAGs derived from pig samples were dereplicated with dRep [34] (parameters: --checkM_method=lineage_wf --length=500 000). All other parameters were set to default. The clusters of similar bins produced by dRep at 95 % identity (primary clusters) and at 99 % identity (secondary clusters) were used for further analysis as described below. siamcat [36] was employed to determine differentially abun- dant GTDB clustered MAGs between groups. The data were normalized by proportions prior to analysis with siamcat. Unsupervised abundance and prevalence filtering is run prior to the association testing. The abundance was normalized, and all GTDB clustered MAGs underwent analysis with siamcat. Associations of species with groups were found by running the siamcat check.associations function, which finds associations of single species with groups using the nonparametric Wilcoxon test and reporting associations and their strength using significance, prevalence shift and 3 3 Gaio et al., Microbial Genomics 2021;7:000501 positive control samples. Distribution of bins over subjects and cohorts are displayed in Fig. S2. generalized fold change (fc) metrics, the latter calculated as the geometric means of the differences between quantiles [37]. The Benjamini–Hochberg method was used to control the false discovery rate (i.e. the expected proportion of false discoveries amongst the rejected hypotheses). Analysis workflow and scripts In order to manage the processing of the data in the high performance computing environment (sequence data processing, pooled assembly, binning, MAGs quality assess- ment), Nextflow [46] was used. For the installation and management of environments, conda [47] was used (v4.7.12). R [48] and the following R packages were used for the data analysis and visualization: ape [49], circlize [50], cluster [51], cowplot [52], data.table [53], dplyr [54], EnvStats [55], facto- extra [56], forcats [57], ggbiplot [58], ggplot2 [58], ggpubr [59], ggrepel [60], gplots [61], gridExtra [62], magrittr [63], matrixStats [64], microbiome [65], openxlsx [66], pheatmap [67], phyloseq [35], plyr [68], purr [69], RColorBrewer [70], readr [71], readxl [72], reshape [73], robCompositions [74], scales [75], seqinr [76], siamcat [36], splitstackshape [77], stringr [78], tidyr [79] and tidyverse [80]. The second method applies clustering via taxonomic assign- ment using the most comprehensive bacterial and archaeal taxonomic database that is currently available. Based on GTDB clustering of MAGs derived from positive control samples (three positive control types; eight replicates within each), MAG taxonomic assignments matched the expected profile and the profile obtained from analysis of the reads with MetaPhlAn2 [82] reported in our previous study [21] (Fig. S4). A schematic workflow of the data processing and analysis is represented in Fig. S1 (available with the online version of this article). Workflows and scripts used for the data processing are available in the GitHub repository (https://github.com/ koadman/metapigs). Scripts used for the data analysis and visualization are available in the GitHub repository (https:// github.com/GaioTransposon/metapigs_dry). High-level taxonomy of the piglet gut microbiome According to taxonomic assignment of the nearly complete genomes with CheckM [30], the average post-weaning piglet gut microbiome is composed of the following phyla in the following proportions: Firmicutes (63.6 %), Bacteroidetes (13.2 %), Tenericutes (9.2 %), Actinobacteria (6.8 %), Proteo- bacteria (2.2 %), Euryarchaeota (2.1 %), Spirochaetes (1.8 %), Chlamydiae (0.8 %) and Synergistetes (0.5 %). CheckM taxo- nomic clustering resolved 91.28 % of the nearly complete MAGs to the order level, the most common taxonomic orders above 1 % being: Clostridiales (56.4 %), Bacteroidales RESULTS A total of 51 170 MAGs from 911 samples were generated in this study, 92.96 % (n=47 569) of which derived from samples of post-weaning piglets, 5.24 % (n=2680) from samples of the piglets’ mothers, and 1.81 % (n=926) from negative and 4 Gaio et al., Microbial Genomics 2021;7:000501 Fig. 1. Quality of MAGs. Distribution of contig counts (a), contig N50 lengths (b), and completeness and contamination rates over MAGs of partial quality (grey) and medium quality (pink), and nearly complete MAGs (blue) (c). Partial and medium quality MAGs have a level of completeness between 60 and 80 %, and between 80 and 90 %, respectively, and a rate of contamination of <10 % and ≤10 %, respectively. MAGs with a ≥90 % completeness and a ≤5 % contamination are considered nearly complete as by the Genomic Standards Consortium MIMAG standards [84]. Fig. 1. Quality of MAGs. Distribution of contig counts (a), contig N50 lengths (b), and completeness and contamination rates over MAGs of partial quality (grey) and medium quality (pink), and nearly complete MAGs (blue) (c). Partial and medium quality MAGs have a level of completeness between 60 and 80 %, and between 80 and 90 %, respectively, and a rate of contamination of <10 % and ≤10 %, respectively. MAGs with a ≥90 % completeness and a ≤5 % contamination are considered nearly complete as by the Genomic Standards Consortium MIMAG standards [84]. (12.9 %), Erysipelotrichales (8.2 %), Coriobacteriales (5.4 %), Lactobacillales (5.4 %), Selenomodales (3.4 %) and Methano- bacteriales (1.8 %). According to taxonomic assessment of MAGs against the GTDB, the genus Prevotella took up the largest proportion, with 10.1 % of the total genera composi- tion. The most common phyla, genera and species of the piglet microbiome are shown in Fig. S5. hosts, independent of treatment, and marked by changes that were clearly associated with particular taxonomic groups (Figs 2 and S8). (12.9 %), Erysipelotrichales (8.2 %), Coriobacteriales (5.4 %), Lactobacillales (5.4 %), Selenomodales (3.4 %) and Methano- bacteriales (1.8 %). According to taxonomic assessment of MAGs against the GTDB, the genus Prevotella took up the largest proportion, with 10.1 % of the total genera composi- tion. The most common phyla, genera and species of the piglet microbiome are shown in Fig. S5. Differential abundance between time points and statis- tical analysis of the changes over time was obtained with siamcat [36]. RESULTS Time points analysed were at consecutive weeks from the start (t0): t0, t2, t4, t6, t8 and t10. The top 10 significantly shifting species in piglets between t0 and t4, and between t4 and t8, are shown in Fig. 3. Correlations with P <0.05 after false discovery rate correction (Benja- mini–Hochberg method) were considered significant. Significance values, generalized fc and other metrics are provided in Table S1. Time trendh The predominant variation we observed in the piglet microbiomes is associated with the aging of the piglets. Both NMDS analysis and network analysis of samples with PhyloSeq [35] showed samples clustering by collection time point (Fig. S6). Samples were the most scattered in PCA at the start of the trial (t0 variance=0.1669, n=122) when piglets were aged between 3 and 4 weeks old, and samples clustered more closely at later time points (t2 variance=0.0625, n=115; t4 variance=0.0405, n=105), shifting most prominently along the first NMDS axis (t-test; Bonferroni adjusted significance: t0–t2 P <0.0001; t2–t4 P <0.0001) (Fig. S6). The temporal shift was evident in the results from all of the various approaches we applied to filter and cluster MAGs into groups; hence, the final number of MAGs included in the analysis (CheckM 12.4×103; dRep 22.4×103; GTDB 51.2×103) (Fig. S7). A total of 143 species were found to significantly shift in abundance (P <0.001; 84 % positive fc) in the piglet population between t0 and t2, (piglets aged 3–4 weeks and 4–5 weeks, respec- tively) (Table S1), among which we found: Blautia_A wexlerae (fc=+1.4), CAG-83 sp003487665 (fc=−0.9), Lactobacillus amylo- vorus (fc=+1.3), CAG-45 sp002299665 (fc=+1.7), CAG-110 sp002437585 (fc=+1.7). Significant shifts in abundance were found for 90 species (P <0.001; 63 % positive fc) between t2 and t4 (Table S1), among which we found: Methanobrevibacter_A smithii (fc=−1.6), Phil1 sp001940855 (fc=−1.3), Prevotella copri (fc=+1.0), Prevotella sp000434515 (fc=+1.3), Lactobacillus amylovorus (fc=+0.7). Fifty-two species significantly shifted in abundance (P <0.001; 85 % positive fc) between t4 and t6 (Table S1), among which we found: Corynebacterium xerosis (fc=+1.4), Blautia_A obeum (fc=+1.1), Blautia_A sp000285855 (fc=+0.8), Remarkable tightly regulated compositional shift We found the aging-associated compositional shift throughout the length of the study to be consistent among 5 Gaio et al., Microbial Genomics 2021;7:000501 Fig. 2. Microbial species abundance profile of the piglet gut over time. Panels represent time points from t0 (piglets aged 3 to 4 weeks old) to t10 (piglets aged 8 to 9 weeks old). Samples were pruned (exclusion of samples with approximately <10 000 read counts) and normalized by rarefaction. Prior to plotting, taxa were filtered to include the most abundant taxa present in at least 20 % of the piglet samples. Of these, the 35 taxa with the highest variance among samples were plotted. Analysis was performed with PhyloSeq. Fig. 2. Microbial species abundance profile of the piglet gut over time. Panels represent time points from t0 (piglets aged 3 to 4 weeks old) to t10 (piglets aged 8 to 9 weeks old). Samples were pruned (exclusion of samples with approximately <10 000 read counts) and Fig. 2. Microbial species abundance profile of the piglet gut over time. Panels represent time points from t0 (piglets aged 3 to 4 weeks old) to t10 (piglets aged 8 to 9 weeks old). Samples were pruned (exclusion of samples with approximately <10 000 read counts) and normalized by rarefaction. Prior to plotting, taxa were filtered to include the most abundant taxa present in at least 20 % of the piglet samples. Of these, the 35 taxa with the highest variance among samples were plotted. Analysis was performed with PhyloSeq. Fig. 2. Microbial species abundance profile of the piglet gut over time. Panels represent time points from t0 (piglets aged 3 to 4 weeks old) to t10 (piglets aged 8 to 9 weeks old). Samples were pruned (exclusion of samples with approximately <10 000 read counts) and normalized by rarefaction. Prior to plotting, taxa were filtered to include the most abundant taxa present in at least 20 % of the piglet samples. Of these, the 35 taxa with the highest variance among samples were plotted. Analysis was performed with PhyloSeq. Diversity Clostridium sp000435835 (fc=+1.0), Clostridium_P ventriculi (fc=+1.4). The least significant shifts in abundance (n=4) were found between t6 and t8 (P<0.05; 50 % positive fc) (Table S1). These were: UBA7748 sp900314535 (fc=−0.9), Clostridium sp000435835 (fc=+0.8), Prevotella copri (fc=+0.3) and Bifido- bacterium boum (fc=−0.8). The number of species significantly shifting in abundance (P <0.001; 32 % positive fc) was again higher (n=28) for the last time interval (t8–t10) when piglets were aged between 7–8 and 8–9 weeks. During this interval, the most significantly changing species were: Clostridium sp000435835 (fc=+1.4), Corynebacterium xerosis (fc=+1.2), Prevotella copri_A (fc=−0.7), Prevotella copri (fc=−0.5), Prevo- tella sp000434515 (fc=−0.5) (Table S1). Notably, of all Prevotella species (n=16) significantly increased in abundance during the first or the second week, nearly all (n=13) decreased during the last time interval (false discovery rate adjusted P <0.05) (Fig. S9). All significance values and confidence intervals are given in Table S1. During the first week (t0–t2) (piglet age range: 3.5±0.5 to 4.5±0.5 weeks) species richness increased (Shannon ∆ t2- t0=0.53±0.12; Bonferroni adjusted P <0.00001) (Fig. S10, Table S1). Between t2 and t4 (piglets age range: 4.5±0.5 to 5.5±0.5 weeks) a loss of species evenness was recorded (Simpson ∆ t4-t2=−0.04±0.02; Bonferroni adjusted P <0.00001). The following week (t4–t6) (piglets age range: 5.5±0.5 to 6.5±0.5 weeks) an increment of species richness (Chao1 ∆ t6- t4=37.20±15.13; Bonferroni adjusted P <0.00001) was observed (Fig. S10; Table S1). Effects of age, breed, co-housing and weight Age and breed were found to correlate with microbial composi- tion (Fig. S11, Table S1). Piglets of the same breed (D×L) and two age groups (3 days difference between the two groups) separated by age at t0 in principal component 1 (PC1) (16.9 % 6 Gaio et al., Microbial Genomics 2021;7:000501 Fig. 3. Significantly changing GTDB species with time. Significant shifts (alpha=0.05) were determined from comparison of GTDB clustered MAGs abundance in samples from distinct time points with siamcat. The Benjamini–Hochberg method was used to control the false discovery rate. (a) Percentages of negative fc and positive fc shifts are reported on top of the bar plots for each time interval, where two consecutive time points were compared. (b) Shown are the top 10 significantly changing GTDB taxonomically clustered MAGs at the species level, between time points 0 and 4 (2 week interval); and (c) between time points 4 and 8 (2 week interval). Points show normalized and log-transformed abundance within each subject and GTDB-predicted species. Fig. 3. Significantly changing GTDB species with time. Significant shifts (alpha=0.05) were determined from comparison of GTDB clustered MAGs abundance in samples from distinct time points with siamcat. The Benjamini–Hochberg method was used to control the false discovery rate. (a) Percentages of negative fc and positive fc shifts are reported on top of the bar plots for each time interval, where two consecutive time points were compared. (b) Shown are the top 10 significantly changing GTDB taxonomically clustered MAGs at the species level, between time points 0 and 4 (2 week interval); and (c) between time points 4 and 8 (2 week interval). Points show normalized and log-transformed abundance within each subject and GTDB-predicted species. variation explained; Dunn test; Bonferroni adjusted P=0.001) and at t2 in PC1 (14.5 % variation explained; Dunn test; Bonfer- roni adjusted P=0.002) and in PC2 (10.0 % variation explained; Dunn test; Bonferroni adjusted P=0.026) (Fig. S11, Table S1). These age groups were compared with siamcat [36] to discover taxa that exhibit an age association in their abundances. A list of taxa that were found to be mildly associated with age at each time point and the significance of these associations are reported in Table S1. P=0.024) (Fig. S11, Table S1). Piglets of the same age and two breed groups were compared with siamcat to find taxa for which abundances were associated with breed. Effects of age, breed, co-housing and weight Neither at 95 % confidence (alpha 0.05) nor 80 % confidence (alpha 0.20) was the association of microbial composition with breed found to be attributable to specific taxa (Table S1). Weak correlations were found between taxon abundances and weight (variance explained <4.3 %; Spearman’s rank rho range −0.349–0.246; P values range 0.003–0.043) (Table S1). Breed was also found to significantly associate with micro- biome composition when piglets of the same age and two breeds (D×L and D×LW) were compared. Significance was reached in PC2 (variation explained: 10.1 %) at t0 (Dunn test; Bonferroni adjusted P=0.001) and in PC1 (variation explained: 12.9 %) at t2 (Dunn test; Bonferroni adjusted Predicted proteome of the piglet Gene prediction from all MAGs (n=51 170) yielded 70 696 284 predicted ORFs. The predicted protein sequences fell into 24.4 million clusters at 100 % amino acid identity and into 6.9 million clusters at 90 % amino acid identity. Homology 7 Gaio et al., Microbial Genomics 2021;7:000501 heterogeneous among the enzyme classes in terms of distri- bution across different species (median=27.0; mean=37.9); therefore, they appeared as scattered across the plot. Also, for this class of enzymes, the majority (n=30) were found among species of the genus Prevotella, and partially shared with Gemmiger (n=8). The genus CAG-269 carries three CBM enzymes neither Prevotella nor Gemmiger carry in their MAGs. searching of all the predicted proteins against the CAZy database with hmmer identified 2 049 008 predicted proteins that are potentially involved in carbohydrate metabolism (E value <1×10−15; coverage median=94.3, mean=87.0). Of these enzymes, 1 023 807 were glycoside hydrolases (GHs), 716 644 glycosyl transferases (GTs), 26 544 carbohydrate binding modules (CBMs), 253 525 carbohydrate esterases (CEs), 16 636 polysaccharide lyases (PLs), 8626 enzymes with auxiliary activity (AA), 3064 S-layer homology domain (SLH) enzymes and 162 were cohesins. We report the sequence identity against the CAZy database for each class of enzymes and the distribu- tion of carbohydrate enzymes across phyla in Fig. S12. Five of the AA enzymes (n=7) were prevalent in the pig population (>90 subjects). AA10 was majorly found in Enterococcus_B hirae (52.9 %), AA2, AA3 and AA6 were found mostly represented by 'Terrisporobacter othiniensis' (28.6%), Methanobrevibacter_A gottschalkii (20.1 %) and Escherichia flexneri (17.5 %), respectively. CAZy species-specificity and distribution across the pig population Cohesin (n=1) and SLH (n=1) were found in 92 and 167 subjects, respectively, and across multiple species. Cohesin was mostly found in Ruminococcus_C sp000433635 (15.4 %), while Ruminiclostridium_C sp000435295 carried the most SLH (13.0 %). Two hundred and ninety-four unique CAZy enzymes were found in our dataset. As each enzyme prediction was associ- ated with a MAG and each MAG was separately classified against the GTDB, the two sources of information were joined as described in Methods. We display the prevalence of each enzyme in the pig population (y-axis), while on the x-axis, we report the highest proportion a distinct species was found for each enzyme ID (Fig. S13). Below, we describe each enzyme class in terms of prevalence in the pig population and its distribution among species. Seven enzymes, present in between 20 and all subjects (n=167, comprising piglets and mothers), were found to primarily derive from a single species (>70 %). These were: CBM44 (Clostridium_P perfringens), GT44 (Chlamydia suis), CBM75 (Ruminococcus_F champanellensis), CBM71 (An172 sp002160515), GH44 (Ruminococcus_C sp000433635), GH54 (RC9 sp000432655), CBM83 (Agathobacter sp900317585). Enzymes of the CE class (n=16) frequently occurred among subjects, with >100 subjects carrying this class of enzymes (median=166; mean=148). These enzymes tend to be species generic (median=5.5; mean=14.2), as they fall on the left side of the plot, showing a broad distribution across multiple species. Out of all genera, Prevotella carried the most of these enzymes (11.5 %). In order of number of genes per genome we found: GT (median=6.3; mean=30.0), CE (median=5.5; mean=25.3), GH (median=3.7; mean=16.2), SLH (median=2.0; mean=12.7), AA (median=2.0; mean=11.2), CBM (median=2.0; mean=6.3), PL (median=1.5; mean=5.7), cohesin (median=1.0; mean=3.5). Gene counts of enzymes per GTDB species are reported in Table S1. Similarly, of the PL enzyme class (n=26), nearly half formed a cluster in the centre left side of the plot, indicating a high prevalence among subjects (median=93; mean=81) and a broad distribution across species (median=20.8; mean=32.2). PL enzymes were carried mostly by the genus Prevotella (40.6%). Piglet carbohydrate proteome across time Of the 294 CAZy enzymes in our dataset, 234 showed a significant change (Bonferroni adjusted P <0.05) (Table S1), when comparing the abundance between any time point by pairwise Wilcoxon test. The abundance shifts of these enzymes at different time points in the piglet population and their abundance in the mothers’ population are reported in Fig. 4 and Table S1. Enzymes of the GT class (n=63) are prevalent among the pig population (median=121; mean=107) and have a broad distribution across species (median=16.0; mean=25.6). The majority of these enzymes (n=47) were found among species of the genus Prevotella, 27 of which were also found in Gemmiger. Species of these genera had the highest variety of enzymes, as well as the highest number of genes (>200) per genome (Fig. S14). A number of enzymes increased in abundance over time in the piglet population to reach similar abundance levels to the mothers’. Among these were: CE13, GH42, GH32, GT53, GT87, GH1, GT85, CBM25, CBM4, CE5, GT101, CBM26, GT103 and GT85. Other enzymes decreased in abundance over time in the piglet population and their abundance in the mothers’ population was as low or lower than the abundance recorded at the last time point for the piglets. Among these were: GH123, GH29, GH110, GH33, GH30, GH109, GH2, GH35, CBM35 and CBM9. A number of enzymes followed an upwards or downwards trend with time in the piglet popula- tion while their abundance in the mothers population was lower or higher, respectively, than the abundance recorded GH enzymes (n=126) were the most represented enzymes in our dataset and over 70 % of these enzymes fell on the top left of the plot, showing a high frequency across subjects (median=154; mean=131) and a high distribution across species (median=11.2; median=20.4). One hundred and thir- teen of the 126 GH enzymes are found in the genus Prevotella, 96 of which were shared with the genus Blautia_A. CBM enzymes (n=54) were moderately prevalent enzymes in the pig population (median=41; mean=65), and the most 8 Gaio et al., Microbial Genomics 2021;7:000501 Fig. 4. Time trend of several enzymes and their species representation. (a) log-transformed normalized abundance of enzymes over time is represented. Shifts in log-transformed abundance over time is visualized from time point 0 (t0; piglets aged 3–4 weeks) to time point 8 (t8; piglets aged 7–8 weeks) for several enzymes. Piglet carbohydrate proteome across time tM represents the abundance of each enzyme in the mothers (single time point); the sample size for piglets and mothers carrying each enzyme is reported next to the boxplots within each panel. (b) Panels report the top three species carrying each enzyme in their MAGs (based on GTDB taxonomic clustering of MAGs). A significant change in abundance was recorded between different time points (Kruskal pairwise comparison with Bonferroni correction) for the enzymes shown. The full list of time trends and percentages of each species per enzyme are reported in Table S1. Fig. 4. Time trend of several enzymes and their species representation. (a) log-transformed normalized abundance of enzymes over time is represented. Shifts in log-transformed abundance over time is visualized from time point 0 (t0; piglets aged 3–4 weeks) to time point 8 (t8; piglets aged 7–8 weeks) for several enzymes. tM represents the abundance of each enzyme in the mothers (single time point); the sample size for piglets and mothers carrying each enzyme is reported next to the boxplots within each panel. (b) Panels report the top three species carrying each enzyme in their MAGs (based on GTDB taxonomic clustering of MAGs). A significant change in abundance was recorded between different time points (Kruskal pairwise comparison with Bonferroni correction) for the enzymes shown. The full list of time trends and percentages of each species per enzyme are reported in Table S1. Fig. 4. Time trend of several enzymes and their species representation. (a) log-transformed normalized abundance of enzymes over time is represented. Shifts in log-transformed abundance over time is visualized from time point 0 (t0; piglets aged 3–4 weeks) to time point 8 (t8; piglets aged 7–8 weeks) for several enzymes. tM represents the abundance of each enzyme in the mothers (single time point); the sample size for piglets and mothers carrying each enzyme is reported next to the boxplots within each panel. (b) Panels report the top three species carrying each enzyme in their MAGs (based on GTDB taxonomic clustering of MAGs). A significant change in abundance was recorded between different time points (Kruskal pairwise comparison with Bonferroni correction) for the enzymes shown. The full list of time trends and percentages of each species per enzyme are reported in Table S1. compositional and functional changes over a 5 week period in 126 post-weaning piglets. at the last time point in the piglet population. Piglet carbohydrate proteome across time Among these were: GT66, CBM51, GH25, GH37, CBM13, CBM67, GH101, CBM44, GT46, GT22 and CBM6. As previously observed [21], we confirm the large time factor driving the compositional shift of post-weaning piglet gut microbiota samples. The temporal shift appears regardless of the approach-dependent grouping of MAGs (CheckM taxo- nomic clustering, 99 % ANI clustering or GTDB taxonomic clustering), or of the filtering criteria (completeness and contamination, length filtering, no filtering, respectively) and, hence, the resulting number of MAGs taking part in the analysis (12.4×103, 22.4×103, 51.2×103, respectively). We also applied a number of normalization methods in parallel, among which we chose median sequencing depth, rarefaction and proportions. The temporal shift was evident when applying any of these normalization methods, although it must be noted that the use of proportions to normalize library size can lead to a higher discovery rate of false correlations (i.e. spurious correlations of ratios) [85–89]. Bias can also arise from the nature of organ- isms in the sample as a consequence of distinct genomic G+C contents [90], causing preferential affinity of the polymerase, thereby influencing the representation of species in the sample. DISCUSSION Similarly to the rumen metagenome [102], half of the enzymes were of the GH class and over a third were of the GT class, the first known to break down sucrose, lactose and starch [103], the latter known to assemble complex carbohydrates from activated sugar donors [104]. The proportions of the most abundant enzymatic classes in this study (50 % GH; 35 % GT; 12 % CE; 1 % PL) roughly matched the proportions suggested by El Kaoutari et al. [103] (57 % GH; 35 % GT; 6 % CE; 2 % PL), who generated a profile of the human carbohydrate repertoire by mapping 177 reference human gut microbial genomes against the CAZy database [103]. We reported the repre- sentation of these CAZymes within each specific taxonomic group at lower (e.g. species) and higher levels (e.g. phylum) based on GTDB taxonomic clustering, and we reported on the carbohydrate enzymes abundance shifts over time in the post-weaning piglet. A number of enzymes were found to change gradually in abundance over time, to reach levels that are similar to those found in the piglets’ mothers. As it could be expected due to the large difference in age between the piglets at the last sampling point and the mothers, the time trend for a minority of the total enzymes changed gradually in the piglets, but did not reach the lower or higher level measured in the mothers. A number of enzymes known to break down animal glycans that are present in milk (GH2, GH20, GH92, GH29, GH95, GH38, GH88) [103] decreased with time in the piglet popula- tion, while, among others, enzymes reported to break down peptidoglycans (GH25, GH73), starch and glycogen (GH13), and sucrose and fructose (GH32) [103] followed an upward trend. After a brief transition period (fourth week; piglets aged between 6 and 8 weeks) during which only a few species are lost and acquired, a microbial community consolidation phase starts, where species start to die off, and a smaller number of dominant taxa remain, reflective of an adult gut [99]. This statement is supported by Thompson et al. [96], who determined that at 6 weeks of age CD8+ T cells infiltrate the intestinal tissue and the mucosal lining resembles that of an adult pig [96]. DISCUSSION Previous efforts have applied metagenomics to survey the diversity of pig gut microbiota [83], but have not focused on the post-weaning microbial community nor have they used an approach that generates MAGs. Bowers et al. (in 2017) described genomes with a completeness of at least 90 % and a contamination rate lower than 5 % as nearly complete genomes [84]. In this study, we generated over 50 000 MAGs from a dataset of 27 billion read pairs, of which 12 486 MAGs are predicted to represent nearly complete genomes of bacte- rial or archaeal organisms. While we previously reported the results obtained from an assembly-free analysis of community structure [21], here we report the composition and predicted carbohydrate proteome of the microbial community using MAGs constructed from the dataset. Using the MAGs and their abundance across the samples, we were able to track 9 Gaio et al., Microbial Genomics 2021;7:000501 Factors such as host weight, breed and small differences in age correlate with microbial composition, as we found by our previous analysis with unassembled metagenomic reads [21] and in this study with the use of MAGs. To assess the correlation of microbial composition and small age differ- ences among the hosts, the dataset was reduced to include only piglets of the same breed, thereby avoiding breed as a confounding factor. The temporal signal in our dataset was in fact sufficiently strong in the first 2 weeks of the trial that piglets differing by just 3 days of age separated cleanly in the first principal component, and a number of GTDB-assigned species were found to be associated with these small age differences. second week (piglets aged between 3 and 6), 12 of which were found to significantly drop in abundance during the last time interval. Previously associated with human milk consumption in infants [91], Bifidobacterium species dropped significantly in abundance during the last 2 weeks, between the third and the fourth week post-weaning, suggesting this shift to be due to the diet change from breast-milk to solid food. Studies have reported the health promoting effects of Lacto- bacillus amylovorus [100, 101], its survival through gastric pH, its ability to adhere to cells and the demonstrated effi- cacy against the growth of certain pathogens [100, 101]. DISCUSSION In this study, Lactobacillus amylovorus follows a specific trend, by gradually increasing in its abundance from piglets aged 3–4 to 5–6 weeks, to then gradually decrease in the hosts, suggesting that the administration of probiotics containing this species, to 6-week-old piglets, may not lead to a successful colonization. Highly structured compositional changes over time were detected. Possible factors that underlie these compositional changes include the exposure to a new environment, the transition from breast-milk to solid food [91–93], which took place upon the start of the trial, and a higher tolerance for new species immediately after weaning [94–96]. Supporting the latter, it is known that during weaning, the immune system undergoes training to recognize pathogens by initially toler- ating a large number of species, so as to develop the necessary antigens upon encounter [97, 98]. Thompson et al. [96] report that the microbiota of piglets of 3 weeks of age is particu- larly dynamic and environmental factors start to be seen at this stage [96]. In fact, over 80 % of the GTDB clustered MAGs that showed significant abundance shifts during the first week of post-weaning were showing positive fc. This is also reflected by the major increase in microbial richness we measured by community composition analysis of the GTDB clustered MAGs, as well as by phylogenetic diversity analysis of the metagenomic reads in our previous study [21]. Nearly 40 % of the species that increased during the first week of the trial (t0–t2) continued to increase during the following week (t2–t4), reflecting the fact that these species kept taking up a larger portion of the microbial community. This is confirmed by the increase of evenness (as measured by balance-weighted phylogenetic diversity) we recorded for this time interval in our previous study [21]. The third week of the trial was the last week in which the majority of the taxonomic shifts were posi- tive fc (>80 %; piglets aged between 6 and 7 weeks), after which species richness stabilized. In summary, in this study, the first 3 weeks (piglets aged between 3 and 7 weeks) represented the establishment of a new microbial community where a large number of species bloomed. Mapping of predicted ORFs generated hits with a high sequence similarity against the CAZy database, spread across eight enzyme classes and containing 294 unique enzymes. References 1. Brown CT, Hug LA, Thomas BC, Sharon I, Castelle CJ et al. Unusual biology across a group comprising more than 15% of domain Bacteria. Nature 2015;523:208–211. 2. Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ et al. A new view of the tree of life. Nat Microbiol 2016;1:16048. 3. Pasolli E, Asnicar F, Manara S, Zolfo M, Karcher N et al. Exten- sive unexplored human microbiome diversity revealed by over 150,000 genomes from metagenomes spanning age, geography, and lifestyle. Cell 2019;176:649–662. 4. Mande SS, Mohammed MH, Ghosh TS. Classification of metagen- omic sequences: methods and challenges. Brief Bioinform 2012;13:669–681. We found enzymes to be either (i) common to many species and to higher level taxonomic assignments, indicating functional redundancy; or (ii) shared among species within the same genus; or (iii) specific to single species. Common enzymes restricted to closely related species were: AA10, found predominantly in the genus Enterococcus, CBM40 in Clostridium, PL31 in Ruminococcus, CE5, GT53, GT85 and GT87 in Corynebacterium, GT21 in Desulfovibrio, GT66 and GT81 in Methanobrevibacter, GT6 in Gemmiger, GH68 in Lactobacillus, PL21, GH54 and GH55 in RC9 genera. Members of the same genus also significantly differed in their enzymatic repertoire, both in the total number of genes encoding enzymes per genome, as well as in the number of different enzymes a genome bears, showing that closely related organisms have evolved to display large differences in behaviour [9, 105]. This was particularly evident among species of the genus Prevotella, potentially suggesting a high degree of specialization in preferred food sources among closely related species. The synthesis of knowledge of enzyme function and substrate-based activity coupled with knowledge of the species carrying these enzymes in their genome can be of high value to the design of probi- otic and prebiotic formulations in the livestock setting (e.g. methane emission control; improvement of substrate energy efficiency), as well as in the human setting (e.g. diet). 5. Dethlefsen L, Relman DA. Incomplete recovery and indi- vidualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc Natl Acad Sci USA 2011;108:4554–4561. 6. Barnes EM, Carter EL, Lewis JD. Predicting microbiome function across space is confounded by strain-level differ- ences and functional redundancy across taxa. Front Microbiol 2020;11:101. 7. 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This project was funded via the Australian Centre for Genomic Epidemiological Microbiology (Ausgem), a collaborative partner- ship between the NSW Department of Primary Industries and the Univer- sity of Technology Sydney. D. G. is a recipient of University of Technology Sydney (UTS) International Research and UTS President’s Scholarships. 15. Cleary B, Brito IL, Huang K, Gevers D, Shea T et al. Detection of low-abundance bacterial strains in metagenomic datasets by eigengenome partitioning. Nat Biotechnol 2015;33:1053–1060. 16. Albertsen M, Hugenholtz P, Skarshewski A, Nielsen KL, Tyson GW et al. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat Biotechnol 2013;31:533–538. Acknowledgements Thank you to Simone Arnold for the insightful discussions on the biological content of this paper, International Animal Health Products for providing access to the probiotic supplements and the Australian Centre for Genomic Epidemiological Microbiology (Ausgem) for finan- cially supporting this study. Thank you John Webster and Dave Wheeler for providing the Department of Primary Industries internal review of this paper. 17. Garza DR, Dutilh BE. From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems. Cell Mol Life Sci 2015;72:4287–4308. 18. Liu M, Darling A. Metagenomic chromosome conformation capture (3C): techniques, applications, and challenges. F1000Res 2015;4:1377. DISCUSSION In the human gut, Prevotella are known to be acquired post-weaning as a consequence of a substrate shift from breast-milk to solid food in infants [91]. Notably, in this study, 16 Prevotella species established during the first and the The data we report in this study address a knowledge gap in the association of carbohydrate active enzymes to microbial species. Similarly to the rumen metagenomic study [102], we found a larger proportion of GT enzymes in the phylum Euryarchaeota, and a larger proportion of CBM and PL enzymes in the phylum Fibrobacterota. However, it must be 10 Gaio et al., Microbial Genomics 2021;7:000501 noted that the method of annotating CAZymes by mapping genomes against a reference database, as for many other reference-based analyses in microbial metagenomics, may suffer bias due to the limited representation of diversity in the database. In the case of CAZymes, the approach may accurately represent the CAZyme profile of better- represented genomes or better-studied enzyme families, while underestimating the CAZyme profile of genomes for which reference genomes are lacking. 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https://openalex.org/W2970097171	https://repositorio.unesp.br/bitstream/11449/183824/1/S1984-29612019005015105.pdf	English	null	Hepatozoon sp. gamonts as an accidental finding in synovial liquid from an injured maned wolf (Chrysocyon brachyurus) in southeastern Brazil	Revista Brasileira de Parasitologia Veterinária/Brazilian Journal of Veterinary Parasitology	2,019	cc-by	4,880	Gamontes de Hepatozoon sp. como achado acidental em líquido sinovial de lobo guará (Chrysocyon brachyurus no sudeste do Brasil Gamontes de Hepatozoon sp. como achado acidental em líquido sinovial de lobo guará (Chrysocyon brachyurus no sudeste do Brasil Lívia Perles1; Guilherme Henrique Fernandes Barranco1; Isabela Maciel Soriano1; Nathan da Rocha Neves Cruz2; Patrícia Jábali Bueno2; Áureo Evangelista Santana2; Rosangela Zacarias Machado1; Karin Werther1; Marcos Rogério André1*  1 Departamento de Patologia Veterinária, Universidade Estadual Paulista Júlio de Mesquita Filho – UNESP, Jaboticabal, SP, Brasil 2 Departamento de Clínica e Cirurgia Veterinária, Universidade Estadual Paulista Júlio de Mesquita Filho – UNESP, Jaboticabal, SP, Brasil 1 Departamento de Patologia Veterinária, Universidade Estadual Paulista Júlio de Mesquita Filho – UNESP, Jabotica 2 Departamento de Clínica e Cirurgia Veterinária, Universidade Estadual Paulista Júlio de Mesquita Filho – UNESP, Jaboticabal, SP, Brasil Received April 15, 2019 Accepted July 04, 2019 Received April 15, 2019 Accepted July 04, 2019 Abstract A free-living, adult male maned wolf (Chrysocyon brachyurus) was referred to the Governador “Laudo Natel” – FCAV/Unesp veterinary hospital after being found with skin lesions and a fracture on the right pelvic limb, which had to be amputated due to compromised integrity. Around 20 days later, bilateral accentuated swollen on humerus- radius-ulna articulation was observed. The synovial liquid was drained and sent to the laboratory for synovial cytology with Rosenfeld staining that revealed predominantly degenerated neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. gametocytes. Blood and synovial liquid samples were submitted to molecular analysis, aiming to amplify the Hepatozoon spp. 18S rRNA gene fragment. Despite the positioning of the found Hepatozoon sequence together with Hepatozoon canis previously detected in domestic carnivores, the BLAST analysis showed only 98% identity with H. canis. To the best of the authors’ knowledge, this is the first time a Hepatozoon was detected in the synovial liquid by clinical pathology and molecular analyses. Keywords: Hepatozoonosis, cytopathology, gametocytes, wild canids. *Corresponding author: Marcos Rogério André. Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias Júlio de Mesquita Filho – UNESP, Campus de Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane, s/n, Zona Rural, CEP 14884-900, Jaboticabal, SP, Brasil. e-mail: marcosandre.fcav@gmail.com ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, in any medium, provided the original work is properly cited. Clinical history Although several drugs have been proposed for treating the infection, there is no parasitological cure and, currently, no established treatment protocol for treating H. canis infection (SAKUMA et al., 2009). There are studies in the literature on treatments with either imidocarb dipropionate only or imidocarb dipropionate associated with doxycycline, toltrazuril, toltrazuril/emodepside, and clindamycin, but none of them proved to be efficient (SAKUMA et al., 2009; SASANELLI et al., 2010; PASA et al., 2011; TOMMASI et al., 2014). It was decided for the amputation of the right pelvic limb due to compromised limb integrity. One month after the procedure, it was noticed a swollen on both thoracic limbs near the humerus‑radius‑ulna articulation. The animal was sedated for physical examination, followed by a trichotomy of both thoracic limbs before synovial liquid sampling performed with a 0.8x25mm needle and 20 mL syringe to collect 50 mL and 22 mL aliquots from the right and left limbs, respectively. Liquid smears were fixed with methanol, stained with Rosenfeld and stored for molecular analyses. Blood smears were also prepared and stained with Fast Panoptic method (Laborclin). The synovial liquid smear was entirely examined, including the feathered and lateral edges, readable and thick areas, under 40x objective. For blood smear analysis, the smear was first examined at the feathered edge for differential leukocyte count under 40x objective, and then scanned for hemoparasites at the feathered edge and monolayer under 100x objective. Additionally, at the day of synovial liquid sampling, the blood sample was also submitted to buffy coat smear analysis. After draining the synovial liquid, the animal was kept with a compressive bandage on both thoracic limbs medicated with meloxicam (0.2mg/kg, IM, SID, for 3 days), nutritional conditions were improved and the cage was modified to shorten the decubitus period after the amputation procedure. Hematological analyses were performed during the entire hospital care. Hepatozoon sp. was detected in the blood of one maned wolf by 18Sr RNA-based PCR for the first time in a Zoo in Sorocaba, southeastern Brazil (ANDRÉ et al., 2010). Interestingly, the found sequence clustered with other sequences detected in wild canids and felids, apart from the H. canis and H. americanum detected in domestic/wild canids (ANDRÉ et al., 2010). Infection by Hepatozoon sp. was also molecularly detected in a free-living, adult female maned wolf in Minas Gerais, southeastern Brazil. In this case, the found 18SrRNA sequence showed 98% identity to H. Clinical history The maned wolf (Chrysocyon brachyurus) is known as the largest wild canid species from South America, occurring in Argentina, Bolivia, Brazil, Paraguay, Peru, and Uruguay (DIETZ, 1984, 1985; RUMIZ & SAINZ, 2002). Currently, the red list of the International Union for Conservation of Nature (IUCN) lists this species as “Near Threatened” (PAULA & DEMATTEO, 2015). Diseases are a potential threat for wildlife conservation, but studies on the occurrence of pathogens in maned wolves are scarce (ANDRÉ et al., 2010; CURI et al., 2012; SILVEIRA et al., 2016). Regarding the occurrence of tick-borne agents, fewer studies have been performed. An intact adult male maned wolf (Chrysocyon brachyurus), weighting 17kg was referred to the Wildlife Medicine Service at the Veterinary Hospital of the Universidade Estadual “Julio de Mesquita Filho” (UNESP) (Jaboticabal, São Paulo, Brazil) after being found injured near the municipality of Barretos (Sao Paulo, southeastern Brazil). The patient was sedated with Ketamine (8 mg/kg, IM) and Midazolam (0.2 mg/kg, IM) for the clinical procedures. The clinical examination indicated pale mucous (oral and eyes), 163 beats/min heart rate, 36 breaths/min respiratory rate, 37.2°C rectal temperature, 3s capillary refill time (CRT), and dehydration estimated in 8% while following complementary laboratory analysis showed 87 mg/dL glucose. Additionally, the right pelvic limb showed a severe bone and muscle damage compromising limb functionality and three wounds were found on the left pelvic limb. Even though a light tick infestation was observed, they were not sampled. p Hepatozoon spp. are apicomplexan parasites that infect a wide variety of hosts, being described in domestic and wild mammals, birds, reptiles, and amphibians (SMITH, 1996; RAMOS et al., 2015). Domestic and wild canids can be infected with at least two Hepatozoon species, Hepatozoon canis and Hepatozoon americanum, which present distinct clinical signs, pathology, biology, and genetic characteristics (O’DWYER et al., 2011). Although Hepatozoon sp. infections in free-living animals are usually subclinical (KOCAN et al., 2000; METZGER et al., 2008), some studies have shown the occurrence of the clinical disease associated with H. americanum in coyotes (Canis latrans) in the United States (KOCAN et al., 2000) and mortality of hyenas (Crocuta crocuta) in Tanzania associated with a Hepatozoon sp. genetically distinct from the H. canis (EAST et al., 2008). These agents may play a role as potential opportunistic pathogens in immunocompromised animals or with concomitant diseases (BANETH et al., 2001; KUBO et al., 2006). Clinical history americanum (SILVEIRA et al., 2016). The present work aims to describe the clinical and laboratory findings of a maned wolf naturally infected by Hepatozoon sp. that was treated at a veterinary hospital in southeastern Brazil. Resumo Um lobo guará (Chrysocyon brachyurus) adulto, macho, de vida livre foi encaminhado para atendimento no hospital veterinário Governador “Laudo Natel” – FCAV/Unesp após ser encontrado com lesões de pele e fratura em membro pélvico direito, sendo amputado devido a comprometimento da integridade do membro. Aproximadamente 20 dias após a chegada ao hospital, foi notado acentuado aumento de volume bilateral em região de articulação úmero‑rádio‑ulnar. O líquido sinovial foi drenado e enviado para análise citológica com coloração de Rosenfeld, revelando a presença de neutrófilos degenerados com cromatina cariolítica associados a inclusões intracelulares sugestivas de gametócitos de Hepatozoon sp. Amostras de sangue e líquido sinovial foram submetidas a análises moleculares visando amplificar um fragmento do gene 18S rRNA de Hepatozoon spp. Apesar da sequência de Hepatozoon detectada se posicionar filogeneticamente no mesmo clado que H. canis previamente detectado em carnívoros domésticos, o resultado da análise do BLAST mostrou somente 98% de identidade com H. canis. De acordo com o conhecimento dos autores, esta é a primeira vez que Hepatozoon foi detectado no líquido sinovial por meio de patologia clínica e análises moleculares. Palavras-chave: Hepatozoonose, citopatologia, gametócitos, canídeos selvagens. Perles, L. et al. 2/7 Braz. J. Vet. Parasitol. Braz. J. Vet. Parasitol. DNA extraction and molecular analyses Both blood and synovial liquid were positive for PCR targeting Hepatozoon sp.-18S rRNA. The 18S rRNA sequence detected in synovial liquid was deposited in the GenBank (MK424119) while BLAST results showed 98% identity with H. canis sequences detected in wild and domestic canids in Brazil and other countries. The phylogenetic inference, estimated by Maximum Likelihood, clustered sequences in six branches: i.) the first group consisted of H. canis sequences detected in domestic carnivores and the maned wolf of this study; ii.) the second, H. canis sequences detected in domestic and wild canids; iii.) the third, H. procyonis, detected in coatis (Nasua nasua); iv.) H. felis and Hepatozoon spp. detected in domestic and wild felids; v.) H. americanum and Hepatozoon spp. detected in domestic and wild canids; vi.) Hepatozoon sp. detected in rodents and reptiles (Figure 2). The clades showed considerable statistical support. Haemogregarina spp. sequences were used as outgroups.h The maned wolf was negative for piroplasmids, hemoplasmas, and Anaplasmataceae agents according the to results of the PCR assays. Results Infection caused by H. canis may be often a subclinical disease, usually associated with low parasitemia, but in some cases, H. canis infection can lead to severe disease that includes muscle lesions, anorexia, weight loss, pale mucous membranes and other clinical sings (BANETH & WEIGLER, 1997; PALUDO et al., 2005). Animals with severe disease symptoms have been shown to have extreme neutrophilia and high levels of parasitemia (BANETH; SHKAP, 2003). H. canis were detected in three Brazilian dogs with persistent parasitemia, causing muscle degeneration and atrophy, and degenerating/regenerating myofibers, similar to the muscle degeneration caused by H. americanum (PALUDO et al., 2005). Although no gametocyte was observed in the peripheral blood DNA extraction and molecular analyses DNA was extracted from 200 µL whole blood with EDTA and from a 200 µL synovial liquid using the DNeasy Blood & Tissue Kit, according to manufacturer instructions. The 18SrRNA of Hepatozoon sp. was amplified based on the PCR protocol described by Ujvari et al. (2004) (targeting a 600 bp fragment). Hepatozoon sp. DNA obtained from a naturally infected Leptodactylus latrans was used as positive control (accession number MK503643). The PCR amplicon obtained from synovial liquid was purified using the Silica Bead DNA gel extraction kit Hepatozoon sp. in Chrysocyon brachyurus 3/7 Ahead of Print, 2019 (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) and ABI PRISM 310DNA Analyzer (Applied Biosystems, Foster City, CA, EUA) (SANGER et al., 1977). Electropherogram quality and a consensus sequence were obtained through analysis of the sense and antisense sequence using the Phred Phrap software (EWING & GREEN, 1998). The identity values were assessed by BLASTn tool (using default parameters), available in the NCBI GenBank database (ALTSCHUL et al., 1990). Sequences deposited in the GenBank used for phylogenetic inferences were selected from BLAST results and from other studies performed in Brazil and in other countries. Maximum likelihood tree inference was performed with IQ-TREE software using TPM2+F+G evolutionary model (TRIFINOPOULOS et al., 2016). The phylogenetic tree edition and rooting were performed using the Treegraph 2.0 beta software (STOVER & MULLER, 2010). The blood samples were also tested for the presence of DNA from other hemoparasites such as Babesia/Rangelia/Theileria targeting the 18SrRNA gene (JEFFERIES et al., 2007), and Mycoplasma sp. (MAGGI et al., 2013) and Anaplasmataceae agents (INOKUMA et al., 2000) targeting the 16S rRNA gene. Blood and synovial liquid smears Blood and synovial liquid smears No gamont suggestive of Hepatozoon spp. infection was observed in the blood smears. The pale red synovial liquid had a low viscosity while the cytology revealed the predominance of degenerate neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. (Figure 1). Additionally, pyogranulomatous inflammation and red blood cells (hemarthrosis) with loss of windrowing, which reflected the low viscosity of the fluid, were also observed. Figure 1. Synovial liquid smear stained with Rosenfeld (100x obj.). (A) Degenerate neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. gametocytes (arrow); (B) Pyogranulomatous inflammation, red blood cells (hemarthrosis) with loss of windrowing and intracellular inclusions suggestive of Hepatozoon sp. gametocytes (arrow). Figure 1. Synovial liquid smear stained with Rosenfeld (100x obj.). (A) Degenerate neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. gametocytes (arrow); (B) Pyogranulomatous inflammation, red blood cells (hemarthrosis) with loss of windrowing and intracellular inclusions suggestive of Hepatozoon sp. gametocytes (arrow). 4/7 Perles, L. et al. Braz. J. Vet. Parasitol. Figure 2. Phylogenetic tree based on the 600 bp fragment (UJVARI et al., 2004) of Hepatozoon spp. 18SrRNA sequences, using the Maximum likelihood inference (ML) method and TPM2+F+G evolutionary model. Numbers at nodes correspond to bootstrap. Accession numbers are indicated in the sequences. The sequence of Hepatozoon spp. detected in the present study is highlighted in blue. The positive control is highlighted in red. Hepatozoon gamonts. It is known that immunosuppression caused by concomitant infections or immunosuppressive medications may influence the pathogenesis of new infection by H. canis or reactivate an existing infection (BANETH et al., 2001). smears, the maned wolf from this study presented neutrophilia with toxic granules, which probably resulted from the infected skin lesions and limb injury, with no relation with Hepatozoon infection. After 59 days, the animal did not present leukocytosis anymore, which indicated that the neutrophilia was associated with bacterial infection, which was resolved after antibiotic therapy. At the day of discharge from hospital (3 months after the entrance of the animal at the Veterinary Hospital), the animal was still positive for Hepatozoon spp. by PCR, which shows that the neutrophilia was not associated with Hepatozoon infection.h g Unlike other tick-borne diseases, hepatozoonosis is transmitted by ingestion of ticks by the intermediary host (BANETH & SHKAP, 2003). In tropical and subtropical regions, including Brazil and the United States, studies showed that H. References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215(3): 403-410. http://dx.doi. org/10.1016/S0022-2836(05)80360-2. PMid:2231712. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215(3): 403-410. http://dx.doi. org/10.1016/S0022-2836(05)80360-2. PMid:2231712. André MR, Adania CH, Teixeira RHF, Vargas GH, Falcade M, Sousa L, et al. Molecular detection of Hepatozoon spp. in Brazilian and exotic wild carnivores. Vet Parasitol 2010; 173(1-2): 134-138. http://dx.doi. org/10.1016/j.vetpar.2010.06.014. PMid:20630658. Baneth G, Samish M, Alekseev E, Aroch I, Shkap V. Transmission of Hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks. J Parasitol 2001; 87(3): 606-611. http:// dx.doi.org/10.1645/0022-3395(2001)087[0606:TOHCTD]2.0.CO;2. PMid:11426725. Baneth G, Shkap V. Monozoic cysts of Hepatozoon canis. J Parasitol 2003; 89(2): 379-381. http://dx.doi.org/10.1645/0022-3395(2003)089[0379:MC OHC]2.0.CO;2. PMid:12760658. Baneth G, Weigler B. Retrospective case-control study of hepatozoonosis in dogs in Israel. J Vet Intern Med 1997; 11(6): 365-370. http://dx.doi. org/10.1111/j.1939-1676.1997.tb00482.x. PMid:9470163. The Hepatozoon sp. epidemiology in wild canids in Brazil has not been fully elucidated yet. Although not producing the clinical disease, Brazilian wild canids apparently have subclinical canine hepatozoonosis, which may be aggravated when associated with other parasites. For instance, Silva et al. (2018) detected co-infection between Rangelia vitalii and H. canis in a free-living L. gymnocercus from the state of Santa Catarina, southern Brazil. Quadros et al. (2015) also reported the co-infection by R. vitalii, H. canis, and Capillaria hepatica in an L. gymnocercus specimen, which developed a fatal illness, in the state of Santa Catarina. In Minas Gerais, southeastern Brazil, Silveira et al. (2016) reported the co-infection by R. vitalii, Hepatozoon sp., Leishmania sp., Entamoeba spp. and intestinal helminths in a maned wolf. In the present case report, co-infections by piroplasmids, hemoplasmas, and Anaplasmataceae agents were not found. Criado-Fornelio A, Ruas JL, Casado N, Farias NA, Soares MP, Müller G, et al. New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain. J Parasitol 2006; 92(1): 93-99. http://dx.doi.org/10.1645/GE-464R.1. PMid:16629322. (Apicomplexa: Adeleorina) from Brazil and Spain. J Parasitol 2006; 92(1): 93-99. http://dx.doi.org/10.1645/GE-464R.1. PMid:16629322. Curi NHA, Coelho CM, Campo MCM, Magni EM, Sábato MA, Araújo AS, et al. Pathogens of Wild Maned Wolves (Chrysocyon brachyurus) in Brazil. J Wildl Dis 2012; 48(4): 1052-1056. http://dx.doi.org/10.7589/2011- 10-304. PMid:23060508. Dietz JM. Ecology and social organization of maned wolf (Chrysocyon brachyurus). Washington: Smithsonian Institution Press; 1984. (Smithsonian Contributions to Zoology; no. 392). https://doi.org/10.5479/ si.00810282.392. Dietz JM. Chrysocyon brachyurus. Blood and synovial liquid smears The increased contact between wild and domestic canids in disturbed environments may facilitate pathogen exchanges, constituting one of the main threats against the maned wolf populations (PAULA et al., 2013; OROZCO et al., 2013). The animal evaluated in this report was found on a road, near a peri-urban area, and its presence in such an area may have facilitated contact with domestic animals and infection by H. canis. Although clustering with H. canis sequences, the amplified sequences showed only 98% of identity with H. canis by BLAST analysis, which may suggest that the maned wolf was infected with another closely related Hepatozoon species. Considering that the 18S rDNA gene has been the only molecular marker used for phylogenetic analyses of Hepatozoon spp. so far, problems for discriminating closely related species might occur due to its high degree of conservation (MORRISON et al., 2004). Recently, the complete genome of H. canis was sequenced, which may allow the use of new target genes, aiming ate achieving better discrimination of Hepatozoon species (LÉVEILLÉ et al., 2019).h carnivores in phylogenetic analyses, BLAST results may indicate that the maned wolf was infected with another closely related Hepatozoon species. Keeping in mind that an increase contact between wild and domestic animals may facilitate pathogen exchange, special attention should be given for vector-borne agents in endangered wild carnivores maintained in captivity. Acknowledgements We are thankful to “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP) for the financial support (Process #2018/02753-0) and “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq – Research Productivity Scholarship - Process number 302420/2017-7) for Researcher Fellowship delivered to MRA. References Mamm Species 1985; 234(234): 1-4. http://dx.doi.org/10.2307/3503796. Blood and synovial liquid smears canis is transmitted by Amblyomma ovale ticks, Haemaphysalis longicornis, Haemaphysalis flava, and Rhipicephalus sanguineus sensu lato (MURATA et al., 1995; FORLANO et al., 2005, 2007; RUBINI et al., 2009). In Brazil, the participation of R. sanguineus s.l. in the transmission of H. canis should be better investigated (GOMES et al., 2016). The maned wolf from this report had a light infestation of ticks, which, unfortunately, were not sampled. To understand better the transmission of hepatozoonosis and other hemoparasites, ectoparasites should always be sampled and identified, and molecular analyses should be performed to identify possible pathogens. The swollen on both thoracic limbs, near the humerous‑radius‑ulna articulation, allowed the detection of Hepatozoon gamonts in synovial liquid smears. In fact, the observed swollen might have been the result of an extensive period of decubitus that the animal showed after the amputation procedure. Results of synovial liquid cytology indicated inflammation, which may have resulted in migration of leukocytes to the region. It is most likely that the migration of leucocytes to humerous-radius-ulna articulation generated an increase of the presence of neutrophils in the area, which in association with immunosuppression due to anesthesia/hospitalization procedures, allowed the detection of Studies in the literature have reported that H. canis has been commonly detected in domestic dogs from all Brazilian regions (RUBINI et al., 2008; SPOLIDORIO et al., 2009; RAMOS et al., Hepatozoon sp. in Chrysocyon brachyurus 5/7 Ahead of Print, 2019 2010, 2015; SOUSA et al., 2017). Hepatozoon phylogenetically related to H. americanum has been recently described in domestic dogs in Belém, northern Brazil (GOMES et al., 2016), while both Hepatozoon species have been detected in wild canids from Brazil. While Hepatozoon sp. related to H. americanum has been detected in maned wolf and crab-eating fox (Cerdocyon thous) (CRIADO‑FORNELIO et al., 2006; ANDRÉ et al., 2010; SOUSA et al., 2017), H. canis was detected in Cerdocyon thous and Pampas fox (Lycalopex gymnocercus) (CRIADO-FORNELIO et al., 2006; SILVA et al., 2018). Additionally, a possible new Hepatozoon species that clustered separately from H. canis and H. americanum based on 18S rRNA phylogenetic inference has been detected in maned wolf, bush dog (Speothus venaticus) and hoary fox (Pseudalopex vetulus) maintained in captivity in Brazil (ANDRÉ et al., 2010). In this study, the molecular and phylogenetic analyses of the Hepatozoon-18SrRNA sequence found in the synovial liquid from the maned wolf clustered with H. canis sequences detected only in domestic carnivores. Conclusion Detection of Ehrlichia platys DNA in brown dog ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan. J Clin Microbiol 2000; 38(11): 4219-4221. PMid:11060094. Quadros RM, Soares JF, Xavier JS, Pilati C, Costa JL, Miotto BA, et al. Natural Infection of the Wild Canid Lycalopex gymnocercus by the Protozoan Rangelia vitalii, the Agent of Canine Rangeliosis. J Wildl Dis 2015; 51(3): 787-789. http://dx.doi.org/10.7589/2014-08-194. PMid:25932667. Jefferies R, Ryan UM, Irwin PJ. PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies. Vet Parasitol 2007; 144(1-2): 20-27. http:// dx.doi.org/10.1016/j.vetpar.2006.09.022. PMid:17127005. Ramos CAN, Babo-Terra VJ, Pedroso TC, Souza AF Fo, Araújo FR, Cleveland HPK. Molecular identification of Hepatozoon canis in dogs from Campo Grande, Mato Grosso do Sul, Brazil. Rev Bras Parasitol Vet 2015; 24(2): 247-250. http://dx.doi.org/10.1590/S1984-29612015019. PMid:26154969. Kocan AA, Cummings CA, Panciera RJ, Mathew JS, Ewing SA, Barker RW. Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma. J Wildl Dis 2000; 36(1): 149- 153. http://dx.doi.org/10.7589/0090-3558-36.1.149. PMid:10682757. Ramos R, Ramos C, Araújo F, Oliveira R, Souza I, Pimentel D, et al. Molecular survey and genetic characterization of tick-borne pathogens in dogs in metropolitan Recife (north-eastern Brazil). Parasitol Res 2010; 107(5): 1115-1120. http://dx.doi.org/10.1007/s00436-010-1979-7. PMid:20680344. Kubo M, Miyoshi N, Yasuda N. Hepatozoonosis in two species of Japanese wild cat. J Vet Med Sci 2006; 68(8): 833-837. http://dx.doi.org/10.1292/ jvms.68.833. PMid:16953084. Léveillé AN, Baneth G, Barta JR. Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): complete apicoplast genome and multiple mitochondrion-associated sequences. Int J Parasitol 2019; 49(5): 375-387. http://dx.doi.org/10.1016/j.ijpara.2018.12.001. PMid:30790556. Léveillé AN, Baneth G, Barta JR. 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Molecular analysis reveals the diversity of Hepatozoon species naturally infecting domestic dogs in a northern region of Brazil. Ticks Tick Borne Dis 2016; 7(6): 1061-1066. http://dx.doi.org/10.1016/j. ttbdis.2016.09.008. PMid:27665264. Paula RC, Rodrigues FHG, Queirolo D, Jorge RPS, Lemos FG, Almeida Rodrigues L. Avaliação do estado de conservação do Lobo-guará Chrysocyon brachyurus (Illiger, 1815) no Brasil. Biodiver Bras 2013; 3: 146-159. Inokuma H, Raoult D, Brouqui P. O’Dwyer LH, Saito ME, Hasegawa MY, Kohayagawa A. Brazilian canine hepatozoonosis. Rev Bras Parasitol Vet 2011; 20(3): 181-193. http:// dx.doi.org/10.1590/S1984-29612011000300002. PMid:21961746. Conclusion (Apicomplexa: Hepatozoidae) in neotropical felids from Brazil. Vet Parasitol 2008; 152(1-2): 28-33. http:// dx.doi.org/10.1016/j.vetpar.2007.12.006. PMid:18243562. Rumiz D, Sainz LA. 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Rev Bras Parasitol Vet 2018; 27(3): 377-383. http://dx.doi.org/10.1590/s1984-296120180018. PMid:29846457. Silva MRL, Mattoso CRS, Costa A, Saito ME, Tchaicka L, O’Dwyer LH. Rangelia vitalii and Hepatozoon canis coinfection in pampas fox Lycalopex Rangelia vitalii and Hepatozoon canis coinfection in pampas fox Lycalopex gymnocercus from Santa Catarina State, Brazil. Rev Bras Parasitol Vet 2018; 27(3): 377-383. http://dx.doi.org/10.1590/s1984-296120180018. PMid:29846457. Stover BC, Muller KF. TreeGraph 2: combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics 2010; 11(1): 7. http://dx.doi.org/10.1186/1471-2105-11-7. PMid:20051126. Silveira JA, D’Elia ML, Oliveira Avelar I, Almeida LR, Santos HÁ, Soares DFM, et al. Rangelia vitalii in a free-ranging maned wolf (Chrysocyon brachyurus) and co-infections. Int J Parasitol Parasites Wildl 2016; 5(3): 280- 285. http://dx.doi.org/10.1016/j.ijppaw.2016.09.003. PMid:27761403. 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https://openalex.org/W2755865808	https://europepmc.org/articles/pmc5602063?pdf=render	English	null	Monotone and fast computation of Euler’s constant	Journal of inequalities and applications	2,017	cc-by	4,533	R ES EA RCH Open Access Monotone and fast computation of Euler’s constant Abstract We construct sequences of ﬁnite sums (˜ln)n≥0 and (˜un)n≥0 converging increasingly and decreasingly, respectively, to the Euler-Mascheroni constant γ at the geometric rate 1/2. Such sequences are easy to compute and satisfy complete monotonicity-type properties. As a consequence, we obtain an inﬁnite product representation for 2γ converging in a monotone and fast way at the same time. We use a probabilistic approach based on a diﬀerentiation formula for the gamma process. MSC: Primary 11M35; 33F05; secondary 60E05 Keywords: Euler-Mascheroni constant; fast computation; inﬁnite product; alternating zeta function; gamma process José A Adell† and Alberto Lekuona† José A Adell† and Alberto Lekuona† José A Adell† and Alberto Lekuona† Correspondence: adell@unizar.es Departamento de Métodos Estadísticos, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna 12, Zaragoza, 50009, Spain †Equal contributors Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 DOI 10.1186/s13660-017-1507-8 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 DOI 10.1186/s13660-017-1507-8 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 DOI 10.1186/s13660-017-1507-8 1 Introduction The Euler-Mascheroni constant γ was ﬁrst introduced by Leonhard Euler (-) as γ = lim n→∞(Hn – logn) = ...., Hn = n k=  k ,n = ,,.... () () () This constant appears in diﬀerent mathematical subjects, such as number theory, spe- cial functions, random matrix theory, random permutations, and mathematical physics, among many others. We refer the interested reader to the survey paper by Lagarias []. As far as we know, two main types of computations of the Euler constant have been developed. The ﬁrst one emphasizes the monotonicity of the corresponding convergent sequences, but the rates of convergence are relatively slow (polynomial rates). The second one emphasizes the speed of convergence (geometric rates), but looses the monotonicity in the approximation. In this paper, we gather both points of view by providing approximating sequences that converge at the geometric rate /and satisfy complete monotonicity-type proper- ties (monotonicity, convexity, etc.). In addition, such approximating sequences are easy to compute. With respect to the ﬁrst type of computations, we mention that Xu and You [] and Lu et al. [, ] have used continued fractions to obtain monotone convergence to γ . For instance, it is shown in [], Theorem , that C (n + )< γ – rn < C n, C⋆ (n + )< r⋆ n – γ < C⋆ n, () () © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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 Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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. equalities and Applications ( 2017) 2017:224 Page 2 of 9 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 2 of 9 Page 2 of 9 where C and C⋆are explicit constants, and (rn)n≥and (r⋆ n)n≥are sequences involving the logarithm of a continued fraction, the ﬁrst one being strictly increasing, and the second one strictly decreasing. Yang [] has found the constants (ai)≤i≤s for a given s = ,,... such that where C and C⋆are explicit constants, and (rn)n≥and (r⋆ n)n≥are sequences involving the logarithm of a continued fraction, the ﬁrst one being strictly increasing, and the second one strictly decreasing. Yang [] has found the constants (ai)≤i≤s for a given s = ,,... such that Hn – log n + s i= ai ni = γ + O  ns+ as n →∞ () () is the fastest sequence converging to γ , giving in this way a constructive answer to a prob- lem posed by Chen and Mortici []. It turns out that, for small values of s, the sequence on the left-hand side in () strictly increases to γ . With regard to the second type of computations, K. and T. Hessami Pilehrood [] have provided a rational approximation pn/qn converging to γ subexponentially. In fact, these authors have shown that pn qn – γ = e–√n π + O n–/ as n →∞, () pn qn – γ = e–√n π + O n–/ as n →∞, () pn qn – γ = e–√n π + O n–/ as n →∞, () where qn = n k= n k  k!, pn = n k= n k  k!(Hn–k – Hk), n = ,,.... Exponential convergence to γ is possible at the price of using logarithms. In this respect, Karatsuba [] showed that γ = – n+ k= (–)k–nk+ (k – )!(k + ) logn –  k +  + O –n , () () whereas Coﬀey [] gave the formula whereas Coﬀey [] gave the formula hereas Coﬀey [] gave the formula γ = log  –  log ∞ k=  k k j= (–)j k j j log(j + ) j +  , () () where the series in () has actually the geometric rate /. On the other hand, Sondow [] obtained the expression γ = An – Ln n n + O –nn–/ as n →∞, () () where, for any n = ,,... , An = n i= n i  Hn+i, Ln =  ≤i<j≤n j–i k= (–)i+j– j – i n i n j log(n + i + k). As mentioned before, the aim of this paper is to compute Euler’s constant in a monotone and fast way at the same time. To achieve this, we combine a formula obtained by Zhang and Williams [] to compute Stieltjes constants (see also Coﬀey []) and a probabilistic Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 3 of 9 ell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 3 of 9 perspective based on a diﬀerentiation formula for expectations of functions of the gamma process (see formula () in Section ). More precisely, let perspective based on a diﬀerentiation formula for expectations of functions of the gamma process (see formula () in Section ). More precisely, let η(z) = ∞ m= (–)m (m + )z , ℜ(z) > , () () be the alternating zeta function. It was shown in Zhang and Williams [], Theorem (see also Coﬀey []) that be the alternating zeta function. It was shown in Zhang and Williams [], Theorem (see also Coﬀey []) that γ = log  + η′() log. () γ = log  + η′() log. () Our computation of η′() is mainly based on the probabilistic representation given in Lemma .(Section ). We point out that some authors have introduced probabilistic tools to deal with diﬀer- ent topics of analytic number theory. For instance, Sun [] described Stirling series in terms of products of uniformly distributed random variables, Srivastava and Vignat [] have given representations of the Bernoulli, Euler, and Gegenbauer polynomials in terms of moments of appropriate random variables, and Ta [] has recently introduced a nice probabilistic approach to study Appell polynomials by connecting them to moments of random variables. Finally, fast computations of the Stieltjes constants using diﬀerentia- tion formulas for linear operators represented by stochastic processes can be found in [] and the references therein. 2 Main results Denote by N the set of nonnegative integers, and let N+ = N \ {}. The mth forward dif- ferences of any sequence (vn)n≥of real numbers are recursively deﬁned by vn = vn, vn = vn+– vn, n ∈N, and mvn =  m–vn = m j= m j (–)m–jvn+j, m ∈N+,n ∈N. Let n ∈N. We consider the coeﬃcients Let n ∈N. We consider the coeﬃcients Let n ∈N. We consider the coeﬃcients an(j) = n k=j k +  j +   k+, j = ,,...,n, () () and deﬁne the following lower and upper approximants of η′(): ln = n j= an(j)(–)j log(j + ) j +  () () ln = j= an(j)(–)j log(j + ) j +  () nd and and un = ln + n +  n+ n+ j= n +  j (–)j log(j + ) (j + )j , () () respectively, where log/:= . With these notations, we enunciate our ﬁrst main result. respectively, where log/:= . With these notations, we enunciate our ﬁrst main result. alities and Applications ( 2017) 2017:224 Page 4 of 9 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 4 of 9 Theorem .Let n ∈N. Then, Theorem .Let n ∈N. Then, Theorem .Let n ∈N. Then, ˜ln := log  + ln log< γ < log  + un log=: ˜un. () n g< γ < log  + un log=: ˜un. () () The sequences (ln)n≥and (un)n≥satisfy the following complete monotonicity-type prop- erties The sequences (ln)n≥and (un)n≥satisfy the following complete monotonicity-type prop- erties (–)m–mln ≥, (–)m–mun ≤, ≤m ≤n + . () () In addition, we have un – ln ≤n +  n+. () un – ln ≤n +  n+. () The sequences (˜ln)n≥and (˜un)n≥in Theorem .provide monotone and fast computa- tions of the Euler-Mascheroni constant γ . Moreover, such sequences are easy to compute. In this regard, let Sj, j ∈N+, be a random variable having the negative binomial distribution with parameters j and /, that is, P(Sj = l) = j – + l j –   j+l , l ∈N. P(Sj = l) = j – + l j –   j+l , l ∈N. The coeﬃcients an(j) in () can be represented as The coeﬃcients an(j) in () can be represented as an(j) = n–j l= j + + l j +   j++l = P(Sj+≤n – j), n ∈N,j = ,...,n. Corollary .We have the inﬁnite product representation γ –log/= lim n→∞ n k= Pk Qk /(k+)k+ , where Pk/Qk > , k ∈N. where Pk/Qk > , k ∈N. Guillera and Sondow [], Example ., have obtained the product formula Guillera and Sondow [], Example ., have obtained the product formula eγ –log/= lim n→∞ n k= Q⋆ k P⋆ k /(k+) , () () where P⋆ k = (even) k+ j= (j + )(k+ j ), Q⋆ k = (odd) k+ j= (j + )(k+ j ), k ∈Z+. () () However, the rate of convergence in Corollary .is faster than that in (). 3 Auxiliary results Let n ∈N. We consider the coeﬃcients Thus, the tail probabilities an(j) can be precomputed, as done in many statistical packages, such as R. Finally, recall that a sequence (vn)n≥is said to be completely monotonic if (–)mmvn ≥, m,n ∈N. This is the reason why the inequalities in () are called complete monotonicity-type properties. The approximating sequences to γ given in () and () are simpler to compute than those in Theorem .. However, the sequences (˜ln)n≥and (˜un)n≥in this theorem converge to γ in a much faster way and enjoy properties such as monotonicity, convexity, and so on. On the other hand, formulas (), (), (), and () provide fast computations of γ at the price of loosing the monotonicity of the corresponding approximating sequences. Certainly, formula () computes γ in a faster way than that in (). However, the sequences (˜ln)n≥ and (˜un)n≥in Theorem .are easier to compute than the main term in (). Denote Denote Pk = (even) k j= (j + )(k+ j+), Qk = (odd) k j= (j + )(k+ j+), k ∈N, () () where (even) (resp. (odd) ) means that the product is extended to those even (resp. odd) integers j running from to k. As a consequence of Theorem ., we give the follow- ing: Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 5 of 9 3 Auxiliary results Let (Xt)t≥be a gamma process (see Çınlar [], pp.-), that is, a stochastic process starting at the origin, having independent stationary increments, and such that for each t > , the random variable Xt has the gamma density ρt(θ) =  (t)θt–e–θ, θ > . () ρt(θ) =  (t)θt–e–θ, θ > . () On the other hand, let V and T be two independent random variables such that V is uniformly distributed on [,] and T has the exponential density ρ(θ) deﬁned in (). We assume that V and T are independent of the gamma process (Xt)t≥. Finally, let f : R+ →R be a diﬀerentiable function such that f⋆(t) := Ef (Xt) < ∞, t ≥, where E stands for mathematical expectation. It has been shown in [], Theorem ., that f ′ ⋆(t) = Ef ′(Xt + VT), t ≥. () f ′ ⋆(t) = Ef ′(Xt + VT), t ≥. () () This formula can be applied to the problem at hand as follows. This formula can be applied to the problem at hand as follows. This formula can be applied to the problem at hand as follows. Lemma .Let η be the alternating zeta function. Then, η′() = Eg(X+ VT), () () η′() = Eg(X+ VT), re the function g is deﬁned in R+ as where the function g is deﬁned in R+ as where the function g is deﬁned in R+ as where the function g is deﬁned in R+ as Lemma .For any n ∈N, we have Lemma .For any n ∈N, we have where the function g is deﬁned in R+ as where the function g is deﬁned in R+ as g(x) = ∞ k= k +  k+ – e–xke–x =  – ∞ k= k k+ – e–xk+. () () Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 6 of 9 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 6 of 9 Proof By () the Laplace transform of Xt is given by Proof By () the Laplace transform of Xt is given by Ee–λXt =  (λ + )t , λ ≥,t ≥. () Ee–λXt =  (λ + )t , λ ≥,t ≥. Ee–λXt =  (λ + )t , λ ≥,t ≥. () () Thus, interchanging the sum with expectation, from () we have Thus, interchanging the sum with expectation, from () we have Thus, interchanging the sum with expectation, from () we have η(t) = ∞ m= E –e–Xtm = Ef (Xt), t ≥, () η(t) = ∞ E –e–Xtm = Ef (Xt), t ≥, () where f (x) =  + e–x = ∞ k= (– e–x)k k+ , x ≥. () () Therefore from () and () we have herefore from () and () we have Therefore from () and () we have Therefore from () and () we have η′() = Ef ′(X+ VT). η′() = Ef ′(X+ VT). This shows (), since f ′ = g, as follows from (). Finally, the second equality in () fol- lows by calculus. The proof is complete. □ This shows (), since f ′ = g, as follows from (). Finally, the second equality in () fol- lows by calculus. The proof is complete. □ In view of Lemma ., we deﬁne, for any n ∈N and x ≥, Ln(x) = n k= k +  k+ – e–xke–x, Un(x) =  – n– k= k k+ – e–xk+. () () It can be checked from () that Ln(x) = Un(x) – n +  n+ – e–xn+. () () Such partial sums allow us to give the following probabilistic representations of the se- quences (ln)n≥and (un)n≥respectively deﬁned in () and (). Such partial sums allow us to give the following probabilistic representations of the se- quences (ln)n≥and (un)n≥respectively deﬁned in () and (). Lemma .For any n ∈N, we have Ln(x) = e–x (n + )τ n+(x) = e–x vn+(). Therefore, the ﬁrst inequality in Lemma .follows from (). Finally, from () and () we have Un(x) = –n τ n+(x) = –τ(x) vn(). Un(x) = –n τ n+(x) = –τ(x) vn(). This, together with (), shows the second inequality in Lemma .and completes the proof. □ Lemma .For any n ∈N, we have ln = ELn(X+ VT), un = EUn(X+ VT). ln = ELn(X+ VT), un = EUn(X+ VT). Proof By Fubini’s theorem the Laplace transform of VT is given by Ee–λVT = E  λV + = log(λ + ) λ , λ ≥. () () Since Xis independent of VT, from () and () we have ELn(X+ VT) = n k= k +  k+ k j= k j (–)jEe–(j+)(X+VT) = n k= k +  k+ k j= k j (–)j log(j + ) (j + )(j + ) = ln, () () alities and Applications ( 2017) 2017:224 Page 7 of 9 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 7 of 9 where the last equality follows from () and () after interchanging the order of summa- tion. Similarly, where the last equality follows from () and () after interchanging the order of summa- tion. Similarly, E – e–(X+VT)n+= n+ j= n +  j (–)j log(j + ) (j + )j . Therefore, the second equality in Lemma .follows from (), (), and (). The proof is complete. □ □ Thanks to Lemma ., the complete monotonicity-type properties of (ln)n≥and (un)n≥ are easily derived from the analogous properties satisﬁed by the sequences of functions (Ln(x))n≥and (Un(x))n≥. Lemma .Let m ∈N+ and n ∈N with m ≤n + . Then, Lemma .Let m ∈N+ and n ∈N with m ≤n + . Then, (–)m–mLn(x) ≥, (–)m–mUn(x) ≤, x ≥. Proof Fix x ≥. For any s ≥, denote Proof Fix x ≥. For any s ≥, denote Proof Fix x ≥. For any s ≥, denote vn(s) = (n + s)τ n(x), τ(x) = – e–x  ,n ∈N. () () Using induction on m, we can check that Using induction on m, we can check that Using induction on m, we can check that Using induction on m, we can check that (–)mmvn(s) = τ n(x) – τ(x) m– n + s – (n + s + m)τ(x) , n,m ∈N, thus implying that (–)mmvn(s) ≥, m,n ∈N,m ≤n + s, () (–)mmvn(s) ≥, m,n ∈N,m ≤n + s, () (–)mmvn(s) ≥, m,n ∈N,m ≤n + s, () since ≤τ(x) ≤/. On the other hand, from () and () we have Ln(x) = e–x (n + )τ n+(x) = e–x vn+(). Authors’ contributions Both authors read and approved the ﬁnal manuscript. Received: 3 May 2017 Accepted: 30 August 2017 References 1. Lagarias, JC: Euler’s constant: Euler’s work and modern developments. Bull. Am. Math. Soc. (N.S.) 50(4), 527-628 (2013). doi:10.1090/S0273-0979-2013-01423-X 2. Xu, H, You, X: Continued fraction inequalities for the Euler-Mascheroni constant. J. Inequal. Appl. 2014, Article ID 343 (2014). doi:10.1186/1029-242X-2014-343 3. Lu, D, Song, L, Yu, Y: Some new continued fraction approximation of Euler’s constant. J. Number Theory 147, 69-80 (2015). doi:10.1016/j.jnt.2014.07.002 4. Lu, D, Song, L, Yu, Y: Some quicker continued fraction approximations and inequalities towards Euler’s constant. J. Number Theory 175, 100-116 (2017). doi:10.1016/j.jnt.2016.11.024 5. Yang, S: On an open problem of Chen and Mortici concerning the Euler-Mascheroni constant. J. Math. Anal. Appl. 396(2), 689-693 (2012). doi:10.1016/j.jmaa.2012.07.007 6. Chen, C-P, Mortici, C: New sequence converging towards the Euler-Mascheroni constant. Comput. Math. Appl. 64(4), 391-398 (2012). doi:10.1016/j.camwa.2011.03.099 7. Hessami Pilehrood, Kh, Hessami Pilehrood, T: On a continued fraction expansion for Euler’s constant. J. Number Theory 133(2), 769-786 (2013). doi:10.1016/j.jnt.2012.08.016 4 The proofs Proof of Theorem .Let n ∈N. By () and Lemmas .and .we get ln = ELn(X+ VT) < Eg(X+ VT) = η′() < EUn(X+ VT) = un. Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 Page 8 of 9 Page 8 of 9 This, in conjunction with (), shows (). On the other hand, let m ∈N+ with ≤m ≤n+. By Lemmas .and .we have This, in conjunction with (), shows (). On the other hand, let m ∈N+ with ≤m ≤n+. By Lemmas .and .we have (–)m–mln = E(–)m–mLn(X+ VT) ≥. The second inequality in () is shown in a similar way. Finally, we see from () and Lemma .that The second inequality in () is shown in a similar way. Finally, we see from () and Lemma .that un – ln = n +  n+E – e–(X+VT)n+≤n +  n+. The proof is complete. □ □ The proof is complete. The proof is complete. Proof of Corollary .Let k ∈N. Using (), (), and (), we can check that Proof of Corollary .Let k ∈N. Using (), (), and (), we can check that log Pk Qk = k j= k +  j +  (–)j log(j + ) log Pk Qk = k j= k +  j +  (–)j log(j + ) = (k + )(k + )E – e–(X+VT)ke–(X+VT) > , = (k + )(k + )E – e–(X+VT)ke–(X+VT) > , which implies that Pk/Qk > . Therefore, for any n ∈N, we have log n k= Pk Qk /(k+)k+ = n k=  k+ k j= k +  j +  (–)j log(j + ) j +  = ln, where the last equality follows from () and (). This, together with () and (), shows the result. □ where the last equality follows from () and (). This, together with () and (), shows the result. □ Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Authors’ contributions Acknowledgements Acknowledgements The authors are partially supported by Research Projects DGA (E-64), MTM2015-67006-P, and by FEDER funds. Publisher’s Note 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 aﬃliation Received: 3 May 2017 Accepted: 30 August 2017 Received: 3 May 2017 Accepted: 30 August 2017 Adell and Lekuona Journal of Inequalities and Applications ( 2017) 2017:224 References 1. Lagarias, JC: Euler’s constant: Euler’s work and modern developments. Bull. Am. Math. Soc. (N.S.) 50(4), 527-628 (2013). doi:10.1090/S0273-0979-2013-01423-X (2013). doi:10.1090/S0273-0979-2013-01423-X 2. Xu, H, You, X: Continued fraction inequaliti (2014). doi:10.1186/1029-242X-2014-343 (2014). doi:10.1186/1029-242X-2014-343 ( ) 3. Lu, D, Song, L, Yu, Y: Some new continued fraction approximation of Euler’s constant. J. Number Theory 147, 69-80 (2015). doi:10.1016/j.jnt.2014.07.002 4. 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Guillera, J, Sondow, J: Double integrals and inﬁnite products for some classical constants v Lerch’s transcendent. Ramanujan J. 16(3), 247-270 (2008). doi:10.1007/s11139-007-9102-0 j 17. Çınlar, E: Probability and Stochastics. Graduate Texts in Mathematics, vol. 261, p. xiv+557. Springer, Berlin (2011). doi:10.1007/978-0-387-87859-1. 17. Çınlar, E: Probability and Stochastics. Graduate Texts in Mathematics, vol. 261, p. xiv+557. S doi:10.1007/978-0-387-87859-1. 18. Adell, JA: Diﬀerential calculus for linear operators represented by ﬁnite signed measures and applications. Acta Math. Hung. 138(1-2), 44-82 (2013). doi:10.1007/s10474-012-0230-7 18. Adell, JA: Diﬀerential calculus for linear operators represented by Hung. 138(1-2), 44-82 (2013). doi:10.1007/s10474-012-0230-7
https://openalex.org/W4385797071	https://zenodo.org/records/8245504/files/Differentiated%20Instruction%20in%20Mathematics%20Its%20Effect%20on%20the%20Level%20of%20Critical%20Thinking%20Skills%20of%20Grade%207%20Students.pdf	English	null	Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	7,885	Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 Asia Pacific Journal of Advanced Education and Technology September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.a Keywords: differentiated instruction, curriculum compacting, flexible grouping, critical thinking skill, problem-solving, reasoning Introduction The learning abilities of individuals vary, even if they are exposed to the same teaching methods and learning environment. Kiley (2011) emphasized that education is unique due to variations in how one studies and learns. Innovations in teaching have been implemented, but some have been inconclusive and had loopholes. In some cases, effective learning is impeded for some students, or their potential is not tapped to the fullest. Among the skills that could enhance the potential of students for life-long learning is critical thinking. (Doyle, A. C. 2019) defined critical thinking as the formulation of reasoned judgment based on the available information. Critical thinking is the ability to distinguish between useful and less useful data, filter relevant research findings, relate important facts, and make objective observations. Inductive and deductive reasoning are essential skills for critical thinking, which can lead to solutions to problems and progress to more sophisticated ones. Critical thinking is a very important skill that needs to be developed among people if they are to lead a good life. It is not only required in employment but also in dealing with day-to-day problems or challenges in life. Mathematics, as a subject, could very well develop the critical thinking skills of students. Solving mathematical problems, especially open-ended and situational ones, requires critical thinking on the part of the students (Henningsen & Stein, 1997 and NCTM, 2000 as cited in (Firdaus, et. al.). Abstract 413 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 414 Objectives of the Study The aims of this study are the following: 1.) To evaluate and compare the efficacy of curriculum compacting and flexible grouping approaches in enhancing the critical thinking abilities of seventh-grade students in the field of mathematics. 2.) To assess the effects of curriculum compacting and flexible grouping techniques on the mathematical multiple intelligences of seventh-grade students; 3.) To assess the efficacy of the McKenzie Multiple Intelligence Survey tool in evaluating the various intelligences of seventh-grade students in the field of mathematics, 4.) To examine the correlation between the various intelligences and critical thinking abilities of seventh-grade students in mathematics, and to ascertain whether this correlation is influenced by the two distinct teaching methodologies. Abstract This research used a quasi-experimental research design, specifically the non-equivalent control group pretest-posttest design. Cluster random sampling was used to determine the respondents of the study. The respondents of the study were Grade 7 students at Sta. Catalina National High School during the school year 2019-2020. Two sections among the Grade 7 students taking mathematics under the researcher for the school year 2019-2020 were selected as respondents – one section was used as curriculum compacting (experiment) and the other one was used as a flexible grouping (experiment). Each section had 30 students each such that the study will have a total of 60 student-respondents. This research study was conducted for five weeks and utilized McKenzie’s Multiple Intelligence Survey instrument in assessing the student’s multiple intelligences. The researcher crafted a 40-item multiple choice test covering the topics of undefined terms in geometry, angles, polygons, and quadrilaterals to assess the level of critical thinking skills of student-respondents. Both independent and dependent t-tests were utilized to determine the significant difference between the pre-test and post-test results of the experimental and control groups. Based on the findings, before exposing students to differentiated instructions, their level of reasoning skill was an apprentice for both flexible grouping and curriculum compacting. However, their levels of problem-solving skills were apprentice and novice, for flexible grouping and curriculum compacting, respectively. After using differentiated instruction, the student’s level of critical thinking skills from both groups was at a proficient level. Findings also showed that the mean pretest and mean posttest scores of each group of respondents were significantly different for both measures of critical thinking skills. This suggested that the two forms of differentiated instruction that were employed in this study are effective in developing students’ critical thinking skills, especially in discussing basic concepts of Geometry. This experimental study found that flexible grouping and curriculum compacting as forms of differentiated instruction can significantly improve the critical thinking skills of the students, then it is recommended that teachers may utilize flexible grouping and curriculum compacting as forms of differentiated instruction in Geometry. Methodology The study utilized a quasi-experimental research design, specifically the nonequivalent control group pretest-posttest design, to investigate the efficacy of differentiated instruction in enhancing students' critical thinking skills (Creswell, J. W. 2018). The utilization of this design was based on the selection of two intact groups from a specific grade level within a public high school located in the province of Quezon. The respondents of the study were Grade 7 students at Sta. Catalina National High School during the school year 2019-2020. Two sections among the Grade 7 students taking mathematics under the researcher for the school year 2019-2020 were selected as respondents – one section was used as curriculum compacting (experiment) and the other one was used as a flexible grouping (experiment). Each section had 30 students each such that the study will have a total of 60 student-respondents. The cluster sampling technique was used in this study. To collect the necessary data, the researcher crafted a 40-item multiple choice test covering the topics of undefined terms in geometry, angles, polygons, and quadrilaterals to assess the level of critical thinking skills of student-respondents. Both independent and dependent t-tests 414 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com were employed to ascertain the statistical significance of the differences between the pre-test and post-test outcomes of the experimental and control groups. Asia Pacific Journal of Advanced Education and Technology Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com f f gy Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www were employed to ascertain the statistical significance of the differences between the pre-test and post-test outcomes of the experimental and control groups. were employed to ascertain the statistical significance of the differences between the pre-test and post-test outcomes of the experimental and control groups. The researcher obtained parental consent, categorized participants into two groups, and employed distinct methods of selection and instructional techniques to optimize the capabilities of the students and the differentiated instruction. The researcher employed Walter McKenzie's Multiple Intelligence Survey instruments to evaluate the primary intelligence among the students in the flexible group, and subsequently selected the subjects for experimentation. Table 1 presents the level of critical thinking skills in terms of the student-respondents’ reasoning before using differentiated instructions. g f f f p y ( ) y Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 415 Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 Jul Methodology According to the results of the Multiple Intelligence Survey, it was found that out of the participants, 9 individuals exhibited a strong inclination towards Musical intelligence, while 8 individuals demonstrated a preference for Mathematical and Logical intelligence. Additionally, 7 individuals displayed a proclivity towards Bodily-Kinesthetic intelligence, and 6 individuals exhibited a preference for Interpersonal intelligence. The participants were categorized based on their level of interest and ability to ensure that they were assigned suitable tasks. The musical ensemble was provided with a piece of music to listen to. The group focused on Mathematics and Logic and engaged in a Cross-Term Puzzle activity. The group with a bodily-kinesthetic learning style was assigned to perform a given figure, while the group with an interpersonal learning style engaged in the game "Circle Time". The researcher fostered a culture of collaboration and mutual support among participants in all undertakings. On the other hand, the researcher considered the needs of the learners in the activities provided in the compacting group. The teacher discussed the lesson, then, asked the students to answer the given activity. Since the students need to master the lesson, their performance in the given activity became the basis if they will advance to the next lesson or will remain on the same topic. Results and Discussion , , , , ff ff f Grade 7 Students, pp. 413 – 425 f f f p y ( ) y R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of 1. Level of Students’ Critical Thinking Skills The succeeding tables present the level of students’ critical thinking skills in terms of reasoning and problem-solving before and after using differentiated instruction. The details are presented in Table 1-3. 1.1. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students 1.1. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students g f f f p y Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of C R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 g f f f p y ( ), y Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 Table 1 Table 1 Level of Critical Thinking Skills in terms of Reasoning of the Student-Respondents before using Differentiated Instruction Scores Flexible Grouping Curriculum Compacting Description F % F % 22 – 28 - - - - Distinguished 15 – 21 - - - - Proficient 8 – 14 19 63.33% 12 40% Apprentice 0 -7 11 36.67% 18 60% Novice Total 30 100% 30 100% cal Thinking Skills in terms of Reasoning of the Student-Respondents before using Differentiated Instruction Table 1 presents the level of critical thinking skills in terms of the student-respondents’ reasoning before using differentiated instructions. 415 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com As shown, the student-respondents in both groups have reasoning skill levels ranging from novice level to apprentice level. Students with the apprentice-level reasoning skills can only answer exactly what the teacher taught for they can only answer through memorization. These students are not using mathematical reasoning skills appropriately when solving the task. For example, question no. 12, “Which description does not guarantee that a quadrilateral is a square?” is a type of question that should be provided to students who are studying Mathematics, more specifically, the properties of the quadrilateral. This question needs to be elaborated properly to find the correct answer. The student has apprentice level of reasoning skills, meaning they have difficulty analyzing mathematical problems and using available information to solve them. The experiment found that these students had deficiencies in their ability to engage in reasoning, apply mathematical principles, and use logical thinking when working with patterns and generalizing and explaining mathematical concepts. 1.2. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Table 2 Level of Critical Thinking Skills in terms of Problem Solving of the Student-Respondents before using Differentiated Instruction Scores Flexible Grouping Curriculum Compacting Description F %f F %f 10 – 12 - - - - Distinguished 7 – 9 6 20% - - Proficient 4 – 6 12 40% 10 33.33% Apprentice 0 -3 12 40% 20 66.67% Novice Total 30 100% 30 100% 1.2. 1.3. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 417 Table 1 416 Asia Pacific Journal of Advanced Education and Technology Asia Pacific Journal of Advanced Education and Technology Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com T bl 3 f f gy Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.a f f gy Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com f f gy Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com bl 3 Table 3 Level of Critical Thinking Skills in terms of Reasoning of the Student-Respondents after using Differentiated Instructions Scores Flexible Grouping Curriculum Compacting Description F %f F %f 22 – 28 2 6.67% 6 20% Distinguished 15 – 21 23 76.67% 22 73.33% Proficient 8 – 14 5 16.67% 2 6.67%% Apprentice 0 -7 - - - - Novice Total 30 100% 30 100% Table 3 Level of Critical Thinking Skills in terms of Reasoning of the Student-Respondents after using Differentia ical Thinking Skills in terms of Reasoning of the Student-Respondents after using Differentiated Instructions Table 3 presents the level of critical thinking skills of the student-respondents in terms of reasoning after using differentiated instructions. Table 3 presents the level of critical thinking skills of the student-respondents in terms of reasoning after using differentiated instructions. As presented, 83.34% of the respondents in flexible grouping have shown at least a proficient level of reasoning skill. Only 16.67% remained to have reasoning skills at the apprentice level. On the other hand, 93.33% of the student-respondents in the curriculum compacting group manifested at least a proficient level of reasoning skill and only 6.67% had an apprentice level. Flexible grouping is a teaching approach that involves diverse learning strategies such as peer interactions and teacher guidance among students. Students with proficient reasoning skills can easily interact and remember what is taught. They can easily answer questions that follow the pattern given by the teacher. However, these students may struggle to explain how they arrived at the correct answer, despite understanding the concepts and ideas of the lessons. In addition, the students with distinguished levels of reasoning skills are active participants in class discussions. Table 1 Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Table 2 Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skill 1.2. Differentiated Instruction in Mathematics: Its Effect on the Level of Cr nking Skills in terms of Problem Solving of the Student-Respondents before using Differentiated Instruction Level of Critical Thinking Skills in terms of Problem Solving of the Student-Respondents before using Differentiated Instruction Scores Flexible Grouping Curriculum Compacting Description F %f F %f 10 – 12 - - - - Distinguished 7 – 9 6 20% - - Proficient 4 – 6 12 40% 10 33.33% Apprentice 0 -3 12 40% 20 66.67% Novice Total 30 100% 30 100% Shown in Table 2 is the level of critical thinking skills in terms of problem-solving of the student- respondents before using differentiated instructions. The findings of the study revealed that a significant majority (80%) of the students who participated in flexible grouping exhibited problem-solving skills at the novice to apprentice level, while a mere minority (20%) demonstrated proficiency in this area. The results indicate that a significant proportion of the student-respondents in the curriculum compacting group exhibited a novice level of problem-solving ability, while 33.33% demonstrated an apprentice level. In a flexible group setting, students who are at the apprentice level of problem-solving skills are limited to recalling information from their memory when answering questions. Individuals at the novice level of problem-solving proficiency exhibit difficulty in recalling the instructional content conveyed by the educator. These students are unable to accurately analyze the provided questions. In question 9, students must possess an understanding of the concept and properties of quadrilaterals to provide a correct explanation and response to the given problem. This low performance in the pre-test of the student-respondents is expected since they don’t have yet the necessary knowledge of the learning competencies. Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 418 Table 1 The students under this level can easily understand and remember the topic; thus, he/she can answer the questions correctly. Moreover, they can give coherent ideas about the topic. For example, question no. 13 needs concepts and ideas about angles to prove and explain the diagram. In this case, the student can easily provide proof and explanation about the given question. g y p p p g q In this instructional approach, students are categorized based on their aptitude, inclination, and proficiency level or subject matter expertise. In this scenario, the provision of explicit instruction is expected to increase as an experienced individual will oversee the learning process. The approach enables learners to advance at their individualized and inherent speed. The students are provided with information regarding the objective they are striving to achieve. Upon successfully achieving the anticipated objective, they proceeded to the subsequent concept or learning target. The engagement and progress of students are maintained due to the appropriate level of difficulty of the work and the clear identification of learning objectives. Ascertaining the learning objective and successfully achieving it can lead to a boost in students' self-assurance, thereby motivating them to continue making progress. Conversely, with regard to student grouping, the responsibility of the grouping process is assumed by the students themselves. (Smith, J. K. 2021) conducted a study to examine the efficacy of various student grouping strategies in fostering self- directed learning. The study centered on the implementation of collaborative groups, performance-based groups, and student pairs, as delineated by (Conklin, J. 2007). The objective of the study was to evaluate and contrast the efficacy of various grouping techniques in fostering self-directed learning, a crucial component of prosperous learning and growth. The study utilized a quantitative methodology, employing a quasi-experimental framework that incorporated pre-and post-tests. The findings suggest that collaborative groups and student pairs are more efficacious in fostering self-directed learning as compared 417 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com f f gy ssue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com f f gy Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / ww to performance-based groups. Table 1 The implications of the study's findings are noteworthy for educators and practitioners who aim to improve self-directed learning and student achievement. to performance-based groups. The implications of the study's findings are noteworthy for educators and practitioners who aim to improve self-directed learning and student achievement. Curriculum compacting is an instructional strategy that can be adjusted based on the learners' needs. Students with proficient reasoning can easily answer questions but may struggle to explain how they arrived at the answer, while students with distinguished reasoning can provide substantial ideas and explanations. By adjusting the curriculum to meet individual needs, students' reasoning skills can be increased, and they can master the lesson before moving on. After the experimentation, there is clear evidence of an increase in the level of students' reasoning skills. They demonstrated proficiency in comprehending mathematical concepts and principles that are intrinsic to various procedures. The individual has developed a set of cognitive processes that have facilitated accurate reasoning and effective problem-solving. According to (Aini, Q.2019) there exists a positive correlation between the level of students' reasoning skills and the rate at which they acquire learning competencies. This experiment suggests that the fundamental ability of mathematics is its reasoning capacity. 1.4. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skill d t Table 4 Table 4 Level of Critical Thinking Skills in terms of Problem Solving of the Student-Respondents after using Diffe Thinking Skills in terms of Problem Solving of the Student-Respondents after using Differentiated Instructions ms of Problem Solving of the Student-Respondents after using Differentiated Instructions Table 4 Level of Critical Thinking Skills in terms of Problem Solving of the Student-Respondents after using Differentiated Instructions Scores Flexible Grouping Curriculum Compacting Description F %f F %f 10 – 12 6 20% 8 26.67%% Distinguished 7 – 9 14 46.67% 14 46.67%% Proficient 4 – 6 10 33.33% 8 26.67% Apprentice 0 -3 - - - - Novice Total 30 100% 30 100% Table 4 shows the level of critical thinking skills of the student-respondents in terms of problem- solving after using differentiated instructions. Out of 30 student respondents in the flexible grouping, 66.67% showed at least a proficient level of problem-solving skill, while only 33.33% have an apprentice level. On the other hand, 73.34% of the student-respondents in curriculum compacting manifested at least a proficient level of problem-solving skill and only 26.67% registered at the apprentice level. The students in flexible grouping with strong problem-solving skills can quickly answer questions, but struggle to explain their answers, while those with limited problem-solving abilities struggle with the order of steps required to solve problems. However, both groups demonstrate proficiency in interpreting data, arriving at solutions, evaluating inquiries, and communicating responses. The implementation of flexible grouping allowed for the development of problem-solving skills based on individual interests and resulted in advanced critical thinking skills demonstrated through substantial arguments and comprehensive explanations. Students with proficient problem-solving skills can accurately respond to questions but may require guidance to provide detailed explanations. Those with high-level problem-solving skills can analyze and provide evidence to support assigned tasks. For item 36, knowledge of polygon properties 418 f f gy Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / ww and the ability to analyze based on the given figure is crucial. Curriculum compacting enhances critical thinking abilities in problem-solving, as evidenced by the rise in post-test scores. After the experiment, many students showed an improved ability to solve problems. They demonstrated proficiency in data interpretation, responding to queries and assertions, analyzing concepts, identifying arguments, engaging in independent problem-solving, and assessing inquiries. Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 419 Table 4 They also showed an understanding of a problem's verbal description and the ability to provide a numerical response by utilizing mathematical connections. Students exhibiting a high level of problem-solving skills demonstrate a proclivity and pleasure in the act of problem-solving, in contrast to their counterparts with lower levels of such skills. They were able to enhance their mathematical proficiency. The individuals utilized the tools to apply their mathematical expertise toward the resolution of both hypothetical and practical problems. nstruction in Mathematics: Its Effect on the Level of Critical Thinking Skills o t II. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Table 5 Students exhibiting a high level of problem-solving skills demonstrate a proclivity and pleasure in the act of problem-solving, in contrast to their counterparts with lower levels of such skills. They were able to enhance their mathematical proficiency. The individuals utilized the tools to apply their mathematical expertise toward the resolution of both hypothetical and practical problems. 2. Difference between Levels of Critical Thinking Skills on the Test Scores of the Two Groups and the difference between the levels of critical thinking skills on the test scores of the two groups. Table 6 2. Difference between Levels of Critical Thinking Skills on the Test Scores of the Two Groups and the difference between the levels of critical thinking skills on the test scores of the two groups. 2. Difference between Levels of Critical Thinking Skills on the Test Scores of the Two Groups and the difference between the levels of critical thinking skills on the test scores of the two groups. Table 6 Test of Difference Between the Levels of Critical Thinking Skills on the Pre-Test Mean Scores of the Two Groups Critical Thinking Skills Flexible Grouping Curriculum Compacting Mean Diff 95% Confidence Interval of Difference T df Sig (2- tailed) M SD M SD L U Reasoning 7.73 2.35 7.53 2.11 0.20 -0.95 1.35 0.347 58 0.730 Problem Solving 4.20 1.90 3.37 1.27 0.83 -0.005 1.67 1.995 58 0.051 Legend: Significant at 0.05 of Difference Between the Levels of Critical Thinking Skills on the Pre-Test Mean Scores of the Two Groups Table 6 presents the results of the test of the difference between the levels of critical thinking skills based on the pre-test mean scores of the two groups. The results of the independent t-test demonstrate that there is no statistically significant distinction in the mean scores of critical thinking skills between the two groups during the pre-test phase (p > 0.05). The findings suggest that students who were placed in the flexible grouping exhibit comparable levels of critical thinking abilities to those who were assigned to the curriculum compacting group. 2.1. Table 5 nking Skills in terms of Problem Solving of the Student-Respondents after using Differentiated Instructions Level of Critical Thinking Skills in terms of Problem Solving of the Student-Respondents after using Differentiated Instructions Scores Flexible Grouping Curriculum Compacting Description F %f F %f 10 – 12 6 20% 8 26.67%% Distinguished 7 – 9 14 46.67% 14 46.67%% Proficient 4 – 6 10 33.33% 8 26.67% Apprentice 0 -3 - - - - Novice Total 30 100% 30 100% Table 5 shows the level of critical thinking skills of the student-respondents in terms of problem- solving after using differentiated instructions. Out of 30 student respondents in the flexible grouping, 66.67% showed at least a proficient level of problem-solving skill, while only 33.33% have an apprentice level. On the other hand, 73.34% of the student-respondents in curriculum compacting manifested at least a proficient level of problem-solving skill and only 26.67% registered at the apprentice level. The students in flexible grouping with strong problem-solving skills can quickly answer questions, but struggle to explain their answers, while those with limited problem-solving abilities struggle with the order of steps required to solve problems. However, both groups demonstrate proficiency in interpreting data, arriving at solutions, evaluating inquiries, and communicating responses. The implementation of flexible grouping allowed for the development of problem-solving skills based on individual interests and resulted in advanced critical thinking skills demonstrated through substantial arguments and comprehensive explanations. p p Students with proficient problem-solving skills can accurately respond to questions but may require guidance to provide detailed explanations. Those with high-level problem-solving skills can analyze and provide evidence to support assigned tasks. For item 36, knowledge of polygon properties and the ability to analyze based on the given figure is crucial. Curriculum compacting enhances critical thinking abilities in problem-solving, as evidenced by the rise in post-test scores. 419 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com After the experiment, many students showed an improved ability to solve problems. They demonstrated proficiency in data interpretation, responding to queries and assertions, analyzing concepts, identifying arguments, engaging in independent problem-solving, and assessing inquiries. They also showed an understanding of a problem's verbal description and the ability to provide a numerical response by utilizing mathematical connections. Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 420 Table 5 Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Table 7 Test of Difference Between the Levels of Critical Thinking Skills in Terms of Reasoning on the Pre-Test and Post-Test Mean Scores of the Two Groups Differentiated Instructions Pretest Posttest Mean Difference 95% Confidence Interval of the Difference t df Sig (2- tailed) M SD M SD Lower Upper Flexible Grouping 7.73 2.35 18.10 3.05 10.37 9.48 11.25 23.95 29 0.000 Curriculum Compacting 7.53 2.11 18.03 3.41 10.50 8.90 12.10 13.41 29 0.000 Legend: Significant at 0.05 Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skill ents 2.1. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 421 Table 7 Legend: Significant at 0.05 Table 7 presents the results of the test of the difference between the levels of critical thinking skills, specifically reasoning skills, as indicated by the mean scores on the pre-test and post-test of the two groups. as revealed by the paired t-test data, demonstrates a statistically significant rise in the scores is significant (p < 0.05). 420 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com This implies that the students performed better in the post-test. Their level of reasoning skills has significantly improved from apprentice to proficient level. Findings also indicated that both flexible grouping and curriculum compacting are effective methods of differentiated instruction in enhancing the reasoning skills of the students. Using two differentiated instructions, the students from apprentice level of reasoning skills improved because they learned according to their abilities and interests. The student’s ability from remembering level improved to reasoning skills where he/she can analyze, synthesize, and present their own opinions about the topic. This implies that the students performed better in the post-test. Their level of reasoning skills has significantly improved from apprentice to proficient level. Findings also indicated that both flexible grouping and curriculum compacting are effective methods of differentiated instruction in enhancing the reasoning skills of the students. Using two differentiated instructions, the students from apprentice level of reasoning skills improved because they learned according to their abilities and interests. The student’s ability from remembering level improved to reasoning skills where he/she can analyze, synthesize, and present their own opinions about the topic. p p p According to Ghanizada’s (2020) research, engaging in argumentative discussions can enhance students' critical thinking abilities, specifically in the domains of analysis, evaluation, and inference. The research underscores the significance of teacher preparation and pedagogical planning in constructing persuasive dialogues that foster analytical reasoning. The author suggests that innovative approaches to instruction necessitate innovative approaches to assessment. This approach will allow students to respond utilizing their analytical reasoning abilities while being presented with rigorous tasks. 2.2. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students Table 8 Table 8 Test of Difference Between the Levels of Critical Thinking Skills in Terms of Problem-Solving on the Pre-Test and Post-Test Mean Scores of the Two Groups Differentiated Instructions Pretest Posttest Mean Difference 95% CID t df Sig(2- tailed) M SD M SD Lower Upper Flexible Grouping 4.20 1.90 7.43 1.87 3.23 2.26 4.18 6.97 29 0.000 Curriculum Compacting 3.37 1.27 7.67 2.25 4.30 3.48 5.12 10.78 29 0.000 Legend: Significant at 0.05 Table 8 Test of Difference Between the Levels of Critical Thinking Skills in Terms of Problem-Solving on the Pre-Tes the Two Groups Table 8 displays the results of the statistical analysis conducted to determine the variance in critical thinking skills between the two groups, as measured by their pre-test and post-test mean scores in problem- solving. The results of the paired t-test demonstrate that the observed data exhibits statistical significance in the increase in scores for both groups (p < 0.05). The data suggests that the problem-solving abilities of the participants have experienced a noteworthy enhancement, progressing from a novice level to a skilled level. The results indicate that the implementation of differentiated instruction is a successful approach to fostering the problem-solving abilities of students. Kamaruddin and Hazni (2010) recognized the importance of developing problem-solving ability among individuals. Thus, they stressed that this skill be developed especially among students since it may improve their learning ability. Moreover, it will equip them in facing and solving problems as human beings. 421 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com The findings of the current undertaking are consistent with what some literature said about differentiated instructions. According to Morin (n.d.), The implementation of flexible grouping has been identified as a potent and efficacious strategy for enhancing the process of learning. This system facilitates the provision of appropriate assistance to students in a timely and effective manner. Incorporating flexible grouping into the classroom regimen allows for the normative occurrence of one or multiple students collaborating with the teacher on a designated task. Students who encounter difficulties do not experience a sense of isolation or social disapproval. Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 422 Table 8 Moreover, according to Gentry (2016), students who received curriculum compacting scored remarkably higher on performance and increasing achievement assessments. Winebrenner, S. (2016). In her book "Teaching Gifted Kids in Today's Classroom: Strategies and Techniques Every Teacher Can Use" (2016), she explains that curriculum compacting involves identifying and eliminating content that students have already mastered so that they can focus on new and challenging material. This strategy allows advanced learners to move at a faster pace and explore topics in greater depth, while still being challenged and engaged in their learning. Winebrenner also emphasizes the importance of differentiating instruction for all learners, including gifted and advanced learners, to meet their individual needs and help them reach their full potential. 2.3. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students 422 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Conclusion The present study aimed to investigate the efficacy of differentiated instruction in Mathematics in enhancing the critical thinking abilities of Grade 7 students. The study employed a quasi-experimental research design, specifically utilizing the non-equivalent control group pretest-posttest design. The groups consisted of students who were deemed to be intact by the public high school in the Province of Quezon. The selection of these groups was carried out using the cluster random sampling technique. Based on the findings, before exposing students to differentiated instructions, their level of reasoning skill was low for both flexible grouping and curriculum compacting. However, their levels of problem-solving skills were low and very low, for flexible grouping and curriculum compacting, respectively. After using differentiated instruction, the students’ level of critical thinking skills from both groups was at a high level. Findings also showed that the mean pretest and mean posttest scores of each group of respondents were significantly different for both measures of critical thinking skills. This suggested that the two forms of differentiated instruction that were employed in this study are effective in developing students’ critical thinking skills, especially in discussing basic concepts of Geometry. The study suggests that the implementation of differentiated instruction techniques such as flexible grouping and curriculum compacting can lead to a significant improvement in students' critical thinking skills. Therefore, it is recommended that Geometry teachers consider utilizing these techniques in their instruction. Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 Recommendations Based on the findings of the study, it is recommended that differentiated instruction in the form of flexible grouping and curriculum compacting be implemented as they have been shown to have a significant positive impact on student's critical thinking abilities. 1. Educators are advised to incorporate flexible grouping and curriculum compacting as means of differentiated instruction, not only in Mathematics but also in other academic domains. 1. Educators are advised to incorporate flexible grouping and curriculum compacting as means of differentiated instruction, not only in Mathematics but also in other academic domains. 2. Educators can explore additional facets of differentiated instruction to effectively execute this approach. 2. Educators can explore additional facets of differentiated instruction to effectively execute this approach. 3. Teachers are encouraged to be more innovative and used a tiered approach to identify if the critical thinking skills are more likely to be flexible grouping. 3. Teachers are encouraged to be more innovative and used a tiered approach to identify if the critical thinking skills are more likely to be flexible grouping. 4. Future researchers are encouraged to replicate this study to three or more groups and use differentiated instruction other than flexible grouping and curriculum compacting. 4. Future researchers are encouraged to replicate this study to three or more groups and use differentiated instruction other than flexible grouping and curriculum compacting. 2.3. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students 2.3. Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students fferentiated Instruction in Mathematics: Its Effect on the Level of Critical T G d 7 S d 2.3. Differentiated Instruction in Mathematics: Its Effect on the Level of Cr of Grade 7 Students Table 9 Test of Difference Between the Levels of Critical Thinking Skills on the Post-Test Mean Scores of the Two Groups Critical Thinking Skills Flexible Grouping Curriculum Compacting Mean Difference 95% CID t df Sig(2- tailed) M SD M SD Lower Upper Reasoning 18.10 3.05 18.03 3.41 0.07 -1.61 1.74 0.080 58 0.937 Problem Solving 7.43 1.87 7.67 2.25 0.24 -1.30 0.84 0.44 58 0.664 Legend: Significant at 0.05 ence Between the Levels of Critical Thinking Skills on the Post-Test Mean Scores of the Two Groups Fl ibl C i l Table 9 presents the results of the test conducted to determine the disparity in critical thinking skills levels between the two groups based on their post-test mean scores. The results of an independent t-test indicate that there is no statistically significant difference in the post-test scores for critical thinking skills, as the p-value is greater than 0.05. The findings suggest that there is no significant difference in critical thinking abilities, specifically in reasoning and problem-solving skills, between students who underwent flexible grouping and those who underwent curriculum compacting. The result suggests that the diligent implementation of differentiated instruction by teachers in mathematics education may lead to an enhancement of students' reasoning and problem-solving abilities, commonly referred to as critical thinking skills. Consequently, this improvement in critical thinking skills may positively impact students' performance in mathematics. The findings are in line with the literature that discusses differentiated instruction. According to (Odicta's 2017) research, students belonging to a differentiated group exhibited elevated levels of critical thinking abilities and achieved notable success in mathematics. Incorporating student interests and preferences in the learning process can enhance relevance and autonomy. (Tomlinson 2017) conducted a study that supports the notion that utilizing diverse instructional strategies to cater to students' readiness, interests, and learning profiles is crucial. She posits that differentiated instruction can enhance student motivation and engagement. Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 Proceedings of the Asia Pacific Conference on Multidisciplinary Research (APCMR), 30 – 31 July 2022 Dailo, A. E., Dailo, R. R., Differentiated Instruction in Mathematics: Its Effect on the Level of Critical Thinking Skills of Grade 7 Students, pp. 413 – 425 References https://www.ascd.org/books/the-differentiated-classroom-responding-to-the-needs-of-all- learners-2nd-edition?variant=108029 424 Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Asia Pacific Journal of Advanced Education and Technology Asia Pacific Journal of Advanced Education and Technology Special Issue, September 2022 / P- ISSN 2815 – 245X / E – ISSN 2815 – 2468 / www.apjaet.com Tomlinson, C. A. (2017). How to differentiate instruction in academically diverse classrooms (3rd ed.). ASCD. https://files.ascd.org/staticfiles/ascd/pdf/siteASCD/publications/books/HowtoDifferentiateInstructioni nAcademicallyDiverseClassrooms-3rdEd.pdf Tomlinson, C. A., Brighton, C., Hertberg, H., Callahan, C. M., Moon, T. R., Brimijoin, K., Conover, L. 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https://openalex.org/W4206196306	https://zenodo.org/record/2565323/files/14.pdf	Latin	null	An overview-analytical document of the international experience of building smart-city	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	6,094	Bashynska I.O., Kaplun A.S. Bashynska I.O., Kaplun A.S. An overview-analytical document of the international experience of building smart-city DOI: 10.5281/zenodo.2565323 UDC Classification: 338.47.656 JEL Classіfіcatіon: R20, F29, I31, O18, O39 Analysis of recent researches and publications Different The analysis of literary sources on the development of smart-cities has shown the lively interest of foreign and Ukrainian scientists. Among them there are works of such actors as: Girman A.P., Kiselev V.R., Zhukovich I.A., Diskina A.A., Goncharenko O.S., Cohen B., Kusuma Adi Ahmad, Lukito Edinugroho, Ahmad Junade, Vidavan, Gil Bettis, Christ J. Kassandras, Carlo Alberto Nucci Amerel Autur, Thierry Burger-Helmchen. Ключові слова: smart-місто,інновації, стратегія, smart- економіка, smart-управління, smart-суспільство, smart- мобільність, smart-довкілля, smart-життя The research of the phenomenon of smart-city was engaged and continues to engage a lot of scientists, however, they mainly focus on narrow aspects, or consider countries and cities locally, so it is necessary to generalize the work of scientists. Bashynska I.O., Kaplun A.S. An overview-analytical document of the international experience of building smart-city. Review article. The article reveals the concept and developed the model of an ideal smart city, analyzed the currently known international rankings, which assess different aspects of the development of smart cities. The advantages and features of ratings as a tool for presentation of the results for evaluation and comparison of smart cities. To construct the ideal model of a smart city, we will use the classical six smart characteristics: economy, governance, society, mobility, environment and life. The success of "smart city" directly depends on effective planning and control over its implementation, which determines the success of the overall restructuring program. The flexible approach to developing a restructuring program is a smart city roadmap, which, according to our proposal, consists of six stages. The aim of the article is to study the international and domestic experience of building smart-city and develop a smart-city roadmap. AN OVERVIEW-ANALYTICAL DOCUMENT OF THE INTERNATIONAL EXPERIENCE OF BUILDING SMART-CITY ОГЛЯДОВО-АНАЛІТИЧНИЙ ДОКУМЕНТ МІЖНАРОДНОГО ДОСВІДУ ПОБУДОВИ SMART-CITY Iryna O. Bashynska, PhD in Economics, Associate Professor Odessa National PolytechnicUniversity, Odessa, Ukraine Scopus Author ID: 57148584000 ORCID: 0000-0002-4143-9277 Email: i.bash@ukr.net Anna S. Kaplun Odessa National PolytechnicUniversity, Odessa, Ukraine ORCID: 0000-0001-7397-6889 Email: kaplunanea@mail.ru Recieved 06.08.2018 Anna S. Kaplun Odessa National PolytechnicUniversity, Odessa, Ukraine ORCID: 0000-0001-7397-6889 Email: kaplunanea@mail.ru Recieved 06.08.2018 Anna S. Kaplun Odessa National PolytechnicUniversity, Odessa, Ukraine ORCID: 0000-0001-7397-6889 Email: kaplunanea@mail.ru Recieved 06.08.2018 M ore than half of the world’s population lives in cities. Cities, metropolitan areas give rise to problems of new species. Difficulties in waste management, resource requirements, air pollution, health hazards, road traffic jams and aging urban infrastructures are among the most important technical, physical and material problems. The urgency of the article is the development of "smart cities", by introducing innovations and improving the efficiency of functioning of the main spheres of life of cities. M Башинська І.О., Каплун А.С. Оглядово-аналітичний документ міжнародного досвіду побудови smart-city. Оглядова стаття. Башинська І.О., Каплун А.С. Оглядово-аналітичний документ міжнародного досвіду побудови smart-city. Оглядова стаття. У статті розкрито поняття та розроблена модель ідеального smart-міста, проаналізовано відомі в даний час міжнародні рейтинги, які дозволяють оцінити різні аспекти розвитку smart- міст. Розглянуто переваги та особливості рейтингів як інструменту подання результатів для оцінювання і порівняння smart-міст. Для побудови ідеальної моделі розумного міста, ми будемо використовувати класичні шість смарт характеристик: економіка, управління, суспільство, мобільність, середовище та життя. Успіх "smart city" безпосередньо залежить від ефективного планування та контролю за його виконанням, що визначає успіх загальної програми реструктуризації. Гнучким підходом до розробки програми реструктуризації є розумна міська дорожня карта, яка, відповідно до нашої пропозиції, складається з шести етапів. Table 1. Definition of abbreviation "Smart" Thus, the development of "smart-city" is a strategic process that requires the novelty of approach, planning, exploitation, networking and management of urban enterprises. In this way, the concept embodied transforms the city’s space into a source of resources for all people who are visiting this city. Thus, the meaning of the term "smart city" indicates the search and implementation of intelligent solutions that help the city to qualitatively and quantitatively increase its own productivity. generation of new ideas with their subsequent implementation in the production of products and services with increased value added and intelligent component [1]; — creation and maintenance of productive business environment for increasing the degree of innovation, optimal use of nature, energy and material saving technologies, etc. The technology of the intelligent network allows solving problems for power companies. SmartGrid is a smart meter, dynamic electrical network management, demand regulation, security enhancement and cost savings. This network will allow you to monitor the energy consumption of each home device and maintain certain rules of behaviour at peak hours and at other times of the day, increase safety and reduce costs. "Smart City" is a new strategy aimed at providing assistance to cities in which there are a number of problems, such as: high population density; increase in the number of vehicles; inefficient use of resources, climate change, deterioration of energy infrastructure. To solve the above-mentioned problems, we propose to develop a model of the ideal "smart-city" (fig. 1), which identifies different sectors of the concept through the implementation of projects for the development of modern urban infrastructure. "Smart-management" means participation in public life, development of public and social services, transparent management, lack of corruption, crowd- sourcing. This is a management that is completely inseparable from the use of intellectual and collective technologies, including crowd-sourcing, knowledge management, BigData [3]. Due to the use of such technologies, in particular, a very effective mechanism for the dissemination of the competencies that are currently required, which will always be highly sought after by the "smart-society", is being formed. To build the perfect smart city model, we will use the classic six characteristics: smart economy, smart- management, smart-society, smart-mobility, smart- environment and smart-life. The need for the transition of "smart-economy" is due to global disturbances in the development and conduct of economic activity of individual cities and the country as a whole. The main part Each year, the number of studies on the impact of innovation on the development of countries, regions and cities is increasing. In many foreign and domestic studies, a methodology for evaluating and comparing the innovative development of the territory is considered. Keywords: smart city, innovation, strategy, smart economy, smart governance, smart society, smart mobility, smart environment, smart living Economic, scientific and cultural potential is concentrated in cities, and therefore they play an 14 №5(39), 2018 ISSN:2226-2172 ECONOMICS: time realities important role in the economic, political and public life of each individual country and of humanity as a whole. The economic and technological changes caused by globalization and integration processes lead to the need for sustainable development and increasing the competitiveness of cities. This problem is related to the need to improve the city in areas such as economy, culture, housing and social conditions, and the environment [1]. One of the ways of the decision was the creation of new cities – "Smart City". y The abbreviation "Smart" in English translates to "intelligent" and stands for: "Specific" – concreteness; "Measurable" – dimensionality; "Attainable" – reach; "Realistic" – realism, "Timebound" – limited time (tab. 1). Table 1. Definition of abbreviation "Smart" S Specific Concreteness the more precise a specific task is described, the higher the probability of obtaining the expected result would be. M Measurable Dimensionality the result must be measurable. To do this, we determine the criteria by which the process will be conducted. A Аttainable Reach this parameter shows the need to discard the knowingly unattainable results, since obtaining the desired result should be at least potentially. R Realistic Realism only this aspect involves not only assessing their presence, but correlating their number with the number necessary to achieve the goal. T Timebound Limited time The purpose setting for this phase involves setting a clear timetable in which it should be implemented. Source: compiled by the authors on the materials [2] Table 1. Definition of abbreviation "Smart" Table 1. Definition of abbreviation "Smart" The main goals of the "smart economy" can be attributed to: The main component of the "smart-society" is the population, so the degree of development of the city directly depends on the level of education of its inhabitants. Therefore, one of the main factors is the level of human qualification. Due to constantly changing technologies, the ability of a person to learn throughout his life was not an important factor in being an active participant in the process of modernization. In principle, factors such as flexibility and agility, creativity, cosmopolitanism, and citizens’ openness to change, as well as their readiness for —recovery of economic growth after the global crisis; —adaptation to changes in the ratio of demand and supply of labour at the expense of high levels of general and vocational education, the activity and mobility of the workforce and changes in regulated working hours; — the formation of an innovation system, the introduction of advanced technologies, smart- networks in all sectors of the economy to provide quick access to information and knowledge, the 15 An overview-analytical document of the international experience of building smart-city Bashynska I.O., Kaplun A.S. Bashynska I.O., Kaplun A.S. active participation in public life, follow from the latter characteristic. technologies (ICTs), sustainable, innovative and safe transport system. Mobility is a key component of the Smart City, and first of all, it is worth paying attention to the inclusion of ICT infrastructure in this characteristic. "Smart-mobility" is a solution to many problems associated with the traffic jams, fares, access to Internet resources in all parts of the city. The main goals of "smart-mobility" are: the ability to easily move around the city, openness and accessibility of the city at the national and international levels, accessibility to information and communication "Smart-environment" – preserves natural resources, as well as orientation towards sustainable development, combating pollution, sustainable resource management. Fig. 1. Table 1. Definition of abbreviation "Smart" Characteristics of "Smart-city" Source: compiled by the authors on the materials [3-14] Smart-city Smart-management Smart-society • Entrepreneurship • Productivity • Flexibility of the labour market • Place in the international system • Competitiveness • Integratedproduction technologies • Innovative approach • SmartGrid (Intelligent Network) • Participation in public life • Social services • Transparent governance • Lack of corruption • Crowd-sourcing • Intelligent government • Level of qualification • Continuing education • Ethnic diversity • Openness • Development of the capital and potential Smart-economy Smart-mobility Smart-environment Smart-Life • Urban transport development • The development of national and international transport • Availability of IT infrastructure • Stability of the transport system • Smart accounting • "Cloud" technology • Environmental conditions • Air quality • Ecological consciousness • Sustainable resource management • Cultural institutions • Medical condition • Security of residence • Quality of housing • Education institutions • Tourist attraction • Economic welfare • Urban transport development • The development of national and international transport • Availability of IT infrastructure • Stability of the transport system • Smart accounting • "Cloud" technology • Environmental conditions • Air quality • Ecological consciousness • Sustainable resource management Fig. 1. Characteristics of "Smart-city" Source: compiled by the authors on the materials [3-14] g y compiled by the authors on the materials [3-14] "Smart Life" is a project devoted to the development of society. The objectives of this project are: the development of cultural space and the presence of cultural objects; developed medicine, high level of individual safety of people, quality of places of living and level of development of system of housing and operating organizations; availability and level of educational institutions; attractiveness of the city for tourists; social cohesion of citizens. about 98% of homes. The system uses excess heat from power generation and redirects it to the heating of buildings. This system allows you to save up to 1400 USD, up to 203 thousand tons of oil annually. Similarly, a large wind power plant built in the city, located at sea 3.5 meters from the coast, produces 4% of the energy for the city. In the second place ranking is the capital of the Netherlands – Amsterdam. B. Table 1. Definition of abbreviation "Smart" For example, every stop in the city is equipped with an electronic screen, which shows the movement of all buses in the district. Traffic is tracked through thousands of webcams, and any one of them can be connected via a mobile phone.  Boston. The first city in the world where the computer system StreetBump was launched: on the wheels of cars installed special sensors that collect information about each pit on the road and send data to the server utilities. Only one year of work of the program has led to the fact that the number of pits on the roads of Boston was reduced almost twice: from 7.8 to 4 thousand. Karamay. The first "smart city" in Xinjiang Uighur Autonomous Region of China. Feature of the Chinese concept of SmartHub: emphasis on combining all devices into a single information network and continuous informing of citizens about all aspects of city life. For example, every stop in the city is equipped with an electronic screen, which shows the movement of all buses in the district. Traffic is tracked through thousands of webcams, and any one of them can be connected via a mobile phone.  San Francisco. In the city there are over 60 computer programs that fully control the operation of public transport and housing and communal services. Also in San Francisco the implementation program of electric vehicles with built-in autopilot is being implemented, for which the densest worldwide network of public charging stations for such cars is already built in the city. It is assumed that in three years in San Francisco, no gasoline car will remain.  San Francisco. In the city there are over 60 computer programs that fully control the operation of public transport and housing and communal services. Also in San Francisco the implementation program of electric vehicles with built-in autopilot is being implemented, for which the densest worldwide network of public charging stations for such cars is already built in the city. It is assumed that in three years in San Francisco, no gasoline car will remain.  Songdo. The first "smart city" in South Korea, which is decided to completely and completely build from scratch. Now Songdo is practically ready for settlement: the city is calculated for 65 thousand people who will be selected by special tests for the level of IQ. Table 1. Definition of abbreviation "Smart" Today in the world about 50 projects of building "smart cities" from the zero cycle are being implemented, but Songdo will become a model for everyone.  Songdo. The first "smart city" in South Korea, which is decided to completely and completely build from scratch. Now Songdo is practically ready for settlement: the city is calculated for 65 thousand people who will be selected by special tests for the level of IQ. Today in the world about 50 projects of building "smart cities" from the zero cycle are being implemented, but Songdo will become a model for everyone.  Tokyo. The Japanese capital initially had a single system of total computer control of the road network, and now became a training ground for the implementation of the "smart city" project, which involves the leading corporations of the y In Ukraine, there are several cities claiming the status of "smart city", including Kiev, Dnipropetrovsk, Kharkiv, Vinnytsia and Lviv. Table 2. The ranking of ten "smart cities" of Europe in 2016 Table 2. The ranking of ten "smart cities" of Europe in 2016 Table 2. The ranking of ten "smart cities" of Europe in 2016 City’s name Ratings of city assessment criteria ∑ Smart- economy Smart- management Smart- society Smart- mobility Smart- environment Smart- life Copenhagen 7 7 1 4 1 2 22 Amsterdam 6 9 2 1 4 4 26 Vienna 4 3 7 6 6 1 27 Barcelona 5 5 5 3 5 6 29 Paris 3 8 4 2 7 9 33 Stockholm 8 4 6 7 2 7 34 London 1 2 3 10 10 10 36 Hamburg 8 10 8 5 3 3 37 Berlin 2 6 10 8 8 5 39 Helsinki 10 1 9 9 9 8 46 Source: compiled by the authors on the materials [4-13] Table 2. Table 1. Definition of abbreviation "Smart" Cohen noted that Amsterdam "is the only city in the world that has more problems than pedestrian and cycling traffic congestion than the congestion of vehicles." Almost 67% of Amsterdam’s residents travel by bike or on foot. Approximately eight out of ten Europeans live in cities, spend 70% of their energy, and the traffic congestion they create in Europe costs roughly 100 billion euro pro year. Therefore, metropolitan areas are constantly looking for ways to become more efficient, resourceful and friendly to the population. Tab. 2 shows the ranking of ten cities that are close to the model of the perfect smart city. In the third place in the rating is Vienna. The relatively small Austrian capital is one of the greenest cities in Europe and provides a high standard of living. More than 15% of the city’s energy resources come from renewable sources, including the use of Europe’s largest biomass power plant. The first place in the 10th "Smart Cities" rating is the prominent business capital of Denmark, Copenhagen. The city has a state-owned enterprise that controls all initiatives within the Smart City program. Also, the company is developing a strategy to reduce CO2 emissions, so that by 2025 become the first carbon-neutral capital. The city has a well- developed centralized heating system, which serves In addition to pan-European ratings, some EU countries carry out national ratings of Smart cities, namely:  New York. One of the first smart cities in which city computer systems began to develop. To date, 16 №5(39), 2018 ISSN:2226-2172 ECONOMICS: time realities it has two "smart" systems at once – Cisco and City24/7, thanks to which each resident has free internet access. It was in New York that a computer program to combat street crime was developed, and now this metropolis has become the safest city in the United States. country. In addition to general computerization, this concept involves gardening the city to create conditions for the harmonious coexistence of man with the environment. For example, in one of the districts of Tokyo adopted a program to replace all pillars by 2015 with live trees.  Karamay. The first "smart city" in Xinjiang Uighur Autonomous Region of China. Feature of the Chinese concept of SmartHub: emphasis on combining all devices into a single information network and continuous informing of citizens about all aspects of city life. Bashynska I.O., Kaplun A.S. An overview-analytical document of the international experience of building smart-city Vinnitsa operates an automated system for managing utilities on a Microsoft cloud platform; an internal portal is created for the city council in which the working documentation is kept, the database of executive bodies is kept, and a number of electronic services are available to the residents of the city: online registration in kindergarten, the possibility to pay utility services via the Internet, search of the necessary public transport route; about 70 cameras were installed on the streets to ensure the safety of the citizens on the streets, information from them goes to the servers of the city council and the police center [14]. applications from leaders of these areas within the city. At the fourth stage, the focus shifts away from the decisions taken on the basis of the creation of primary smart city services and applications, the training of smart data (intelligent data) and feedback from users, and, through feedback, the definition of business changes and technologies, development of architecture for a longer term and strategic decisions. Finally, when smart city services are catching up with the critical mass, the program is being upgraded to a wider range of smart city projects, completing the transition to a complete strategic IT platform becomes necessary to ensure future maneuverability as changes and business priorities to customers. In Lviv, on the basis of the cloud platform, Microsoft Azure operates a portal of open data, which makes information on the tourism industry automated and displayed in a visual fashion; a tender for the purchase of a single electronic ticket system was announced; operates a traffic control center and dispatching a vehicle to which 150 traffic lights are connected. Some of them are intelligent traffic lights; a smart tourist route with QR-tags will soon be created in the city center [14]. A traditional operating model for the city was founded around functionally-oriented service providers that work as non-interconnected vertical silos, which are often not built around the needs of users. Smart cities should develop new operating models, stimulate innovation and cooperation between these vertical silos. Bashynska I.O., Kaplun A.S. Decision making and service delivery have been built into a vertically integrated supply chain within cities – implementation silos that are built around a function that does not belong to the needs of users: Cities and their transformation in smart cities are not in the empty space, but in the context of the global transformation of the world economy into the digital economy. And practically everything that is happening in this or that country is related to the "capital" of the digital economy – a smart city. The success of the "smart-city" project depends directly on effective planning and control of its implementation. Therefore, the planning and control of the implementation of the change in the structure of the project are important components that determine the success of the restructuring program as a whole. A smart approach to developing a restructuring program is the smart-city roadmap. A roadmap is a set of methodologies and tools for preparing real organizational documents for each particular city with its diverse peculiarities. Work begins with the problem statement and moves to finding the optimal solution. — a single citizen or business must deal with each silos individually: establishing a connection for themselves, and not receiving seamless and connected services that are appropriate to their needs; — data and information has typically been locked in these silos, limiting the potential for collaboration and innovation throughout the city, and limiting the speed of city-wide change. Table 1. Definition of abbreviation "Smart" The ranking of ten smart cities of Europe in 2016 City’s name Ratings of city assessment criteria ∑ Smart- economy Smart- management Smart- society Smart- mobility Smart- environment Smart- life Copenhagen 7 7 1 4 1 2 22 Amsterdam 6 9 2 1 4 4 26 Vienna 4 3 7 6 6 1 27 Barcelona 5 5 5 3 5 6 29 Paris 3 8 4 2 7 9 33 Stockholm 8 4 6 7 2 7 34 London 1 2 3 10 10 10 36 Hamburg 8 10 8 5 3 3 37 Berlin 2 6 10 8 8 5 39 Helsinki 10 1 9 9 9 8 46 Source: compiled by the authors on the materials [4-13] is planned to introduce a single electronic ticket in the subway, trams, trolleybuses and buses. Estimated purchase amount – 300 million UAH. These funds are expected to cost 1.500 on-board computers for buses, trams and trolleybuses, as well as 5500 electronic validators. The KyivPass hotel project is already presented – the only ticket for a tourist that will allow you to visit interesting places and move around the city on the road. Kyiv is the first city in Ukraine, integrating smart technologies and building digital infrastructure. To date, there are 4100 special cameras HikVision and Huawei. It is planned to cover all entrances and exits from the city (61), entrances/exits to the subway, 120 traffic lights. Plan for the end of the year – another 4.000 cameras. Also, cameras will have the main places of accumulation of people on the level of city districts, the main transport interchanges. Similarly, it is planned to install Wi-Fi in tourist places and medical institutions. The private company will cover the main locations: Bessarabskaya Square, Khreshchatyk, Independence Square, European and Mikhailovskaya Square, Andriyivsky Uzviz, Postal and Kontraktova Square, Leo Tolstoy Square and Shevchenko Park [14]. Until the beginning of 2018, it In Dnipropetrovsk’s plans are to install smart city- wide stops, equipped with heaters, charging for mobile phones, Wi-Fi, information boards, fixtures; introduce a single electronic ticket to the metro and tram; to establish a system of reasonable traffic lights, which will allow remotely control the traffic of streets [14]. 17 Список літератури: 1. Гірман А.П. Кисельов В.Р. Перспективи організації праці в контексті розвитку smart-економіки / А.П. Гірман, В.Р. Кисельов // Східна Європа: економіка, бізнес та управління – 3 (08), 2017. – С. 137-141 1. Гірман А.П. Кисельов В.Р. Перспективи організації праці в контексті розвитку smart-економіки / А.П. Гірман, В.Р. Кисельов // Східна Європа: економіка, бізнес та управління – 3 (08), 2017. – С. 137-141 2. Vito Albino, Umberto Berardi & Rosa Maria Dangelico (2015).Smart Cities: Definitions, Dimensions, Performance, and Initiatives, Journal of Urban Technology, 22:1, 3-21, DOI: 10.1080/10630732.2014.942092 2. Vito Albino, Umberto Berardi & Rosa Maria Dangelico (2015).Smart Cities: Definitions, Dimensions, Performance, and Initiatives, Journal of Urban Technology, 22:1, 3-21, DOI: 10.1080/10630732.2014.942092 3. Bashynska I. RiskManagement.Lecturecourse: textbook // Bashynska I., Filyppova S. – Харків: вид-во «Діса плюс», 2017. – 101 с. 3. Bashynska I. RiskManagement.Lecturecourse: textbook // Bashynska I., Filyppova S. – Харків: вид-во «Діса плюс», 2017. – 101 с. 4. Жукович І. А. Міжнародний досвід оцінювання та порівняння smart-міст / І. А. Жукович // Статистика України – 2015 р. – С. 16-22 – [Електронний ресурс]. – Режим доступу: 194.44.12.92:8080/jspui/bitstream/123456789/1780/1...vych I. A._16-22.pdf/ 4. Жукович І. А. Міжнародний досвід оцінювання та порівняння smart-міст / І. А. Жукович // Статистика України – 2015 р. – С. 16-22 – [Електронний ресурс]. – Режим доступу: 194.44.12.92:8080/jspui/bitstream/123456789/1780/1...vych I. A._16-22.pdf/ j p y p 5. European smart cities version 3.0 (2014) – [Електронний ресурс]. – Режим доступу: http://www.smart-citie.eu/?cid=2& ver=3. j p y p 5. European smart cities version 3.0 (2014) – [Електронний ресурс]. – Режим доступу: http://www.smart-citie.eu/?cid=2& ver=3. p 6. Bashynska I. Smart-innovationsintheurbanpassengertransportinthecontextof smart-cityconcept / Економіка. Фінанси. Право. – Київ, 2017. – №11/2, С. 4-6 p 6. Bashynska I. Smart-innovationsintheurbanpassengertransportinthecontextof smart-cityconcept / Економіка. Фінанси. Право. – Київ, 2017. – №11/2, С. 4-6 7. Дискіна А.А. Пропозиції щодо впровадження системи смарт-обліку в міському пасажирському транспорті з використанням новітніх інформаційних технологій [Електронний ресурс] / А.А.Дискіна // Економіка та суспільство. – 2017. – № 13. – Режим доступу до журналу: http://economyandsociety.in.ua. 7. Дискіна А.А. Пропозиції щодо впровадження системи смарт-обліку в міському пасажирському транспорті з використанням новітніх інформаційних технологій [Електронний ресурс] / А.А.Дискіна // Економіка та суспільство. – 2017. – № 13. – Режим доступу до журналу: http://economyandsociety.in.ua. p y y 8. Goncharenko O.S. Ecological and economic efects from implementation of the model of solidarityuse of motortransport in the context of smart city creation [Електронний ресурс] / O.S. Goncharenko, O.V. Hrynevych // Економіка: реалії часу. Науковий журнал. – 2017. – № 6(34). Abstract Introduction. More than half of the world's population lives in cities. Cities, metropolitan areas give rise to problems of new species. Difficulties in waste management, resource requirements, air pollution, health hazards, road traffic jams and aging urban infrastructures are among the most important technical, physical and material problems. The urgency of the article is the development of "smart cities", by introducing innovations and improving the efficiency of functioning of the main spheres of life of cities. p g y g p Purpose. The article is to study the international and domestic experience of building smart-city and develop a smart-city roadmap. y p Research results. "Smart City" is a new strategy aimed at providing assistance to cities in which there are a number of problems, such as: high population density; increase in the number of vehicles; inefficient use of resources, climate change, deterioration of energy infrastructure. To solve the above problems, we propose to develop a model of the ideal "smart-city". To build the perfect smart city model, we will use the classic six characteristics: smart economy, smart-management, smart-society, smart-mobility, smart-environment and smart-life. Cities and their transformation in smart cities are not in the empty space, but in the context of the global transformation of the world economy into the digital economy. And practically everything that is happening in this or that country is related to the "capital" of the digital economy - a smart city. The success of the "smart-city" project depends directly on effective planning and control of its implementation. Therefore, planning and controlling the implementation of the change in the structure of the project are important components that determine the success of the overall restructuring program. A flexible approach to developing a restructuring program is the smart-city roadmap, which, according to our proposal, consists of six stages. Conclusion. There is currently no single methodology for evaluating and comparing smart cities. Researchers working in this area use rating agencies as the tool for presenting survey results. The construction of "Smart City" is a complex of measures aimed at modernizing all areas of city management. Each city, as a rule, has its own tasks. Some solve security-related problems, others will improve the functioning of municipal transport, while others reduce energy losses in homes. None of these tasks will be addressed effectively if the city does not have a basic infrastructure. Conclusions The conducted research makes it possible to draw some conclusions. There is currently no single methodology for evaluating and comparing smart cities. Researchers working in this area use rating agencies as the tool for presenting survey results. The construction of "Smart City" is a complex of measures aimed at modernizing all areas of city management. Each city, as a rule, has its own tasks. Some solve security-related problems, others will improve the functioning of municipal transport, while others reduce energy losses in homes. None of these tasks will be addressed effectively if the city does not have a basic infrastructure. The first stage is the preparation and planning, it is necessary to develop, taking into account the road map of the city, in order to ensure that the business case is fully formulated and that all key stakeholders in it are foreseen. At the second stage of development, the emphasis is on creating a maximum impulse for implementing a roadmap at a minimal risk of implementation. This means, in particular, targeting: quick victories to demonstrate progress and early benefits, with low expense, in order to strengthen faith and trust through the interested city parties; the introduction of a roadmap in the governance structures and processes that will be needed to create an environment for informing all future investments. With efficient organization, smart cities have a lot of advantages:  Development with a long-term perspective. Implementation of IT-technologies allows the municipal authorities to optimize the cost of resources: water, fuel, electricity and even household waste.  The city. Implementation of IT technologies contributes to the formation of a well-informed and healthy society. At the third stage, some of the most significant investments are starting to emerge, for example, an open Data Platform for supporting small and medium businesses and an innovative community with city data for the creation of community services. Also, here comes the first wave of intelligent services and  Expansion. Intelligent cities not only save municipal funds and improve the quality of life of the inhabitants. 18 ISSN:2226-2172 №5(39), 2018 ECONOMICS: time realities References: References: 1. Girman, A.P. & Kyselov, V.R. (2017). Prospects for the organization of work in the context of the development of smart economy. Eastern Europe: Economics, Business and Management, 3 (08), 137- 141 [in Ukrainian]. [ ] 2. 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Світові досягнення у сфері смарт-інновацій / А. А. Дискіна // Економічний журнал Одеськогополітехнічногоуніверситету. – 2018. – № 1 (3). – С. 19-31. – Режим доступу до журн.: http://economics.opu.ua/ejopu/2018/No1/19.pdf. DOI: 10.5281/zenodo.1303994. 12. Дискіна А.А. Світові досягнення у сфері смарт-інновацій / А. А. Дискіна // Економічний журнал Одеськогополітехнічногоуніверситету. – 2018. – № 1 (3). – С. 19-31. – Режим доступу до журн.: http://economics.opu.ua/ejopu/2018/No1/19.pdf. DOI: 10.5281/zenodo.1303994. p p j p p 13. Amel Attour, Thierry Burger-Helmchen (2015) Guest editorial., Journal of Strategy and Management 8 (3). http://dx.doi.org/10.1108/JSMA-06-2015-0046 [in English] p p j p p 13. Amel Attour, Thierry Burger-Helmchen (2015) Guest editorial., Journal of Strategy and Management 8 (3). http://dx.doi.org/10.1108/JSMA-06-2015-0046 [in English] 14. Українські Smart City. [Електронний ресурс]. – Режим доступу:https://sites.google.com/site/ocakivsmartcity/10-klucovih-oznak-rozumnogo-mista/realizacia- proektu-v-5-naselenih-punktah-ukraieni. 14. Українські Smart City. [Електронний ресурс]. – Режим доступу:https://sites.google.com/site/ocakivsmartcity/10-klucovih-oznak-rozumnogo-mista/realizacia- proektu-v-5-naselenih-punktah-ukraieni. Reference a Journal Article: Bashynska I .O. An overview-analytical document of the international experience of building smart-city / I. O. Bashynska, A. S. Kaplun // Economics: time realities. Scientific journal. – 2018. – № 5 (39). – Р. 14-20. – Retrieved from https://economics.opu.ua/files/archive/2018/No5/14.pdf. DOI: 10.5281/zenodo.2565323. This is an open access journal and all published articles are licensed under a Creative Commons «Attribution» 4.0. Посилання на статтю: Bashynska I .O. An overview-analytical document of the international experience of building smart-city / I. O. Bashynska, A. S. Kaplun // Економіка: реалії часу. Науковий журнал. – 2018. – № 5 (39). – C. 14-20. – Режим доступу до журналу: https://economics.opu.ua/files/archive/2018/No5/14.pdf. DOI: 10.5281/zenodo.2565323. References: Retrieved from http://economics.opu.ua/ejopu/2018/No1/19.pdf [in English]. g 13. Amel Attour, Thierry Burger-Helmchen (2015). Guest editorial., Journal of Strategy and Management 8 (3). Retrieved from http://dx.doi.org/10.1108/JSMA-06-2015-0046 [in English]. 14. Ukrainian Smart City. Retrieved from https://sites.google.com/site/ocakivsmartcity/10-klucovih-oznak- rozumnogo-mista/realizacia-proektu-v-5-naselenih-punktah-ukraieni [in Ukrainian]. Посилання на статтю: Bashynska I .O. An overview-analytical document of the international experience of building smart-city / I. O. Bashynska, A. S. Kaplun // Економіка: реалії часу. Науковий журнал. – 2018. – № 5 (39). – C. 14-20. – Режим доступу до журналу: https://economics.opu.ua/files/archive/2018/No5/14.pdf. DOI: 10.5281/zenodo.2565323. Reference a Journal Article: Bashynska I .O. An overview-analytical document of the international experience of building smart-city / I. O. Bashynska, A. S. Kaplun // Economics: time realities. Scientific journal. – 2018. – № 5 (39). – Р. 14-20. – Retrieved from https://economics.opu.ua/files/archive/2018/No5/14.pdf. DOI: 10.5281/zenodo.2565323. 20
https://openalex.org/W2895835269	https://aip.scitation.org/doi/pdf/10.1063/1.5053100	English	null	Photoluminescence, infrared, and Raman spectra of co-doped Si nanoparticles from first principles	Journal of chemical physics online/The Journal of chemical physics/Journal of chemical physics	2,018	cc-by	11,988	RESEARCH ARTICLE \| OCTOBER 18 2018 Photoluminescence, infrared, and Raman spectra of co- doped Si nanoparticles from first principles RESEARCH ARTICLE \| OCTOBER 18 2018 J. Chem. Phys. 149, 154702 (2018) https://doi.org/10.1063/1.5053100 Articles You May Be Interested In Zinc cysteine active sites of metalloproteins: A density functional theory and x-ray absorption fine structure study J Ch Ph (M h 2008) J. Chem. Phys. (March 2008) Mechanisms for pressure-induced crystal-crystal transition, amorphization, and devitrification of SnI4 J. Chem. Phys. (October 2015) Silicon 1s near edge X-ray absorption fine structure spectroscopy of functionalized silicon nanocrystals J. Chem. Phys. (October 2016) I. INTRODUCTION falls beyond the bandgap of bulk Si.2 Overall, heavily co- doped Si NPs are very promising for the realization of novel optoelectronics applications. Few nanometers-sized Si nanoparticles (NPs) have been in the focus of nanoscience as their size-dependent bandgap enables the tuning of their electrical and optical properties.1 Due to the abundance and environmental friendliness of ele- mental Si, and the accumulation of knowledge from the semi- conductor industry, Si NP-based solutions are a very attrac- tive and promising platform over similar but less developed technologies based on compound semiconductors. An advan- tageous feature of Si NPs is that they can be doped by various dopants. In particular, Si NPs can be co-doped by boron and phosphorus beyond the bulk solubility of dopant atoms.2–10 Theseheavilyco-dopedSiNPscanbefabricatedbytheanneal- ing borophosposilicate glass (BPSG) matrix.7 The size of the nanoparticles can be controlled by the annealing temperature in the range of 1–14 nm.7 The resulting nanoparticles can be extracted from the BPSG matrix by HF etching. According to scanning transmission electron microscopy (STEM)2,11 and X-ray diffraction measurements,12 the extracted co-doped Si NPs have highly crystalline core encapsulated by an amor- phous layer.11 The concentration of B and P in the nanocrys- tals determines their dispersibility in solution.13 The resulting co-doped Si NPs exhibit numerous advantageous properties compared to undoped Si NPs: they are stable, resistant against oxidation, and dispersible in aqueous environments.13,14 Sim- ilarly to undoped Si NPs, they exhibit size-dependent pho- toluminescence (PL) in the visible and near-infrared (NIR), but the absorption edge and PL peak are generally red- shifted compared to their non-doped counterparts at identical NP diameters.10 For larger diameters, the luminescence peak p pp Electron energy loss spectroscopy (EELS) can be uti- lized13 to conﬁrm the presence of dopants in Si NPs. The dopant concentration of heavily doped Si NPs can be estimated by inductively coupled plasma atomic emission spectrome- try (ICP-AES) measurements,13,15 or atom probe tomography (APT),16 which also offers some insight into the distribution of dopants within the Si NPs. According to ICP-AES mea- surements,10,13 the concentration of dopants can be controlled by the boron and phosphorous concentration of the BPSG matrix. Photoluminescence, infrared, and Raman spectra of co-doped Si nanoparticles from ﬁrst principles B´alint Somogyi,1,a) Emilie Bruyer,1 and Adam Gali1,2,b) 1Wigner Research Centre for Physics, Institute for Solid State Physics and Optics, Hungarian Academy of Sciences, P.O. Box 49, H-1525 Budapest, Hungary 2Department of Atomic Physics, Budapest University of Technology and Economics, Budafoki ´ut 8., H-1111 Budapest, Hungary (Received 21 August 2018; accepted 3 October 2018; published online 18 October 2018) (Received 21 August 2018; accepted 3 October 2018; published online 18 October 2018) Co-doped silicon nanoparticles (NPs) are promising for the realization of novel biological and opto- electronic applications. Despite the scientiﬁc and technological interest, the structure of heavily co-doped Si NPs is still not very well understood. By means of ﬁrst principles simulations, various spectroscopic quantities can be computed and compared to the corresponding experimental data. In this paper, we demonstrate that the calculated infrared spectra, photoluminescence spectra, and Raman spectracanprovidevaluableinsightsintotheatomisticstructureofco-dopedSiNPs.©2018Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5053100 THE JOURNAL OF CHEMICAL PHYSICS 149, 154702 (2018) a)somogyi.balint@wigner.mta.hu b)gali.adam@wigner.mta.hu I. INTRODUCTION The B and P concentration of extracted Si NPs gener- ally exceeds the dopant concentration of the BPSG matrix,13 and the co-doped Si NPs typically contain signiﬁcantly more B than P.10 It was also established that the annealing temperature does not have a signiﬁcant impact on the dopant concen- tration.10 APT analysis of co-doped Si NPs can offer addi- tional insight about the dopant distribution within the Si NPs. According to a recent study16 of Si NPs embedded in BPSG, P atoms are more likely to be found in the inner region of the Si NPs whereas B accumulates near the NP-BPSG boundary. By contrast with ICP-AES results, the P concentration was found to exceed the concentration of B. While this contradic- tion might be resolved by undetected B counts,17 it is not clear how these ﬁndings translate to the case of freestanding Si NPs. In addition to the radial distribution of dopants, the same study also established the abundance of B–P and P–P clusters within the Si NPs. X-ray photoelectron spectroscopy (XPS) measure- ments provide further information about the chemical conﬁg- urations of the dopant atoms.8,18 In particular, the presence of non-oxidized B and P atoms in the proximity of the surface was established.8 24 October 2024 04:41:14 a)somogyi.balint@wigner.mta.hu b)gali.adam@wigner.mta.hu 0021-9606/2018/149(15)/154702/12 Infrared (IR) spectroscopy and Raman spectroscopy mea- surements can shed further light on the atomistic structure © Author(s) 2018 149, 154702-1 149, 154702-1 0021-9606/2018/149(15)/154702/12 Somogyi, Bruyer, and Gali J. Chem. Phys. 149, 154702 (2018) 154702-2 154702-2 of Si NPs. Raman spectroscopy offers insights about the lower energy vibrations (300-800 cm−1),8 where IR spec- troscopy is ineffective. IR spectroscopy measurements can complement these results by the identiﬁcation of oxygen and hydrogen related vibrational bands in the 700-3500 cm−1 region.11,13,19 Limited amount of structural information can be also deducted by analyzing the photoluminescence (PL) spec- tra. Traditionally spectroscopy measurements are performed on large ensembles of nanoparticles; thus, the obtained PL spectra are featureless2–4,6,10,13 due to the slightly varying zero-phonon line (ZPL) of the different NPs. However, it is also possible to measure the PL signal of individual Si NPs,20 where the shape of the PL spectra shows some char- acteristic features at low temperatures. Comparing the PL spectrum of individual nanoparticles at different temperatures or single NP measurements with ensemble measurements, one can partition the broadening of PL spectra into contri- butions from zero-point vibrations, thermal broadening, and inhomogeneous broadening. I. INTRODUCTION Finally, the emission line shape can be obtained as as I(ω) = nω3 3ϵ0πc3ℏ\|µ\|2 ∞ −∞ G(t)eiωt−γ(t)dt, (9) (9) where n is the refractive index of the media (that is unity for simulation of Si NPs in the gas phase), c is the speed of light, where n is the refractive index of the media (that is unity for simulation of Si NPs in the gas phase), c is the speed of light, µ is the transition dipole moment between the ground and excited electronic states, and γ(t) can be chosen appropri- ately to describe the inhomogeneous broadening of the spectral lines. The choices of γ(t) = γ1\|t\| and γ(t) = γ2t2 results in Lorentzian and Gaussian-broadening, respectively.24,25 The description of inhomogeneous broadening is outside of the scope of this work; thus, we present our results with small, artiﬁcially chosen Gaussian-broadening (2-5 meV), in order to improve the clarity of our PL plots. I. INTRODUCTION Sk = ωk q2 k 2ℏ, (3) (3) where Sk is the partial Huang-Rhys factor of the kth mode, ωk is the energy of the kth mode, and qk is given by qk = X i √mi(Rex i −Rgr i )uk;i, (4) (4) (4) where Rgr and Rex are the atomic coordinates in the ground and excited state, m is the atomic mass of the correspond- ing atom, and uk is the kth normal mode. We neglect the effects of the Duschinsky rotation, and thus ωk and uk cor- respond to equilibrium coordinates of the electronic ground state. From the partial Huang-Rhys factors, the emission spectra can be obtained (see Ref. 23) by constructing the so-called electron-phonon spectral function S(ω) = X k Skδ(ω −ωk) (5) (5) Despite the signiﬁcant experimental and computational effort, the structure of heavily co-doped Si NPs is still not understood at the atomic level to a satisfactory degree. and two auxiliary functions deﬁned as f ±(t) = ∞ −∞ S(ω) ( nω nω + 1 ) e±iωtdω, (6) (6) Raman, infrared, and photoluminescence spectra can also be calculated utilizing ﬁrst-principles computational methods. Calculating the vibrational density of states (VDOS) can be considered as routine and offers valuable information about the vibrational energies of molecules or bulk material. The IR spectrum of a system can be obtained with little additional cost by calculating the effective Born charges, and the result offers insight not only about the characteristic vibrational ener- gies but the relative intensities of the IR bands. Our goal was to predict the experimental signals of certain structural features of co-doped Si NPs in order to improve the current understanding of their atomic structure. After the vibrational signature of clusters of few atoms is identiﬁed, the results can be compared to the experiments and the gained insight can be utilized to build accurate atomic models for co-doped Si NPs. where nω is the Bose-Einstein statistics nω(T) = [exp(ℏω/kbT) −1]−1. At T = 0 K, Eq. (6) simpliﬁes to f (t) = ∞ −∞ S(ω)e−iωtdω. (7) (7) With these auxiliary functions, the generating function G(t) can be constructed as G(t) = eiE0t/ℏ−(f +(t)+f −(t)−f +(0)−f −(0)), (8) (8) where E0 is the energy of the no-phonon transition or the ZPL energy. II. THEORY Before calculating the vibrational energies and normal modes, the structure was further relaxed to <0.001 eV/Å in order to achieve accurate vibrational energies. The dynamic matrix was constructed utilizing the ﬁnite-differences method with 0.015 Å displacements. Born effective charges were calculated utilizing density functional perturbation the- ory (DFPT).34 The vertical excitation energies and transition dipole moments were obtained from linear-response TDDFT calculations with the PBE035 hybrid-functional in the adia- batic TDDFT kernel. Excited state geometries were calcu- lated by forcing non-Aufbau occupation numbers according to the ﬁrst optical excitation. We utilized the plane-wave Vienna Ab initio Simulation Package (VASP)36,37 code for the ground state and excited state structure relaxation, the determination of vibrational energies and normal modes, and the computation of effective Born charges. VASP uti- lizes a plane-wave basis set where the core electrons were taken into account within the projector augmented wave (PAW)38 framework. Because of the periodic boundary con- ditions, at least a 10 Å vacuum was ensured between the nanoparticles and their mirror images, in order to mini- mize the spurious nanoparticle-nanoparticle interaction. The TDDFT calculations were carried out utilizing the Turbomole code,39,40 with a gaussian-type double-zeta basis set with additional polarization (DZP) orbitals and the valence elec- trons were taken into account utilizing electric core potentials (ECPs). where ωI is frequency of the incident light, ωj, ωk are the frequencies of the corresponding vibrational modes, and ∆k is the dimensionless displacement between the ground and excited electronic state minima that is related to the par- tial Huang-Rhys factors deﬁned in Eqs. (3) and (4) by Si = ∆2 i /2. The approximation in Eq. (10) holds when ωI ≫ωj, ωk. Generally, the Savin formula is expected to give a rea- sonable estimation for the resonance Raman spectra, when (i) short time dynamics govern the Raman process,24,29 (ii) the Duschinsky rotation can be neglected, (iii) the ground state vibrational frequencies are good approximates for the excited state ones, (iv) and the overtones in the Raman spectra can be neglected (true if Si ≪1). Similar com- putational approach to obtain the resonant Raman spectra was already utilized in the context of CdSe nanocrystals30 and silicon nanocrystals covered with an amorphous surface layer.31 In addition, we comment on the excitation wavelengths in our simulation and its implications when compared to exper- imental data. II. THEORY To calculate the resonance Raman spectra, the ground and excited state geometriesand the vibrational proper- tiesaredetermined fromﬁrstprinciplescalculations.Indensity functional theory (DFT) based calculations, the relaxation of the geometry in excited electronic states is very challenging. Time-dependent density functional theory (TDDFT) can be utilized to obtain excited state properties of molecules,32 but the structural relaxation of nanocrystals with ∼1000 electrons is extremely time consuming in the computations. Instead, we utilized the ∆SCF method where excited state electronic structure was mimicked by forcing non-Aufbau electron occu- pations. While this method is justiﬁed for geometry relaxation in the lowest lying excited state, it fails for higher excita- tion. This is not an issue for the calculation of PL spec- tra, where the excited system quickly relaxes to the lowest lying excited electronic state (Kasha’s rule) via vibrational relaxation; thus, the lowest lying electronic excited state par- ticipates in the emission. By contrast, the higher excited electronic states should be considered to calculate the reso- nance Raman spectra, unless the laser frequency coincides with the ZPL. This is not the case in a realistic experimen- tal scenario, where the laser frequency is usually signiﬁcantly higher than the ZPL. This may result in a larger error in the calculated Raman spectra than that in the calculated PL spectra. We found that our ﬁrst principles methodology did not always give accurate results for the vibrational energies. Most notably, our calculations underestimate the energy of the trans- verse optical (TO) phonon peak of silicon which is a prevalent signal in Raman studies of Si NPs. On the other hand, we foundthatthecalculatedfrequencyofthesubstitutionalBatom (618 cm−1) is in very good agreement with the experimental value41 for B11, 620 cm−1. As there are two common iso- topes of B, one would have to consider both 10B and 11B in the simulation process. For clusters containing multiple B atoms, the number of possible combinations scales exponen- tially with the number of B atoms drastically increasing the complexity of the calculations. To simplify our task, we per- formedthecalculationsusingtheabundance-weightedaverage standard atomic weight for B (10.811), so the local mode energies of 10B and 11B atoms are expected to slightly dif- fer from our results. II. THEORY g p ), p g , µ is the transition dipole moment between the ground and excited electronic states, and γ(t) can be chosen appropri- ately to describe the inhomogeneous broadening of the spectral lines. The choices of γ(t) = γ1\|t\| and γ(t) = γ2t2 results in Lorentzian and Gaussian-broadening, respectively.24,25 The description of inhomogeneous broadening is outside of the scope of this work; thus, we present our results with small, artiﬁcially chosen Gaussian-broadening (2-5 meV), in order to improve the clarity of our PL plots. The IR spectra are calculated from the effective Born charge tensors and the normal modes as Iν IR = X µ X l Z∗ l,µuν l 2 , (1) (1) where l is the atomic index, uν is the νth normal mode and the Born effective charges are deﬁned as Z∗ l,µλ = − ∂2Etot ∂ul,µ∂Eλ ul,µ=0,Eλ=0 , (2) In this approximation, one has to obtain the following quantities to calculate the PL emission spectra: the ground and excited state geometries, the vibrational energies and normal modes, and the ZPL energy. This method has been proven to reliable for the description of the PL spectrum emitted by color centers in solids.26,27 (2) whereEtot isthetotalenergyofthesystemand Eλ istheelectric ﬁeld applied in the λ = x, y, z direction. We calculate the PL spectra within the Franck-Condon approximation using the generating function method devel- oped by Kubo and Toyozawa21 and Lax.22 The emission spectra are calculated using the so-called partial Huang-Rhys factors deﬁned by Resonant Raman spectra was calculated using the Savin28,29 formula, where the relative intensities of fundamen- tal Raman intensities of totally symmetric vibrational modes are given by 154702-3 Somogyi, Bruyer, and Gali J. Chem. Phys. 149, 154702 (2018) J. Chem. Phys. 149, 154702 (2018) Ij,10 Ik,10 = (ωI −ωj)3(ℏωj∆j)2 (ωI −ωk)3(ℏωk∆k)2 ≈ ω2 j Sj ω2 kSk , Raman spectra can be decomposed as these spectral quantities are calculated as a sum over the normal modes. (10) We utilized density functional theory (DFT) and time- dependent density functional theory (TDDFT) to study selected nanostructure models. We calculated the relaxation of structures and the vibrational energies by the use of the semi-local Perdew-Burke-Ernzerhof (PBE)33 exchange- correlation functional. During the structural optimization, the atoms were relaxed until all of the forces fell below 0.02 eV/Å. II. THEORY The ratio of the vibrational frequencies of 10B and 11B in bulk Si is ≈1.03941 and the 1.03-1.05 ratios were found to hold for isotopically pure complexes of 10B/11B too.42 As a result, we expect that our results slightly overestimate the vibrational frequencies of 11B (<1% error), while underestimate the vibrational frequencies of 10B (3%-4% error). In our work, we employed various projection techniques, to gain more insight about correlations between structural and vibrational properties of co-doped Si NPs. In this approach, we examine which normal modes are localized on a given set of atoms (e.g., a given atomic species, or atoms at the NP surface) by calculating Pi,S = X α,k∈S \|ui,k,α\|2, (11) (11) where α = {x, y, z}, k is the atomic index, S is the chosen set of atoms, and Pi,S is the weight of the ith normal mode projected to the atoms of set S. After the calculation of the projected weights for each normal mode, the VDOS, IR spectra, and J. Chem. Phys. 149, 154702 (2018) 154702-4 Somogyi, Bruyer, and Gali A. Properties of undoped Si NPs First, we investigate the (projected) vibrational density of states of a model Si NP that is terminated by H atoms but does not contain B or P dopants. Figure 1 shows the pro- jected vibrational density of states (PVDOS) of a 1.6 nm Si NP model. Utilizing projection to the Si, (Si)–H, (Si)–H2, we can analyze the nature of IR bands. Apart from the charac- teristic stretching modes around 2100-2200 cm−1, the rolling, rocking, and twisting modes of the (Si)–H, (Si)–H2 moieties can interfere with the local modes of dopant-containing struc- tures. The measurements indicate13,19 that H is either absent or present in diminishing quantities in co-doped Si NPs; however, we terminate the surface of our Si NP models with hydrogen in order to reduce the number of electrons entering our sim- ulations. Fortunately, the calculation of PVDOS allows us to draw conclusions about the contributions of the local modes of speciﬁc atomic species. This is especially important for the Si–H/Si–H2 related IR band around 620 cm−1 which happens to coincide with the important Raman signal of substitutional B. Even after subtracting the H contribution from the VDOS, FIG. 2. The calculated photoluminescence emission spectra and resonant Raman spectra of hydrogen-terminated, pristine Si nanocrystals with diam- eters in the region of 1.1-2.8 nm. For the PL spectra, black and red curves correspond to 0 K and 300 K, respectively. For the Raman spectra, black curves correspond to the full spectra, while green curves correspond to the projected Raman spectra where the outermost layer of Si atoms and H atoms is excluded from the projection. 24 October 2024 04:41:14 FIG. 1. Vibrational density of states of a hydrogen terminated, 1.6 nm Si NP built up from 147 Si atoms. The VDOS was decomposed to show the contribution of surface (Si–H and Si–H2) and bulk (Si–Si) vibrations. FIG. 2. The calculated photoluminescence emission spectra and resonant Raman spectra of hydrogen-terminated, pristine Si nanocrystals with diam- eters in the region of 1.1-2.8 nm. For the PL spectra, black and red curves correspond to 0 K and 300 K, respectively. For the Raman spectra, black curves correspond to the full spectra, while green curves correspond to the projected Raman spectra where the outermost layer of Si atoms and H atoms is excluded from the projection. FIG. 1. III. RESULTS AND DISCUSSION a small peak remains signaling that the Si atoms of the out- ermost layer vibrate differently compared to the Si atoms of the bulklike region. In the experiments, this IR peak is absent for undoped Si NPs, which can be explained by the smaller surface/volume ratio of experimental NP ensembles (usually 3-7 nm), the oxidation of the NP surface, or the Raman inactivity of such modes. First, we discuss our results regarding the the character- istic local mode energies of certain boron and phosphorous related clusters that are expected to occur in co-doped Si nanoparticles. During this study, our goal was not to cre- ate realistic atomistic models of co-doped nanocrystals, but to study the vibrational properties of small, few atom-sized atomic structures embedded in the Si NP, e. g., substitu- tional dopants, and small dopant clusters. This approach can be justiﬁed by the observation that the global structure of the Si NPs have only small effect on the vibrational ener- gies belonging to the covalent bonds of the investigated clusters. We also calculated the PL spectra of H-terminated undoped Si NPs with diameteres in the range 1.1–2.8 nm and plotted the results in Fig. 2. The quantum conﬁnement effect (QCE)43 is signiﬁcant in this NP size range, as the PL peak shifts from 3.2 eV to 2.3 eV with increasing NP size. In addi- tion, the vibration induced broadening is also size-dependent. For the smaller NPs, the calculated emission spectra show no distinguishable ZPL due to the strong vibration induced broadening. As the size of the NPs increases, the Debye- Waller factor increases, and the PL is dominated by the ZPL at low temperatures, in good agreement with the experimen- tal data.20 We also calculated the Raman spectra, where the size-dependence of the transverse optical (TO) phonon peak of the Si crystal can be identiﬁed in accordance with earlier experimental44–47 and theoretical48–50 results that also con- ﬁrmed that the shift of the TO-peak towards lower energies While it is impossible to investigate every possible structure that might possess a unique vibrational footprint in the experimental characterization of co-doped Si NPs, we believe that our extensive study is able to assist and guide the experiments in the interpretation of the observed spectra. B. Eﬀect of boron doping in the vibrational properties of Si NPs The most essential vibrational signal related to the point defects is the characteristic local modes of substitutional phos- phorous and boron atoms. From an experimental viewpoint, their signiﬁcance is different as the phosphorous related sig- nal appears as a shoulder on the lower energy side of the silicon TO phonon peak;7 thus, it is rather difﬁcult to deter- mine its intensity. While the TO related peak could be easily subtracted from the total spectra for bulk Si, the picture is more complicated for nanocrystals as the shape of the TO peak becomes size and structural dependent. On the other hand, the local modes of substitutional boron produces a well distinguishable peak in the Raman spectra7,8 for which the intensity and shape seem to vary with doping concentration.11 Because of this fact, we focused on energy and intensity of the substitutional boron as a promising indicator of atomic-scale properties. FIG. 3. Boron-projected vibrational density of states of a 2.4 nm sized Si NPs containing a single substitutional B atom with different boron positions indicatedinthecorrespondingpictograms.TheBatomsarenegativelycharged and four-fold coordinated except from the uppermost model, where the B atom sitsatthesurfaceoftheSiNP,isthreefoldcoordinated,andhasaneutralcharge state. The numbers above the spectra correspond to the B atoms’ distance from the outermost layer of Si atoms. First, we investigated the case of a single, isolated boron atom inside a hydrogen terminated Si NP. Our goal was to determine whether the position of B atoms within the NP affects the energy of its local modes. As experiments indicate that co-doped Si NPs are compensated, we gave an overall negative charge to the B containing Si NP. Figure 3 shows the boron-projected vibrational density of states for a single B containing, 2.4 nm sized Si NP. It is clear that the position of the B atom has a signiﬁcant impact on the vibrational energy of the B–Si related normal mode. Si NPs indicates that they are compensated; thus, the excess B must be inactive. This is seemingly consistent over a relatively broad range of B and P concentration, raising the questions about the nature of inactive B. Neutral, triply coordinated B atoms are the simplest B defects that do not introduce acceptor states. In contrast to substitutional B in bulk Si (where neutral B always introduces an acceptor state), it is possible to introduce B atoms to the outermost Si layer of Si NPs that are inactive. A. Properties of undoped Si NPs Vibrational density of states of a hydrogen terminated, 1.6 nm Si NP built up from 147 Si atoms. The VDOS was decomposed to show the contribution of surface (Si–H and Si–H2) and bulk (Si–Si) vibrations. Somogyi, Bruyer, and Gali J. Chem. Phys. 149, 154702 (2018) 154702-5 154702-5 FIG. 3. Boron-projected vibrational density of states of a 2.4 nm sized Si NPs containing a single substitutional B atom with different boron positions indicatedinthecorrespondingpictograms.TheBatomsarenegativelycharged and four-fold coordinated except from the uppermost model, where the B atom sitsatthesurfaceoftheSiNP,isthreefoldcoordinated,andhasaneutralcharge state. The numbers above the spectra correspond to the B atoms’ distance from the outermost layer of Si atoms. is caused by QCE. It is also notable that the shape of the cal- culated Raman spectra also changes with the diameter. For the smallest Si NP models, there is a signiﬁcant feature in the 600-700 cm−1 region that gradually disappears with increasing NP size. This indicates that the 600-700 cm−1 feature is related to the surface of the NPs, which we conﬁrmed by calculating a projected Raman spectra where the projection excluded the outermost Si layer and the H atoms. B. Eﬀect of boron doping in the vibrational properties of Si NPs Because of the signiﬁcance of triply coordinated B atoms of the outermost atomic layer, we also investigated clusters of triply coordi- nated B atoms on the NP surface (see Fig. 4). These surface clusters are formed by second neighbor three fold coordinated boron atoms. Introducing a cluster of 3 three fold coordinated B atoms to the surface, the B related vibrational modes fall between 710 and 760 cm−1. This IR band becomes even wider as one of the facets of the Si NPs is fully terminated by three fold coordinated B atoms, while the shape of the TO peak remains preserved. Finally, the model where surface Si atoms are randomly substituted by three-fold coordinated B atoms at 20% total B concentration was considered. This model exhibits an even broader, featureless band falling in the 640-860 cm−1 region. For B atoms far from the surface, the B related local modes are degenerate and fall around 615 cm−1. By contrast, the B atom in the proximity of the surface exhibits signiﬁ- cantly higher vibrational energies with broken degeneracy in the region of 630-660 cm−1. The most extreme examples are the B atoms introduced to the outermost and the second outer- most atomic layer of the Si NP. For the second outermost layer, the B related peak is split to a 765 cm−1 mode and several lower energy modes in the 600-700 cm−1 region. For the out- ermost Si layer, the B atom is only three-fold coordinated and is in a neutral charge state. This atomic conﬁguration results in a vibrational energy of 735 cm−1 which is much higher than local modes of the four-fold coordinated B residing in the bulk region of Si NPs. We ﬁnd that the transition point between bulk-like and surface-like local modes of substitutional B falls between 3.8 and 5.4 Å distance measured from the outermost Si layer. The case of B proximate to the NP surface is particu- larly important, as experiments indicate a strongly B enriched, amorphous layer encapsulating the crystalline NP core.11,51 In addition, the concentration of B in B/P co-doped Si NPs is usually higher, while the electronic structure of the co-doped C. Vibrational properties of boron and phosphorus co-doped Si NPs Next we turn our attention towards B–P complexes within Si NPs. As the local modes of P are expected to be masked J. Chem. Phys. 149, 154702 (2018) J. Chem. Phys. 149, 154702 (2018) 154702-6 154702-6 154702-6 Somogyi, Bruyer, and Gali Somogyi, Bruyer, and Gali FIG. 4. Silicon- and boron-projected vibrational density of states of Si NP models containing three fold coordinated substitutional B atoms at their sur- face. The hexagonal shape of the Si NP silhouettes emphasizes the faceted nature of the model Si NPs. From the top to the bottom: fully B terminated Si NP, Si NP with one of its facets terminated by B atoms, Si NP with 3 B atom on one of its facets, and ﬁnally a Si NP with a single B atom on its surface. The B-projected VDOS is scaled up to 25% concentration equivalent for sake of clarity. still feasible to occur due to the high concentration of B dopants). p Figure 5 shows the B-, P-, and Si-projected vibrational density of states of various Si NP models containing B and P atoms. Figures 5(a) and 5(b) show the case of individual B and P, respectively, whereas (c) and (d) are the case of separated and ﬁrst neighbor B–P pair, respectively. For the single B and P dopants, a total charge of −1 and +1 was applied, respectively, in order to compensate the extra hole/electron. Figure 5(g) shows the projected vibrational density of states of a Si NP model containing a ring-like B–P cluster, while (h) shows the PVDOS of a Si NP model for which the innermost 26 atoms were substituted by a boron phospide core. Finally, Fig. 5(i) is the PVDOS of a model containing 3 fourfold coordinated P and B atoms and 3 threefold coordinated B atoms at the NP surface. The B- and P-projected VDOS of the BP pair approx- imates the B- and P-projected VDOS of the single-dopant model, with minor differences. For the nearest neighbor pair, a B-P mode appears around 560 cm−1, well distinguishable from the 620 cm−1 B–Si modes. For the distant B–P pair, this softer mode does not occur, but the B related modes are shifted towards higher energies due to the proximity of the NP surface. C. Vibrational properties of boron and phosphorus co-doped Si NPs The P-projected VDOS remains scattered in a broad energy range, rendering the P-related bump on the low wavenumber side of the TO-peak less valuable for quantitative analysis. Even the introduction of a simple B–B–P cluster induces a rel- atively complex B-related signal in the vibrational spectrum [see Fig. 5(e)]. FIG. 4. Silicon- and boron-projected vibrational density of states of Si NP models containing three fold coordinated substitutional B atoms at their sur- face. The hexagonal shape of the Si NP silhouettes emphasizes the faceted nature of the model Si NPs. From the top to the bottom: fully B terminated Si NP, Si NP with one of its facets terminated by B atoms, Si NP with 3 B atom on one of its facets, and ﬁnally a Si NP with a single B atom on its surface. The B-projected VDOS is scaled up to 25% concentration equivalent for sake of clarity. FIG. 4. Silicon- and boron-projected vibrational density of states of Si NP models containing three fold coordinated substitutional B atoms at their sur- face. The hexagonal shape of the Si NP silhouettes emphasizes the faceted nature of the model Si NPs. From the top to the bottom: fully B terminated Si NP, Si NP with one of its facets terminated by B atoms, Si NP with 3 B atom on one of its facets, and ﬁnally a Si NP with a single B atom on its surface. The B-projected VDOS is scaled up to 25% concentration equivalent for sake of clarity. by the TO peak of the Si NP, our main focus is to deter- mine how the proximity of P atoms affects the local modes of B. Our previous work indicated that the formation of neighboring B–P pairs is energetically preferred; thus, the vibrational energies of small B–P cluster are of interest. The concentration of B always exceeds the concentration of P in experimental samples of co-doped Si NPs, and thus we only considered B–P cluster (which are favored energetically) and B–B clusters (which are energetically not favored, but by the TO peak of the Si NP, our main focus is to deter- mine how the proximity of P atoms affects the local modes of B. Our previous work indicated that the formation of neighboring B–P pairs is energetically preferred; thus, the vibrational energies of small B–P cluster are of interest. C. Vibrational properties of boron and phosphorus co-doped Si NPs The concentration of B always exceeds the concentration of P in experimental samples of co-doped Si NPs, and thus we only considered B–P cluster (which are favored energetically) and B–B clusters (which are energetically not favored, but By calculating the B-projected VDOS and investigating the normal modes that contribute the most to the B-projected VDOS, we can identify the following vibrations: the stretching mode of the B–B bond around 850 cm−1, the rocking mode of the B–B bond around 700 cm−1, and the mode that is local- ized on the B atoms, qualitatively similar to the normal mode of a single fold coordinated B atom. We also ﬁnd a mode FIG. 5. Silicon-, boron-, and phosphorus-projected vibrational density of states with a pictogram representing the dopant conﬁgurations of the corresponding co-doped Si NPs models. Green and orange dots with black rectangles represent four fold-coordinated substitutional B and P atoms, while green dots with black triangles represent threefold coordinated B atoms at the surface of the Si NPs. Adjacent dots represent covalent bonds between the relevant dopant atoms whose remaining bonds are connected to adjacent silicon atoms. For the sake of clarity, we scaled up the B- and P-projected VDOS (to an equivalent intensity of 25 B/P per Si NP) meaning that the PVDOS intensities between different subplots are not comparable. FIG. 5. Silicon-, boron-, and phosphorus-projected vibrational density of states with a pictogram representing the dopant conﬁgurations of the corresponding co-doped Si NPs models. Green and orange dots with black rectangles represent four fold-coordinated substitutional B and P atoms, while green dots with black triangles represent threefold coordinated B atoms at the surface of the Si NPs. Adjacent dots represent covalent bonds between the relevant dopant atoms whose remaining bonds are connected to adjacent silicon atoms. For the sake of clarity, we scaled up the B- and P-projected VDOS (to an equivalent intensity of 25 B/P per Si NP) meaning that the PVDOS intensities between different subplots are not comparable. Somogyi, Bruyer, and Gali J. Chem. Phys. 149, 154702 (2018) 154702-7 FIG. 6. Calculated IR spectra for two different sized Si NP models, with and without a B–P pair. The contribution of the surface (H atoms and the outermost Si layer) is subtracted in order to relate the calculated spectra to the experimental data. C. Vibrational properties of boron and phosphorus co-doped Si NPs Black, green, and orange curves correspond to the contributions of the Si atoms (without the outermost layer), B atoms, and P atoms, respectively. Note that the B- and P-related contributions to the IR spectra were scaled up for the sake of clarity. that is localized on the B, P, and neighbor Si atoms around 633 cm−1 and two modes that are localized in the B atoms and the proximate Si atoms at 606 and 607 cm−1. At 588 cm−1, there is a mode that is localized on the B–P bond, some neigh- bor Si atoms, and also hybridized with the bending modes of the Si–H bonds at the surface. The 526 cm−1 mode is localized on the B, P dopants and the proximate Si atoms. The broad IR band below 440 cm−1 can be attributed to B and P related vibrations hybridized by rather delocalized, almost bulk-like vibrations of the host Si NP. In the case of the B–P–B cluster [see Fig. 5(f)], the B–B related local mode is missing, and the most distinct IR band (720-750 cm−1) is related to the vibration of the threefold coordinated B atom bonding to a P atom and two Si atoms. The inner B atom contributed to the VDOS in the 580-670 cm−1 region where the vibrations are localized in the ﬁrst neighbor atoms. FIG. 6. Calculated IR spectra for two different sized Si NP models, with and without a B–P pair. The contribution of the surface (H atoms and the outermost Si layer) is subtracted in order to relate the calculated spectra to the experimental data. Black, green, and orange curves correspond to the contributions of the Si atoms (without the outermost layer), B atoms, and P atoms, respectively. Note that the B- and P-related contributions to the IR spectra were scaled up for the sake of clarity. The 6-atomic ring-like BP cluster [Fig. 5(g)] introduces a rather broad IR band between 520 and 690 cm−1. The intro- duction of a small cubic boron cluster in the center of the Si NP results in a similar broad band from 530 to 730 cm−1. C. Vibrational properties of boron and phosphorus co-doped Si NPs Inter- estingly, the characteristic TO and longitudinal optical (LO) lines (799 and 829 cm−1, respectively52) of cubic boron phos- phide are absent from the PVDOS, indicating that the stress induced by the mismatch between the lattice constant of boron phosphide (4.54 Å) and Si (5.43 Å) changes the vibrational properties of the BP core. The lack of >800 cm−1 frequencies indicate negative pressure on the BP core53 which is consistent with differences between the lattice constants. A surprising result is that the introduction of the BP pair signiﬁcantly changes the overall intensity and shape of the IR spectra in addition to the appearance of the B-related peak. In particular, the IR signal of the bulk-like region in the <500 cm−1 of the Si NP becomes an order of magnitude smaller. Furthermore, the contribution from the B-related local modes are negligible for the case of adjacent B–P pair. The case of the distant B–P pair is different, as the donor-acceptor pair (DAP) acts as a huge dipole, resulting in a signiﬁ- cant contribution from the B-related local modes to the IR spectra. Finally, we investigate the case of the surface-bound BP cluster [Fig. 5(i)] that is expected to occur due to the concentra- tion of dopants on the NP surface. In this cluster, the presence of B in both of its three- and fourfold coordinated conﬁgura- tions results in complex vibrational signal in the 520-800 cm−1 region. The calculated PVDOS is somewhat similar to the B–B–P cluster, with the notable exception of the missing 850cm−1 asthismodeldoesnothaveB–Bbonds.Theseresults indicate that even relatively simple B–P complexes can induce rather complex IR signals in Si NPs. The calculated IR spectra for the other model structures are plotted in Fig. 7. The IR signal of the threefold coordinated B-atomisratherweak[noticethe10×factorinFig.7(a)].There is also signiﬁcant difference between the IR intensities of P–B–B and B–P–B complexes. For the B–P–P cluster [Fig. 7(b)], the IR peaks related to the stretching and rock- ing modes of the B–B bond (at ≈850 cm−1 and ≈700 cm−1, respectively) are both relatively stronger compared to the IR intensity related to B–Si vibrations in the 600-635 cm−1 region. For the B–P–B cluster [Fig. C. Vibrational properties of boron and phosphorus co-doped Si NPs 7(c)], the intensity of the threefold coordinated B atom’s local modes (at ≈730 cm−1) and the vibrations related to the bonds between the inner B atom and the neighboring Si atoms and P atom (≈590-670 cm−1) contribute the most to the B-projected IR intensity. E. Raman and PL spectra of boron and phosphorus co-doped Si NPs We also calculated the Raman and PL spectra of these model structures and plotted them in Fig. 8. The PL spectra of the various models do not contain substantial information about the structural properties. The Si NP models with a single B or P dopant resulted in relatively sharp emission spectra, but the spectra of the rest of the models could be only distinguished by the energy of the PL peak. For a single DAP, the absorption and emission energies depend on the donor-acceptor separa- tion distance, resulting in lower emission energies for the NP model where the B and P are relatively far from each other. The threefold coordinated B atoms on the surface signiﬁcantly reduce the emission energy for each Si NP model where they occurred [(e), (f), (i)] which is also in contradiction with exper- imental results. The PL spectra of the Si NP model containing a small (g) and larger (h) BP cluster is rather similar to the case of a single BP pair. On the other hand, the calculated Raman spectra of model (f) seems compatible with the experimental results with a strong TO-peak and a rather weak and broad B-related IR band between 580 and 730 cm−1. Finally we turn our attention towards the model with a surface-bound B–P cluster (i). The calculated Raman spectra for this model shows the usual TO-peak and B-related band, but exhibits two addition bands around 520-560 cm−1 and 740-780 cm−1. The latter can be identiﬁed as the characteristic local mode of the threefold coordinated B-atoms at the surface, while the former is a collective mode involving the 3 P atoms, 3 fourfold coordinated B atoms, and some neighboring Si atoms to a lesser degree. We note that the B and P concentration of these mod- els differs from the experimental co-doped Si NPs. Models (a)–(f) have lower while model (h) has higher B/P concentra- tion compared to experimental samples. This means that the relative intensity of the TO-peak and the B-related band should be scaled accordingly when compared to experiments. One other question is that how these different characteristic dopant conﬁgurations interplay in the same Si NP. While the VDOS By contrast, the Raman spectra calculated for the differ- ent Si NP models differ signiﬁcantly, indicating that Raman spectroscopy is a more useful experimental tool for struc- tural characterization. D. IR spectra of boron and phosphorus co-doped Si NPs As the vibrational density of states cannot be directly mea- sured, it is beneﬁcial to calculate the IR spectra, in order to provide a basis for comparisons with the experiments. Since bulk Si is a nonpolar crystal, we expect the IR signal of pris- tine Si NPs to be rather weak. Our calculations indicate that the IR spectra of the Si core do not vanish, but it is masked by the signal of the Si–H bonds. As a consequence, we sub- tract the contribution of the H atoms and the outermost layer of Si atoms for a better approximation of realistic Si NPs. Figure 6 shows the case of a single B–P pair introduced into two different sized host NPs (1.6 nm, 2.1 nm). It is apparent that a small peak in the 600-700 cm−1 region remains even after the subtraction of the contributions from the NP sur- face. The relative intensity of this small peak is expected to decrease with increasing NP size, similarly to the VDOS (see Fig. 2). FIG. 7. The calculated IR absorption spectra for the depicted Si NP models containing a few B/P atoms decomposed for the different elements. In the case of Si, the outermost layer of Si atoms is excluded from the projection. FIG. 7. The calculated IR absorption spectra for the depicted Si NP models containing a few B/P atoms decomposed for the different elements. In the case of Si, the outermost layer of Si atoms is excluded from the projection. Somogyi, Bruyer, and Gali J. Chem. Phys. 149, 154702 (2018) 154702-8 The case of the ring-like BP cluster [Fig. 7(d))] introduces a broad band in the 600-700 cm−1 region that is localized on B–Si vibrations. The model with a large BP cluster introduced into the center of a Si NP [Fig. 7(e)] has a rather broad IR band in the 520-720 cm−1 region. The absolute intensity of this band is stronger compared to the other model systems; however, the IR intensity normalized for the dopant concentration is rather small. The Si NP with a single P dopant shows qualitatively sim- ilar Raman spectra to the undoped Si NP, while the models containing a single B–P pair or a single B atom show the char- acteristic B-related Raman peak around 600-640 cm−1 after the surface atoms are projected out. D. IR spectra of boron and phosphorus co-doped Si NPs The Raman spectra of the Si NP model with a small, ring- like B-P cluster is also quite reminiscent of the aforementioned case, but the B-related band is more diffuse with a prominent B–P peak at ≈550 cm−1. Finally, we discuss the case of the BP cluster introduced to the NP surface [Fig. 7(e)]. The threefold coordinated B atoms once again induce a distinct peak in the calculated IR spectra (≈780 cm−1), while the bonds between the inner B atoms and their neighboring P and Si atoms are manifested as a broad band in the 590-680 cm−1 region. On the other hand, the Raman spectra of the Si NP model with a large BP cluster in its center (h) differs signiﬁcantly from the experiments. The B-related band extends from 500 to 720 cm−1, and the shape and relative intensity of the TO-peak is very different from that is observed in the experiments. Finally, we discuss the case of Si NP models that contain threefold coordinated surface B atoms. The model with adja- cent B-B atoms (e) is exceptional as the 850 cm−1 stretching mode of the B–B bond dominates the Raman spectra. The lack of such signals in Raman measurements could be a good indi- cation that this conﬁguration is indeed very rare or does not occur at all. E. Raman and PL spectra of boron and phosphorus co-doped Si NPs In order to differentiate between the dopant related and surface related signals, we utilize the pro- jection technique introduced earlier. This is important as we wish to model larger Si NPs with our 1.6-2.4 nm Si NPs (see Fig. 2). FIG. 8. Calculated resonance Raman and PL spectra for co-doped Si NP models depicted in Fig. 5. In case of the Raman spectra, black and green lines correspond to the full Raman spectra and the Raman spectra projected to the atoms in the bulk-like region of the nanocrystal, respectively. For the PL spectra, black and red lines correspond to spectra calculated for 0 K and 300 K temperatures, respectively. FIG. 8. Calculated resonance Raman and PL spectra for co-doped Si NP models depicted in Fig. 5. In case of the Raman spectra, black and green lines correspond to the full Raman spectra and the Raman spectra projected to the atoms in the bulk-like region of the nanocrystal, respectively. For the PL spectra, black and red lines correspond to spectra calculated for 0 K and 300 K temperatures, respectively. J. Chem. Phys. 149, 154702 (2018) Somogyi, Bruyer, and Gali 154702-9 and IR spectra are expected to be superposition of the char- acteristic atom clusters’ signals due to the local nature of the characteristic vibrations, the situation is more complicated for Raman spectroscopy. Resonance Raman spectroscopy probes the vibrational properties by projecting the normal modes to the geometry distortion between the ground state structure and the structure of excited electronic states. The relaxation of the structure upon electronic excitation depends on the nature of the electron-hole pair induced by the absorbed photon; thus, the prediction of resonant Raman spectra for systems that contain qualitatively different dopant clusters is not straight- forward. It is possible that the induced electron-hole pair (at a given excitation wavelength) becomes localized on the bulk regionoftheSiNP;thus,themeasurementbecomesinsensitive to the surface region (or vice versa). related to B atoms in the proximity of the NP surface; thus, we calculated the correlation between the normal modes’ energy and the position of the involved B atoms. From Fig. 9(b), it is apparent that the smaller, high energy B-related IR band is indeed related to B atoms close to the surface. While the outermost layer of Si atoms were not substituted by dopants during the generation of these random structures, the layer below that was allowed to be substituted. 2. Raman spectra The calculated resonance Raman spectra averaged for the 10 co-doped Si NP models is shown in Fig. 10(b) compared to the experimental Raman spectra taken from Ref. 11. The B and P concentration for sample A was 0.80% and 0.29%, respectively, while 0.86% and 0.62% for sam- ple B. After eliminating the Si–H related vibration from the Raman spectra, the result shows reasonable agreement with the observations.7,8,11 Notable differences between the simu- lated and measured Raman spectra are the underestimation of the TO peak energy, the raggedness of the B-related band, the small 790 cm−1 peak that is absent in the experiments, and the overall shift of the B-related band towards higher energies. The simulated B-related band falls around 680 cm−1, whereas the measured B-related band is at 635 cm−1 and 650 cm−1 for sample A and B, respectively. Finally, the relative inten- sity of the B-related band is slightly overestimated for our models. FIG. 9. (a) The PVDOS and IR spectra averaged over 10 energetically stable, B– and P–codoped Si NPs. For the sake of clarity, the B– and P–projected VDOS were multiplied by a factor of 5 in the plot. The 1.6 nm sized host Si NP is H-terminated, and the concentration of B and P is 3% for both dopants. (b) A heatmap representation of the B-projected VDOS, where a second pro- jection was applied which represents the involved B atoms’ distance from the NP surface. By integrating the 2D function in the y-direction, the original B-projected VDOS is obtained. This heatmap offers some insight about the correlation between the B-related normal modes’ energy and the B-atoms’ position within the Si NP. FIG. 9. (a) The PVDOS and IR spectra averaged over 10 energetically stable, B– and P–codoped Si NPs. For the sake of clarity, the B– and P–projected VDOS were multiplied by a factor of 5 in the plot. The 1.6 nm sized host Si NP is H-terminated, and the concentration of B and P is 3% for both dopants. (b) A heatmap representation of the B-projected VDOS, where a second pro- jection was applied which represents the involved B atoms’ distance from the NP surface. By integrating the 2D function in the y-direction, the original B-projected VDOS is obtained. This heatmap offers some insight about the correlation between the B-related normal modes’ energy and the B-atoms’ position within the Si NP. 1. IR spectra We also calculated the averaged IR intensities projected to B, P, and the Si atoms (excluding the outermost atomic layer). The B related IR peak is much more intense than the TO-peak, similarlytothecaseofsingleB–Ppairs(seeFig.6).Inaddition, the relative IR intensity of the higher energy B-Si vibrations is diminished compared to the VDOS, which might explain that this high energy peak does not appear in experimental IR spec- tra,11,13 even though there is signiﬁcant evidence suggesting thatthesurfaceofco-dopedSiNPscontainsBinveryhighcon- centration. Alternatively, the amorphous B-rich surface layer encapsulating the crystalline core may alter the vibrational energies of surface-bound substitutional B atoms. E. Raman and PL spectra of boron and phosphorus co-doped Si NPs According to our previous result (see Fig. 3), B atoms substituting the second- outermost Si layer introduce this higher energy band in the VDOS. 24 October 2024 04:41:14 The B-related peak between 570 and 720 cm−1 is famil- iar with the Raman measurements of co-doped Si NPs, with the exception that our results indicate a second, smaller inten- sity peak around 780 cm−1. We suspected that this IR band is F. Properties of randomly generated boron and phosphorus co-doped Si NPs Next, we analyze the PVDOS of a couple of randomly generated B and P co-doped Si NPs. The size of the NPs is once again 1.6 nm, and we substituted 5-5 of the 147 Si atoms by B and P atoms, respectively. Our previous study demonstrated54 that the formation energy and optical prop- erties of co-doped Si NPs are greatly inﬂuenced by the dis- tribution of dopants even at a given doping concentration. Because of this, we have chosen the 5 most stable dopant conﬁgurations from our randomly generated ensemble of 395 co-doped Si NP models.54 In addition, we generated another 5 energetically stable conﬁgurations utilizing the empirical formula we established between the formation energy and mean dopant-dopant distances within the NP. Figure 9(a) show the calculated PVDOS averaged over the 10 different dopant conﬁgurations. We note that some IR measurements11 of co-doped Si NPs do exhibit small absorption peaks around ≈850 cm−1 and ≈900 cm−1 that were assigned to Si–H vibrations. Given that the presence of Si–H bonds was established, this assignment is plausible, but it cannot be ruled out that B atoms also con- tribute to (one of) the IR peak(s) (see Fig. 1). IR measurements performed on aged samples also show a broad absorption band in the 800-900 cm−1 region,13,19 but the source of this signal could be possibly oxygen related. 3. PL spectra The broader TO peak can be explained by the rather small NP size, while the higher relative intensity of the B-related peak is the consequence of the larger dopant con- centration. Due to the relatively small size of our NP models and the low formation energies of our sample [the dopants are typically accumulated near the surface because of the effec- tive repulsion between B–B and P–P pairs,54 see Fig. 9(b)], B-atoms that are in the proximity of the surface are proba- bly overrepresented compared to those in the larger NPs of the Raman measurements. This can explain the shift of the B-related band towards higher energies in our simulations. On the other hand, the experimental B-related band cuts off around 700-725 cm−1, that is, the local modes belonging to B atoms at the two outermost atomic layers of the Si NPs are missing from the Raman spectra. This might be explained by the amorphous dopant-rich layer encapsulating the crystalline core11 that could affect vibrational frequencies of these modes. It is possible, that the induced electron-hole pair is conﬁned in the inner region of the Si NP; thus, the vibrational signature of B atoms that are proximate to the surface is not manifested in the measured Raman spectra. The most signiﬁcant deviation of our results from the experiment is the energy of the PL peak. Our 10 NP ensemble has a PL peak around 2.4 eV compared to the 1.6 eV peak of the experimental sample from Ref. 57. As these Si NPs are larger than our NP models, the quantum conﬁnement effect can be partially responsible for this discrepancy. Si NPs similar to the size of our models were also characterized by PL spectroscopy exhibiting emission peak around 1.8 eV.10 While it is plausi- ble that the applied PBE0 TDDFT kernel is not sufﬁciently accurate, it is difﬁcult to assess the error originating from the TDDFT approximation as PBE0 is known to underestimate the excitation energy of very small Si NPs,58 but overestimates the bandgap of bulk Si.59,60 On the other hand, it is possible that our Si NP mod- els are too oversimpliﬁed for direct comparison to experi- mental data. Admittedly, our treatment of the NP surface is rather crude and encapsulating the crystalline core with the experimentally observed amorphous dopant-rich layer might lead to improved absolute emission energies. 3. PL spectra We also calculated the PL spectra for the aforementioned 10 Si NP models and plotted the results in Fig. 10(d). The shape of the individual PL peaks is rather similar for all 10 NPs, but the ZPL energies show a signiﬁcant variance. The full width at half maximum (FWHM) of the calculated 10 spectra is 0.22 ± 0.03 eV at room temperature, while the FWHM of the 10 particle ensemble is 0.36 eV. In comparison, the FWHM of experimental samples grown at ∼900 ◦C (corresponding to 1.2 nm particle size) is 0.35-0.40 eV in the BPSG matrix and 0.5-0.6 eV after etching.10 Individual co-doped Si NPs have also been characterized recently.57 The investigated co-doped Si NPs were grown at 1000-1100 ◦C corresponding to ∼2-4 nm particle sizes, and the single-dot PL measurements were per- formed at 77 K. Kanno et al. found that the mean FWHM of single co-doped Si NPs is between 0.22 and 0.29 eV, in contrast to the 0.39-0.43 eV FWHM of the ensemble. They also found that the distribution of the PL FWHM changes with the growth temperature (or the NP diameter) with the smallest Si NPs showing the widest distribution of PL FWHM (75-360 meV), while the PL FWHM of the larger NPs show somewhat less variance (110-350 meV). FIG. 10. (a) The experimental Raman spectra of two different co-doped Si NP ensembles (data taken from Ref. 11) compared to the (b) averaged full– and projected–resonance Raman spectra calculated for the 10 randomly generated Si NP models. (c) Measured PL spectra for a co-doped Si NP ensemble (black line) and individual NPs (colored lines) (data taken from Ref. 57). (d) The cal- culated PL spectra for the individual co-doped Si NP models (blue/orange thin lines), and the averaged PL spectra (black/red thick lines) at 0 K (blue/black lines) and 300 K (orange/red lines). 24 October 2024 04:41:14 higher dopant concentration. The sample size of our simula- tion is rather small, and only contains Si NPs with very low formation energies, while experimental samples are expected to contain less stable conﬁgurations due to entropy arguments (see Ref. 54). The underestimation of the TO peak energy may be attributed to the smaller NP size and the error of the DFT method. 2. Raman spectra Before further discussion of the differences between the simulated and experimental spectra, it is important to high- light the known differences between our Si NP models and the experimental NPs. Our models are signiﬁcantly smaller than the experimental NPs (1.6 nm vs. 7.0 ± 1.4 nm8), with J. Chem. Phys. 149, 154702 (2018) 154702-10 Somogyi, Bruyer, and Gali Somogyi, Bruyer, and Gali 154702-10 FIG. 10. (a) The experimental Raman spectra of two different co-doped Si NP ensembles (data taken from Ref. 11) compared to the (b) averaged full– and projected–resonance Raman spectra calculated for the 10 randomly generated Si NP models. (c) Measured PL spectra for a co-doped Si NP ensemble (black line) and individual NPs (colored lines) (data taken from Ref. 57). (d) The cal- culated PL spectra for the individual co-doped Si NP models (blue/orange thin lines), and the averaged PL spectra (black/red thick lines) at 0 K (blue/black lines) and 300 K (orange/red lines). which fact is inconsistent with the electronic density of states (DOS) measurements performed for co-doped Si NPs56 where no such deep levels were found. On the other hand, a recent work12 studied the Auger interactions in the nonlinear exci- tation regime of co-doped Si NPs and concluded that the nonradiative recombination can be attributed to dopant-related deep defect states. The lower energy tail of the broad B-related band is most likely the signature of existing B–P bonds in the NPs, as our calculations indicate that the vibrational energy of the B-P inside a Si NP ∼560 cm−1, while larger BP clusters introduce broader bands in the 550-700 cm−1 interval, cov- ering the whole experimentally observed B-related band (see Fig. 5). 3. PL spectra We also note that we observed a very strong correlation between the co- doped Si NPs formation energy and the optical gap in our We also note that boron clusters containing interstitial B or Si atoms could be also responsible for vibrational signals in the 600-720 cm−1 region.42,55 While we cannot disregard this scenario for Si NPs too, we note that most of these clusters introduce deep defect states inside the Si bandgap56 Somogyi, Bruyer, and Gali J. Chem. Phys. 149, 154702 (2018) 154702-11 Somogyi, Bruyer, and Gali 154702-11 earlier study.54 Due to the expensive nature of the calcula- tion of the vibrational properties, we restricted our simula- tions to co-doped Si NPs with very low formation energies. We showed in our previous work54 that not only these low energy conﬁgurations occur in realistic ensembles due to the very low entropy of the stable conﬁgurations. As our sam- ple selection favored dopant conﬁgurations with very low formation energies, we obtained a narrower, higher energy emission spectra compared to what we expect for an ensem- ble with broader distribution of formation energies. Extend- ing our calculations to include less stable dopant conﬁgura- tions could potentially lead to larger variation of the FWHMs of individual Si NPs and a larger FWHM of the simulated ensemble. earlier study.54 Due to the expensive nature of the calcula- tion of the vibrational properties, we restricted our simula- tions to co-doped Si NPs with very low formation energies. We showed in our previous work54 that not only these low energy conﬁgurations occur in realistic ensembles due to the very low entropy of the stable conﬁgurations. As our sam- ple selection favored dopant conﬁgurations with very low formation energies, we obtained a narrower, higher energy emission spectra compared to what we expect for an ensem- ble with broader distribution of formation energies. Extend- ing our calculations to include less stable dopant conﬁgura- tions could potentially lead to larger variation of the FWHMs of individual Si NPs and a larger FWHM of the simulated ensemble. IV. SUMMARY 1A. P. Alivisatos, Science 271, 933 (1996), http://science.sciencemag. org/content/271/5251/933.full.pdf. 2 We studied the vibrational properties of B and P co-doped Si NPs by means of ﬁrst principles simulations, in order to characterize co-doped Si NPs. In this work, we focused in the bulk region of the Si NPs, as the introduction of B–P clus- ters already creates a rather challenging problem even without considering the various surface related issues. To this end, we terminated the NP surface by H atoms, in order to reduce the computational cost. However, this compromise complicated our job as the rolling vibrational modes of the Si–H bonds overlap with the characteristic B-related local modes. In order to quantify the effect of dopants on the vibrational properties of Si NPs, we applied a projection technique to ﬁlter out the contributions of the Si–H bonds. This was especially important as most of our model Si NPs were rather small (1.6 nm) with larger surface/volume ratios compared to experimental sam- ples. Despite of this compromise, we did identify quantiﬁable effects that are related to the proximity of the NP surface. We found that the vibrational signal of substitutional B is depen- dent on the dopants position within the NP. 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Imakita, Nanotechnology 27, 262001 (2016). 8 j g gy ( ) 8M. Fujii, H. Sugimoto, M. Hasegawa, and K. Imakita, J. Appl. ACKNOWLEDGMENTS A.G. acknowledges the support from the National Research Development and Innovation Ofﬁce of Hungary (NKFIH) Grant No. NN118161 that ﬁnances Visegrad Group (V4) and the Japan Joint Research Project on Advanced Materials (NamSeN project). This work was also ﬁnanced by NKFIH within the Quantum Technology National Excel- lence Program (Project No. 2017-1.2.1-NKP-2017-00001). A.G. acknowledges the support from KIF ¨U Supercomputer Center Grant No. 1090. Overall, our results compare well with the experiments apart from the absolute energy of the PL peak, despite being smaller than the experimentally studied ones and our simplistic treatment of the NP surface. 3. 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W2085256329.txt	https://www.frontiersin.org/articles/10.3389/fenvs.2015.00027/pdf	en		Monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted Uncaria tomentosa root cultures	Frontiers in environmental science	2,015	cc-by	8,217	ORIGINAL RESEARCH published: 09 April 2015 doi: 10.3389/fenvs.2015.00027 Monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted Uncaria tomentosa root cultures Ileana Vera-Reyes 1 , Ariana A. Huerta-Heredia 1 , Teresa Ponce-Noyola 1 , Carlos M. Cerda-García-Rojas 2 , Gabriela Trejo-Tapia 3 and Ana C. Ramos-Valdivia 1* Edited by: Rene Kizek, Central European Institute of Technology in Brno, Czech Republic Reviewed by: Naser A. Anjum, University of Aveiro, Portugal Zbynek Heger, Central European Institute of Technology in Brno, Czech Republic Ondrej Zitka, Mendel University in Brno, Czech Republic Correspondence: Ana C. Ramos-Valdivia, Departamento de Biotecnología y Biongeniería, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, A. P. 14-740, México, D. F. 07000, Mexico aramos@cinvestav.mx Specialty section: This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science Received: 06 January 2015 Accepted: 20 March 2015 Published: 09 April 2015 Citation: Vera-Reyes I, Huerta-Heredia AA, Ponce-Noyola T, Cerda-García-Rojas CM, Trejo-Tapia G and Ramos-Valdivia AC (2015) Monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted Uncaria tomentosa root cultures. Front. Environ. Sci. 3:27. doi: 10.3389/fenvs.2015.00027 1 Departamento de Biotecnología y Biongeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, D. F., Mexico, 2 Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, D. F., Mexico, 3 Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Yautepec, Mexico Plants cells sense their environment through oxidative signaling responses and make appropriate adjustments to gene expression, physiology and metabolic defense. Root cultures of Uncaria tomentosa, a native plant of the Amazon rainforest, were exposed to stressful conditions by combined addition of the glutathione inhibitor, buthionine sulfoximine (0.8 mM) and 0.2 mM jasmonic acid. This procedure induced a synchronized two-fold increase of hydrogen peroxide and guaiacol peroxidases, while the glutathione content and glutathione reductase activity were reduced. Likewise, in elicited cultures, production of the antioxidant secondary metabolites, monoterpenoid oxindole, and glucoindole alkaloids, were 2.1 and 5.5-fold stimulated (704.0 ± 14.9 and 845.5 ± 13.0 µg/g DW, respectively) after 12 h, while phenols were three times increased. Upon elicitation, the activities and mRNA transcript levels of two enzymes involved in the alkaloid biosynthesis, strictosidine synthase and strictosidine β-glucosidase, were also enhanced. Differential proteome analysis performed by two-dimensional polyacrylamide gel electrophoresis of elicited and control root cultures showed that after elicitation several new protein spots appeared. Two of them were identified as thiol-related enzymes, namely cysteine synthase and methionine synthase. Proteins associated with antioxidant and stress responses, including two strictosidine synthase isoforms, were identified as well, together with others as caffeic acid O-methyltransferase. Our results propose that in U. tomentosa roots a signaling network involving hydrogen peroxide and jasmonate derivatives coordinately regulates the antioxidant response and secondary metabolic defense via transcriptional and protein activation. Keywords: oxidative stress, Uncaria tomentosa, proteome, antioxidant responses, glutathione Introduction Oxidative stress arises from disruption in redox balance due that the amount of reactive oxygen species (ROS) exceeds the ability of the cell to accomplish an effective antioxidant response. Unlike other ROS, hydrogen peroxide (H2 O2 ) is a non-radical species, containing no Frontiers in Environmental Science \| www.frontiersin.org 1 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion (Huerta-Heredia et al., 2009), a glucoindole alkaloid with hypotensive and antioxidant activities (Endo et al., 1983) and dolichantoside (Luna-Palencia et al., 2013), a N-β-methylated strictosidine with potent anti-malarial effect (Frédérich et al., 2000). Moreover, the antioxidant response and alkaloid production stimulation have been correlated with oxidative stress (Trejo-Tapia et al., 2007) triggered by H2 O2 treatment (HuertaHeredia et al., 2009; Vera-Reyes et al., 2013) and by combined addition of the glutathione inhibitor, buthionine sulfoximine and jasmonic acid (Vera-Reyes et al., 2013). It has been suggested that monoterpenoid indole alkaloids (MIA) are precursors of MOA whose transformation may take place through oxidation of the indole ring system. The central precursor of the MIA pathway is the glycosylated indole alkaloid strictosidine, which is formed through the condensation of the indole precursor tryptamine with secologanin catalyzed by the enzyme strictosidine synthase (STR; EC 4.3.3.2). Then, strictosidine β-D-glucosidase (SGD; EC 3.2.1.105) hydrolyzes the glucose moiety present in strictosidine forming an aglycone, which is rapidly converted to a dialdehyde intermediate. In some plants such as Catharanthus roseus, this substance is reduced by NADPH to ajmalicine or their isomers through cathenamine (Kutchan, 1995). Strictosidine also participates in the biosynthesis of other glucoindole alkaloids characteristic of the Rubiaceae family such as isodihydrocadambine (Szabó, 2008). Both STR and SGD are encoded by single genes (McKnight et al., 1990), even though the STR from C. roseus has shown several isoforms due to post-translational modifications (De Waal et al., 1995; Jacobs et al., 2005). Vera-Reyes et al. (2013) reported that in U. tomentosa root cultures, the increase of oxindole and glucoindole alkaloids observed under oxidative stress, is provoked by the regulatory mechanisms at the level of enzyme activities and gene expression of STR and SGD. Thus, proteomics provides a promising approach for the study of the protein response to oxidative stress in general and its relation with the secondary metabolism production (Ramos-Valdivia et al., 2012). Particularly, comparative proteomic studies based on contrasting plant cultures on stressed and non-stressed conditions are essential for understanding the stress-related defense mechanisms. In order the study the regulatory mechanisms functioning in the monoterpenoid indole alkaloid production in U. tomentosa root cultures, activities and mRNA transcript levels of two enzymes involved its alkaloid biosynthesis, antioxidant defense and comparative proteome analysis in response to oxidative stress were examined. net charge, with a relatively long half-life. Because of these properties, H2 O2 acts as a long-distance signaling molecule and is a physiological indicator of the intensity of biotic and/or abiotic stress (Apel and Hirt, 2004). In turn, to prevent the harmful effects of ROS, plants have evolved coordinate antioxidant mechanisms that include superoxide dismutase, peroxidases, the ascorbate-glutathione cycle, and other antioxidant responses (Noctor and Foyer, 1998). Glutathione is a low molecular weight tripeptide useful in protecting plant cells from oxidative injury due to its redox buffering capacity and relative abundance. In response to environmental stress through the ascorbate–glutathione pathway, the redox potential of the reduced glutathione (GSH) pool is altered and converted to the disulfide form (GSSG) without net consumption (Meyer and Fricker, 2002). It has been reported that H2 O2 , produced in response against various stimuli, would be acting as a signaling molecule, regulating the expression of selected genes, including those involved in the defense pathways and participating in the crosstalk between other metabolic signals (Quan et al., 2008). Several studies suggest that, as the result of adaptation responses of plants to oxidative stress, changes occur not only in the primary defense mechanisms but also in the profile of secondary metabolism (Apel and Hirt, 2004). Alkaloids represent one of the most active natural product groups against a wide range of organisms. The main role of these substances is generally linked to plant defense mechanisms from predators, besides the important ecological factors associated to them. However, the close relationship between alkaloids and the oxido-reduction processes in plants containing them strongly suggests that these compounds play a fundamental role in protecting plants when they are subjected to oxidative stress (Ramos-Valdivia et al., 2012). Furthermore, polyphenols are the most abundant and widely distributed group of naturally occurring compounds. Their functions are critical to the maintenance of the plant, being relevant in the defense against herbivores, for protection to different types of biotic or abiotic stress, as well as signals in interactions either with other plants or with microbes (Buer et al., 2010). GSH deficit may occur in plants as a consequence of increased cellular consumption and/or due to biosynthetic disorders. However, GSH depletion of GSH can occur by addition of L-buthionine-(S,R)-sulphoximine (BSO). This nontoxic substance is a specific inhibitor of γ-glutamylcysteine synthetase (Ruegsegger et al., 1990; May and Leaver, 1993). Treatment of plant tissue with BSO has been used as an elicitor of secondary metabolites since this substance weakens the antioxidant defense mechanisms, provoking endogenous accumulation of H2 O2 and oxidative stress (Berglund and Ohlsson, 1993; Guo et al., 1993; Vera-Reyes et al., 2013). Uncaria tomentosa, which belongs to the Rubiaceae family, is an Amazon rainforest species known as cat’s claw. This plant produces the highly oxidized monoterpenoid oxindole alkaloids (MOA) isopteropodine, mitraphylline, isomitraphylline and rhynchophylline, which exhibits immunomodulatory, antiAIDS, cytotoxic, and antileukemic properties (Laus, 2004). In previous work, it was found that root suspension cultures of this species produced MOA and accumulated 3α-dihydrocadambine Frontiers in Environmental Science \| www.frontiersin.org Materials and Methods Root Cultures and Elicitation Root cultures of U. tomentosa (line Utr-3) arising from micropropagated plantlets (Luna-Palencia et al., 2013) were grown in 250-mL Erlenmeyer flasks (covered with aluminum foil) with 100-mL of MS medium (Murashige and Skoog, 1962), 2% sucrose without plant growth regulators and pH 6.4 adjusted prior to sterilization. The cultures were incubated at 25 ± 2◦ C, using orbital agitation at 110 rpm, and under continuous light intensity 2 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion 13 µmol m−2 s−1 . The cultures were sub-cultivated every 20 days and uniform inocula for the experiments were developed in 1000mL Erlenmeyer flasks containing 400-mL of culture medium. A selection of 20-days-old roots were cut in pieces of ∼5 cm length and kept in deionized water until they were inoculated (2 g FW) into 250-mL shaken flasks containing 100-mL culture medium. Roots were elicited at day 13 with simultaneous addition of 0.8 mM BSO and 0.2 mM jasmonic acid (BSO-JA) and were incubated as indicated above. Three control cultures and three elicited flask cultures were harvested after 12 h. four gels for each sample. Only those spots that showed significant and reproducible changes (at least 1.3-fold) were taken in to account as differentially expressed proteins, ANOVA (p < 0.05). The Scaffold program (version 4.0.6.1 from Proteome Software Inc., Portland, OR) was employed for protein identification. The validation was done if the probability was greater than 99.0% and contained at least 2 identified peptides. Protein probabilities were allocated by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that could not be differentiated based on MS/MS analysis were grouped to satisfy the principles of parsimony. The estimated experimental Mr /pI was useful to rise the identification confidence. Extraction of Total Proteins for 2D SDS-PAGE Ten grams of frozen roots were ground using a mortar and a pestle and were cooled with liquid N2 . A solution (20 mL) of cold (−20◦ C) 10% TCA in acetone with 0.07% β-mercaptoethanol was poured over the sample (Jacobs et al., 2005). The mixture was kept at 20◦ C overnight to enable a complete precipitation. After centrifugation for 15 min at 3000 g, samples were washed twice with a cold solution (−20◦ C) of acetone and 0.07% βmercaptoethanol for removing TCA. The precipitate was solubilized in ReadyPrep rehydration/sample buffer BioRad [8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT), 0.2% (w/v) Bio-Lyte R 3/10 ampholytes, and bromophenol blue (trace)] completed with 2 M thiourea. The mixture was vortexed and centrifuged (5 min, 16,000 g) several times during 1 h. The supernatant was recovered and cleaned up using a Micro Bio-Spin R column (BioRad, USA) and stored at −80◦ C. The concentration of protein was measured with a 2D Quant kit (Amersham Biosciences, USA). In-Gel Digestion, MALDI-TOF MS and Database Search Excised SYPRO R Ruby (BioRad)-stained protein gel spots following 2D SDS-PAGE were digested with trypsin (10 µg/mL) at 37◦ C for 12 h. Tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) analysis of in-gel trypsin digestedproteins (Shevchenko et al., 1996) was performed in a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) furnished with an Advion nanomate ESI source (Advion, Ithaca, NY). ZipTip (Millipore, Billerica, MA) C18 sample cleanup was achieved as indicated in the manufacturer’s instructions. The peptide fraction was eluted from a C18 precolumn of 100µm id × 2 cm (Thermo Fisher Scientific) and loaded onto an analytical C18 column of 75-µm ID × 10 cm C18 (Thermo Fisher Scientific) eluting with solvent A (water and 0.1% formic acid) and a 5–10% gradient of solvent B (acetonitrile, 0.1% formic acid) for 5 min, followed by a 10–35% gradient of solvent B for 35 min, 35–50% gradient of solvent B for 20 min, 50–95% gradient of solvent B for 5 min, and 95% solvent B for 5 min, all elutions were run at a flow rate of 400 nl/min. Data dependent scanning (m/z 400–1600) was carried out in the Orbitrap analyzer, followed by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) of the 14 most intense ions in the linear ion trap analyzer using the Xcalibur v 2.1.0 software (Andon et al., 2002) and a mass scan of 60,000 resolution. The precursor ions were chosen by the monoisotopic precursor selection (MIPS) setting the acceptance or rejection of ions thought a ±10 ppm window. Dynamic exclusion was established to place any selected m/z peak on an exclusion list for 45 s after a single MS/MS. All MS/MS spectra were explored against asterids proteins downloaded from Uniprot or from NCBI on October 09, 2012 or June 20, 2013, respectively, using Thermo Proteome Discoverer 1.3 (Thermo Fisher Scientific). The UniprotKB protein database of all species was also used in searching the data independently. Variable modifications considered during the search included methionine oxidation, adding 15.995 Da, and/or cysteine carbamidomethylation, adding 57.021 Da. At the time of the search, asterids database from Uniprot or NCBI contained 65,406 and 102,843 entries, respectively (UniprotKB database contained 452,768 entries as of October 10, 2012). Proteins were identified with a confidence level of 99% with XCorr score cut-offs (Qian et al., 2005) as determined by a reversed database search. The results were displayed with the Scaffold program v 3.6.1 (Proteome Software Inc., Portland OR) that depends on various 2D-PAGE About 250 µg of protein was loaded into 11-cm strips with a pH gradient between 4 and 7 (IPG, immobilized pH gradient, Bio-Rad) by in-gel rehydration during 12 h. Isoelectric focusing (IEF) was carried out on a Protean IEF apparatus (Bio-Rad, USA) at 20◦ C by application of a voltage gradient from 0 to 250 V for 1 h, 250 to 500 V for 1 h, 1000 to 8000 V for 1 h, from 8000 to 20,000 V for 2 h, and 500 V for 2 h. The protein IPG strips were equilibrated before applying a sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) procedure using an equilibration buffer I (50 mM Tris HCl [pH 8], 8 M urea, 30% glycerol, 2% SDS, and 0.3% DTT) for 10 min. The strips were then soaked for 10 min in the equilibration buffer II containing 50 mM Tris HCl (pH 8), 8 M urea, 30% glycerol, 2% SDS, and 4.5% iodoacetamide. SDS-PAGE was done using polyacrylamide 12% acrylamide gels. Electrophoresis was carried out at 25 mA for 45 min and 35 mA for 2.5 h (SE 600 Ruby ; GE Healthcare Life Science, USA). Protein samples were visualized by staining with Sypro Ruby (BioRad, USA). ™ Gel Analysis At least three independent 2-D experiments were repeated at minimum four times to confirm reproducibility. Image analysis was achieved by visual inspection and the observed changes were qualitative using Melanie 7.0 gel analysis platform (GE Healthcare). The volume of each spot was normalized as a relative volume to compensate for the variability in gel staining. Manual editing was carried out after the automated detection and matching for each spot, achieving this procedure with a minimum of Frontiers in Environmental Science \| www.frontiersin.org 3 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion Antioxidant Enzyme Assays search engines (Sequest, X!Tandem, MASCOT) using Bayesian statistics (Keller et al., 2002; Nesvizhskii et al., 2003). Guaiacol peroxidases were measured as oxidation of guaiacol (8.26 mM, ∈ = 26.6 mM−1 cm−1 ) according to Pütter (1974). Enzyme extract was incubated in 100 mM phosphate buffer pH 6.0 containing 3 mM H2 O2 . The reaction was started by addition of 15 mM guaiacol and the absorption was measured for 2 min at 470 nm using a Beckmann spectrophotometer (DU 7500, Munich). Rates were corrected by chemical control experiments. Peroxide activity was determined as the amount of protein that produces 1 µmol of oxidized guaiacol. The activity of glutathione reductase was measured using the Glutathione Reductase Assay Kit (Sigma-Aldrich, St. Louis, USA), which was determined by the absorbance decrease caused by NADPH oxidation at 340 nm. One enzyme unit (U) catalyzes the oxidation of 1 µmol of NADPH per min at 25◦ C. Quantification of Phenolic Compounds Powdered roots (0.20 g) were frozen in liquid N2 , pulverized and sonically extracted with 5 mL of methanol-water (8:2 v/v) and centrifuged. A supernatant aliquot of 0.2 mL was mixed with 0.2-mL Folin-Ciocalteu reagent diluted 1:1 (v/v) with water, 0.6 mL of sodium carbonate (Na2 CO3 ) saturated solution and 4 mL of deionized water. The mixture was intensively shaken, left at room temperature for 25 min, and centrifuged at 5000 rpm for 10 min. The absorbance of supernatant was registered at 725 nm in a Genesys 10V spectrophotometer (Thermo Scientific). Total phenols were expressed in terms of D-catechin equivalents. Quantification of individual phenols was done by HPLC analysis according (Pavei et al., 2010) using a 3-caffeoylquinic acid (chlorogenic acid) calibration curve. Strictosidine-Related Enzyme Assays The assay of strictosidine synthase (STR) activity depends on the enzymatic condensation of secologanin and tryptamine to produce strictosidine. Strictosidine formation was quantified by HPLC using a strictosidine standard (Phytoconsult, The Netherlands) for constructing the calibration curve. Strictosidine glucosidase (SGD) activity was determined by measuring the glucose release using Amplex Red R (Invitrogen) assay kit. Both enzyme assays were previously described (Vera-Reyes et al., 2013). Extraction and Quantification of Alkaloids Alkaloid extraction and quantification were performed as described previously (Vera-Reyes et al., 2013). Briefly, frozen roots (liquid N2 ) were pulverized and sonically extracted with 5% hydrochloric acid. Alkaloids from the acid-solutions or culture media were extracted twice with chloroform adjusting the pH to 8-9 using a NH4 OH solution. The organic layer was vacuum evaporated and the solid residue was dissolved in a 9:11 mixture of acetonitrile and 10 mM phosphate buffer at pH 7. The solutions were filtered and injected into a Varian ProStar 333 HPLC system equipped with a photodiode array detector (Varian, Walnut Creek, CA) using a reverse-phase C18 column (Waters Spherisorb 5 mm ODS2 of 250 mm length 4.6 mm i.d.). Elution was carried out with the same 9:11 mixture of acetonitrile and phosphate buffer at 0.7 mL/min flow rate and detecting at 244 nm. For quantification of MOA and glucoindole alkaloids, mitraphylline and 3α-dihydrocadambine respectively, were used as the standard compound to determine the calibration curve. mRNA Extraction, cDNA Synthesis and Semiquantitative RT-PCR Analysis RNA isolation, DNA treatment, reverse transcription, and semiquantitative-PCR amplification were achieves as reported previously (Vera-Reyes et al., 2013), as well as the primers used for the genes: STR (strictosidine synthase), SGD (strictosidine glucosidase and the control 18S rRNA. The relative gene expression was analyzed using a Kodak Image Station 2200R, DU R 730 equipped with Molecular Imaging Software version 1.4 (Kodak) on a 1.2% agarose gel. The gene expression analysis is represented in arbitrary units employing average values of semi-quantitative RT-PCR assays in triplicate with respect to the corresponding non-treated cultures. Statistical Analysis All measurements were done in triplicate and the statistical evaluation was achieved with Anova, taking p ≤ 0.05 as significant. Protein Extracts and Enzyme Assays Determination of H2 O2 Roots (1 g) were homogenized in a pre-chilled mortar under liquid N2 with 1–2% (w/w) polyvinylpyrrolidone. Extraction buffer (0.1 M potassium phosphate pH 6.3, containing 3 mM EDTA and 6 mM DTT) was added in a 1:1 ratio (v/w) shaking to obtain a homogeneous mixture. For GR assay, the extraction buffer was 0.1 M potassium phosphate pH 7.5, with 1 mM EDTA. Centrifugation at 18,000 g was done for 10 min at 4◦ C and the supernatant was collected and desalted on Bio-Rad Micro Bio-Spin R P-30 columns. The eluted samples were employed for the enzymatic assays. The protein fractions were kept frozen at −20◦ C until use. The total protein content was determined following the procedure described by Peterson (1977) with bovine serum albumin as the standard. Roots (500 mg) were frozen and pulverized under liquid N2 . The powder was extracted with 5 mL of 0.1% TCA (w/v), mixed with ice for 5 min, and pelleted by centrifugation at 10,000 g at 4◦ C for 10 min. The supernatant was neutralized with 0.2 M NH4 OH to pH 8.0 and was centrifuged at 3000 g for 2 min to sediment the insoluble material. The quantification of H2 O2 in the extracts was done with the Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes, Invitrogen), according to the manufacturer instructions. A total of 50 µL of extract was combined with an equal volume of 50 mM sodium phosphate buffer pH 7.4 containing 0.1 U/mL of horseradish peroxidase and incubated for 1 h at room temperature, measuring the absorbance at 560 nm. The H2 O2 concentration for each sample was determined with a standard curve obtained with known concentrations of H2 O2 . Frontiers in Environmental Science \| www.frontiersin.org 4 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion Glutathione Assay concentration was significantly reduced in a 55%, while the GR activity was slightly lower (17%) than non-treated roots (Figures 1B,D). Noteworthy, biomass concentration (6.33 ± 0.20 g DW/L) and viability of roots after the elicitation remained essentially the same as in controls. The levels of total glutathione (GSH + GSSG) were determined with a glutathione assay kit (Sigma) following the manufacturer’s protocol. Roots were frozen in liquid N2 and pulverized until obtaining fine particles. A solution of 5% 5-sulfosalicylic acid (500 µL) was added to 0.1 g of the powder to deproteinize the sample. Glutathione was measured in a kinetic assay based on the reduction of 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) to yellow TNB, which was spectrophotometrically measured at 412 nm. The amount of total glutathione was determined with a standard curve of reduced glutathione. Activities of Strictosidine-Related Enzymes, mRNA Expression Levels, and Production of Phenols and Alkaloids in Response to BSO-JA Elicitation After 12 h of elicitor treatment, MOA, 3α-dihydrocadambine and dolichantoside production (Figure 2A) were rapidly increased by 2.1-, 5.5-, and 2.6-fold, respectively, compared with control cultures (329.7 ± 39.8 µg/g DW; 152.4 ± 27.9 µg/g DW; 14.0 ± 1.8 µg/g DW). Concurrently, BSO-JA treatment increased STR activity by three times in relation to untreated roots (38.7 ± 4.0 pKat/mg protein), while SGD activity had 4.2 times more activity than the control (65.8 ± 2.9 pKat/mg protein) (Figure 2B). Upon elicitation, STR and SGD transcripts increased during the first 12 h after treatment reaching 5.8- and 9.7-fold higher, respectively, compared to the control levels (Figures 2C,D). Results Hydrogen Peroxide and Antioxidant Response to BSO-JA Elicitation U. tomentosa roots induce their antioxidant defense to scavenge excess of ROS in response to combined addition of BSO-JA. After 12 h of elicitation, a two-fold increase of H2 O2 concentration (from 0.48 ± 0.05 to 0.96 ± 0.03 µmol/g FW) and POD activity (from 243.9 ± 15.4 to 370.8 ± 8.9 µM/mg.min protein) were found (Figures 1A,C). In these elicited cultures, glutathione peroxidase (POD) and (D) glutathione reductase (GR). The elicitor was added to 13 days-old root cultures (exponential growth phase). Error bars indicate standard deviation from the mean (n = 3). FIGURE 1 \| Responses of Uncaria tomentosa root cultures after 12 h of BSO-JA elicitor addition, (A) Changes in peroxide hydrogen (B) glutathione concentration. Antioxidant enzyme activities of (C) guaiacol Frontiers in Environmental Science \| www.frontiersin.org 5 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion FIGURE 2 \| Effect of BSO-JA elicitation on U. tomentosa root cultures growing in Erlenmeyer flasks after elicitor addition (black bars). (A) Production of MOA, DHC, and dolichantoside. (B) Specific activities of strictosidine synthase (STR) and strictosidine β-glucosidase (SGD). Error bars indicate standard deviation from the mean (n = 3). Semiquantitative RT-PCR analysis of the elicitor effects in the STR and SGD mRNA transcript levels (C) Representative expression profile (The amplification products were analyzed by agarose gel electrophoresis (lane 1 control, lane 2 elicited) stained with ethidium bromide, and visualized by UV transillumination (306 nm). An inverse image of the stained gel is shown. (D) Schematic presentation of the RT-PCR results in arbitrary units. The analysis of gene expression is represented in the bar chart using average values of semiquantitative RT-PCR triplicate analysis. The results are relative to non-treated root cultures. extracts from 12 h after BSO-JA addition were compared with the same number of replicates from non-treated root cultures. Although the gels showed the same profile, the control gels exhibited more proteins than the elicited ones. The control gel with higher protein spots (480) was used for the analysis as standard reference gel. An 87% of the protein spots on the other three gels from untreated roots coincided with those found in the reference gel, whereas those from elicited extracts were 85% coincident. The new proteins that appeared after elicitation and those proteins from the region pI 5–6 and 30–35 kDa (Figure 3) that, as previously reported correspond to alkaloid biosynthesis enzymes (Jacobs, 2005), were selected for sequenciation. The 14 identified proteins (Table 2 and Supplementary Table 1) can be classified into several functional categories, including energy metabolism and photosynthesis: two triosephosphate isomerases (chloroplastic and cytoplasmic; spots 1, 2, and 8), as the same protein in multiple spots differing in pI and Mr , and ribulose 1,5-bisphosphate carboxylase (Rubisco) large chain (spot 4). Protein synthesis: some proteins involved in the sulpur amino acid biosynthesis such as cysteine synthase (spot 11) and methionine synthase (spot 10) were up-regulated in BSO-JA conditions. Secondary metabolism: oxidative stress increased the expression of protein spots 7 and 9, identified as strictosidine synthase (STR) isoforms, and spot 12 identified as caffeic acid O-methyltransferase. ROS TABLE 1 \| Polyphenols accumulation in Uncaria tomentosa root cultures growing in Erlenmeyer flasks 12 h after BSO-JA elicitor addition. Compound 3-Caffeoylquinic acid Caffeic acid Control Elicited* µg/g DW µg/g DW 315.1 ± 14.7 978.7 ± 44.7 38.0 ± 4.6 57.4 ± 6.8 Catechin 123.9 ± 4.7 150.8 ± 8.0 Epicatechin 189.8 ± 8.6 301.5 ± 19.1 Values are the mean of three replicates ± standard error of means. In correlation with the alkaloid induction after BSO-JA addition, total polyphenol content in U. tomentosa root cultures increased from 3.40 ± 0.12 mg/g to 11.45 ± 0.02 mg/g DW. In these elicited roots, the content of 3-caffeoylquinic acid, caffeic acid, catechin, and epicatechin were increased by 210.5, 50.8, 21.7, and 58.8%, respectively (Table 1). Detection of Differentially Expressed Proteins after BSO-JA Elicitation One of the key approaches of proteomic analysis is to identify differential protein expression between control and experimental samples. Hence, four replicate gels of U. tomentosa protein Frontiers in Environmental Science \| www.frontiersin.org 6 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion Nevertheless, the non-induction of GR activity at 12 h after elicitation could be caused by the increase in polyphenol content as previously reported (Zhang et al., 1997). Under the assayed conditions, BSO-JA addition did not affect the biomass concentration and root viability, probably because jasmonic acid would be inducing the biosynthesis and activity of other defense responses (Sasaki-Sekimoto et al., 2005) offsetting the antioxidant diminishing caused by BSO. Relations among Activities of Strictosidine-Related Enzymes, mRNA Expression Levels, and Production of Phenols and Alkaloids in BSO-JA Elicited Roots In cell or plant cultures, a synergistic effect of elicitors on secondary metabolites production may occur (Zhao et al., 2005). It has been reported that BSO induces oxidative stress by depletion of glutathione (Noctor and Foyer, 1998), JA can induce ROS production, and JA signaling is important for oxidative stress tolerance (Sasaki-Sekimoto et al., 2005; Pauwels et al., 2008). Separate application of JA or BSO in U. tomentosa roots also elicited the production of alkaloids but in smaller quantities (Vera-Reyes et al., 2013). An increase in secondary metabolite production was also obtained in carrot cells when BSO was used alone or in combination with a yeast glucan elicitor, stimulating an increase in the H2 O2 at cellular level (Guo and Ohta, 1993). In U. tomentosa cell cultures growing in bioreactors, a positive correlation among the increment of endogenous H2 O2 level, activities of NAD(P)H oxidase and peroxidases, and MOA production was reported (Trejo-Tapia et al., 2007). Moreover, H2 O2 treatment induced oxidative stress and alkaloid production in U. tomentosa roots (Huerta-Heredia et al., 2009; Vera-Reyes et al., 2013). In C. roseus, STR and strictosidine are confined inside the vacuole (McKnight et al., 1990) separated from the activity of the nuclear localized SGD (Guirimand et al., 2010). In U. tomentosa root cultures, a probable cell compartmentalization for alkaloids has been suggested (Vera-Reyes et al., 2013) as MOA were mainly found in the culture medium, while the glucoindole alkaloids 3αdihydrocadambine and dolichantoside were always found inside the roots. Furthermore, alkaloid biosynthesis includes multiple oxidations catalyzed in a stereo- and regiospecific fashion, indicating that specific oxidases are involved in the in vivo biosynthesis. It has been found that peroxidases, microsomal cytochrome P-450-dependent enzymes, 2-oxoglutarate dependent dioxygenases and flavoproteins catalyze some of these oxidations with high substrate specificity enzymes (Kutchan, 1995). However, in vitro studies have revealed the ability of plant peroxidases to accept alkaloids as substrates as well as a number of vacuolar metabolites, such as phenols and flavonoids (Sottomayor et al., 2004; Takahama, 2004). In response to the BSO-JA elicitation, polyphenols production in U. tomentosa root cultures, mainly 3-O-caffeoylquinic acid and catechins, was highly stimulated due to the prevailing oxidative stress. Therefore, polyphenols, as flavonols and phenylpropanoids present in vacuoles and the apoplast, can metabolize H2 O2 as an electron donor for phenol peroxidases. This change results in the formation of the respective phenoxyl radicals, which can be regenerated by a nonenzymatic reaction with ascorbate (Figure 4). Thus, in C. roseus it FIGURE 3 \| Representation of two-dimensional electrophoresis (2-D SDS-PAGE) profiles of the soluble proteins extracted from Uncaria tomentosa root cultures (250 µg). The proteins were separated on a pH 4-7 linear IPG strip, followed by 12% SDS-PAGE. The gel was visualized by Sypro Ruby staining. Number indicates the protein spots identified by MS analysis. scavenging, defense and stress: abundance of defense-related proteins as ascorbate peroxidase (spot 3), proteasome alpha subunit (spot 6), universal stress protein (spot 13), and pathogenesisrelated protein (spot 14) were altered during oxidative stress condition. Discussion Induction of Hydrogen Peroxide and Antioxidant Responses by BSO-JA Elicitation The high increase in H2 O2 combined with reduction of glutathione concentration in U. tomentosa roots 12 h after addition of BSO-JA may reflect that oxidative stress conditions were present. The plant cell protection of reduced glutathione (GSH) against the oxidative injury is established by its redox buffering activity and abundance. Therefore, treatment of plant cell or tissue with the glutathione biosynthesis inhibitor BSO can lead to weakened antioxidative defenses; an increase in the concentration of endogenous H2 O2 ; and secondary metabolites stimulation (Berglund and Ohlsson, 1993; Guo and Ohta, 1993; Guo et al., 1993). Furthermore, H2 O2 is a secondary messenger that mediates hormonal responses, biotic/abiotic environmental stresses, and developmental signals (Neill et al., 2002). Thus, the jasmonate signaling is mediated by H2 O2 (Orozco-Cárdenas et al., 2001), and is controlled via a suitable antioxidant response to neutralize its adverse effects. The increase of peroxidase activity in elicited cell and plant cultures has been found as a primary response to oxidative stress (Quan et al., 2008), whereas glutathione reductase plays a key role in the antioxidant defense processes by reducing oxidized glutathione (GSSG) to glutathione (GSH), thus allowing the maintenance of a high GSH/GSSG ratio (Foyer and Noctor, 2005). Roots grown in the presence of BSO would be unprotected by the glutathione diminution. Frontiers in Environmental Science \| www.frontiersin.org 7 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion TABLE 2 \| Protein identification through MALDI-TOF from Uncaria tomentosa root cultures under BSO-JA treatment. Spot no. Identified protein Organism Accession no. ENERGY METABOLISM 1 Triosephosphate isomerase chloroplastic Secale cereale P46225 2 Triosephosphate isomerase cytosolic Oryza sativa subsp. japonica P48494 8 Triosephosphate isomerase Solanum tuberosum Q3HRV9 Rubisco large chain Uncaria tomentosa D8V9G4 PHOTOSYNTHESIS 4 PROTEIN SYNTHESIS 5 Putative glycine-rich RNA binding protein Catharanthus roseus Q9M6A1 10 Methionine synthase Solanum tuberosum Q9LM03 11 Cluster of putative chloroplast cysteine synthase Nicotiana tabacum Q3LAG6 SECONDARY METABOLISM 7 Strictosidine synthase Ophiorrhiza pumila Q94LW9 9 Strictosidine synthase Mitragyna speciosa B7SFV7 12 Caffeic acid O-methyltransferase Coffea canephora Q8LL87 ROS SCAVENGING, DEFENSE, STRESS RELATED 3 Ascorbate peroxidase Nicotiana tabacum Q40589 6 Proteasome alpha subunit Solanum tuberosum Q38HT0 13 Universal stress protein Salvia miltiorrhiza KOA1Y2 14 Pathogenesis-related protein Olea europea J7FNN9 necessary for degradation of damaged proteins and for maintaining cellular protein homeostasis (Kurepa et al., 2009). Evidence obtained in U. tomentosa BSO-JA elicited cultures indicates posttranslational modifications of STR proteins in correlation with the three times increase in the STR enzyme activity. Six isoforms of the glycosylated enzyme STR have been detected in C. roseus (De Waal et al., 1995), while in these cell cultures five STR isoforms were induced after elicitation with P. aphanidermun (Jacobs et al., 2005). It is known that jasmonic acid acts as a signal for the biosynthesis of MIA, and is involved in the activation of transcription factors such as ORCA, which have shown to activate transcription of the STR (Peebles et al., 2009). Another interesting protein identified as up-accumulated in the present study was caffeic acid-O-methyl transferase, one of the key enzymes that catalyzes O-methylation of the hydroxyl group at C5 in phenolic rings (Tu et al., 2010). In general, most methyltransferases possess a broad substrate permissiveness, which also includes several alkaloid N-methyltransferases (Zubieta et al., 2003; Nomura and Kutchan, 2010). The ascorbate peroxidase, which constitutes one of the most important antioxidant systems for removal of H2 O2 generated in the cell, was also up-expressed by BSO-JA addition. Deficiency of cytosolic ascorbate peroxidase occasioned accumulation of H2 O2 and consequently damage in specific proteins of leaf cells (Davletova et al., 2005). In U. tomentosa root cultures, BSO-JA elicitation induced intracellular JA and H2 O2 accumulation by glutathione depletion (Figure 4). They act as a signal transducers and secondary messengers, triggering signaling cascades and activating certain late genes that regulate the activity of detoxifying enzymes associated with antioxidant compounds. Therefore, production of alkaloids and specific phenylpropanoids is also activated, has been suggested that vacuolar alkaloids, peroxidases, and phenolic derivatives can function as a hydrogen peroxide scavenging system (Ferreres et al., 2011). Differentially Expressed Proteins after BSO-JA Elicitation Identification of proteins that differ in stressed and control plants has revealed groups of proteins that respond to oxidative stress conditions with different roles. Nevertheless, the crucial limitation for protein identification using mass spectrometry analysis is the lack of the sequence data of genes and proteins of U. tomentosa. The SWISS-PROT database (November 2014) only contains five protein entries for this species. Consequently, identification of proteins from 2D-gels requires the knowledge of the sequence data and not relying solely on peptide masses. Several studies reported that oxidative stress provoked different responses such as induction or more often repression of the enzymes involved in carbon metabolism. Therefore, plants must be required to make an economical use of their metabolites and energy to deal with adverse environments (Zhang et al., 2012). It has also been reported that under conditions of oxidative stress, Rubisco was differentially regulated even though its activity decreased having transcriptional and translational repression thereof caused by jasmonates (Weidhase et al., 1987). Moreover, JA stimulates the glutathione, ascorbate and cysteine accumulation while increases dehydroascorbate reductase activity. This last is a relevant enzyme involved in the ascorbate recycling system (Sasaki-Sekimoto et al., 2005). Cysteine synthase is a key enzyme in cysteine biosynthesis, which constitutes one of the significant factors limiting GSH biosynthesis in plants (Vierling, 1991). Proteolysis-related proteins like proteasome alpha subunit were also more abundant in stressed conditions because they are Frontiers in Environmental Science \| www.frontiersin.org 8 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion ascorbate or GSH are unable to be regenerated into the cell (Guo and Ohta, 1993). In order to maintain the redox equilibrium in the cell, production of antioxidant compounds as polyphenols and alkaloids (secondary metabolites) could be induced (Ramos-Valdivia et al., 2012). Polyphenols (Ph) can detoxify H2 O2 as electron donors by peroxidases (PDO), which results in formation of the respective phenolic radical (Ph•). This species can be regenerated by a non-enzymatic reaction with ascorbate. Dehydroascorbate (DHA) may be reduced back to ascorbate by a dehydroascorbic acid reductase (DHAR) (Ferreres et al., 2011). Superoxide dismutase (SOD); glutathione peroxidase (GPx). FIGURE 4 \| Schematic network of alkaloid and polyphenol activation through signal transduction caused by oxidative stress in Uncaria tomentosa root cultures. The elicitation with BSO-JA activates a signal cascade into the cell which include Ca2+ influx, cytosolic free Ca2+ , stimulation of peroxidases, NADPH oxidases, and phospholipases, which further generates other signaling messengers, such as ROS (Quan et al., 2008) and JA together with inducible secondary metabolites production (Zhao et al., 2005; Peebles et al., 2009). Inhibition of GSH biosynthesis by BSO could provoke an increase in ROS (H2 O2 ) due to non-enzymatic scavengers such as protecting roots from oxidative stress damage. Identification of proteins with diverse roles that are present in oxidative stress conditions evidences the complexity of the responses. This approach contributes to the understanding of the metabolic mechanisms operating in U. tomentosa subjected to oxidative stress and the manner how this plant produces the appropriate adjustments for tolerating them. Fontaine for technical support. We appreciate the proteomics analytical support of Dr. George Tsaprailis (Arizona Proteomics Consortium). Mass spectrometry and proteomics data were acquired by the Arizona Proteomics Consortium supported by NIEHS grant ES06694 to the SWEHSC, NIH/NCI grant CA023074 to the UA Cancer Center and by the BIO5 Institute of the University of Arizona. Acknowledgments Supplementary Material This research was financed by CINVESTAV-IPN and CONACYT-Mexico (222097). IV thank CONACYT-Mexico for a doctoral (173034) fellowship. Authors wish to thank C. The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fenvs. 2015.00027/abstract Frontiers in Environmental Science \| www.frontiersin.org 9 April 2015 \| Volume 3 \| Article 27 Vera-Reyes et al. Secondary metabolites and glutathione depletion References micropropagated plantlets and root cultures. Biotechnol. Lett. 35, 791–797. doi: 10.1007/s10529-012-1128-8 May, M. J., and Leaver, C. J. (1993). Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiol. 103, 621–627. McKnight, T. D., Roessner, C. A., Devagupta, R., Scott, A. I., and Nessler, C. L. (1990). Nucleotide sequence of a cDNA encoding the vacuolar protein strictosidine synthase from Catharanthus roseus. Nucleic Acids Res. 18, 4939–4939. doi: 10.1093/nar/18.16.4939 Meyer, A. J., and Fricker, M. (2002). Control of demand-driven biosynthesis of glutathione in green Arabidopsis suspension culture cells. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 11 April 2015 \| Volume 3 \| Article 27
https://openalex.org/W3047245569	http://dirros.openscience.si/Dokument.php?id=10086&dn=	English	null	Mesto imunoterapije pri zdravljenju raka	DOAJ (DOAJ: Directory of Open Access Journals)	2,017	cc-by	3,610	ABSTRACT The immunotherapy era began in 1893. Nearly a hundred years later, the first immunotherapy drugs were approved, for instance interferon and interleukin. The role of the immune system in cancer is best represented by the cancer-immunity cycle. In the first step of the cycle, tumor neo-antigens are released and, subsequently, captured by the antigen-presenting cells. In the second step, neo-antigens are presented to the T-cells in the lymph nodes. But additional co-stimulatory signals are needed for the priming and the activation of T cells. At that level, the potential inhibitory signals inhibit or prevent the hyperactivation of T cells and auto immunity. The activated T cells migrate through circulation into the tumor tissue. A tumor cell is detected by a T-cell receptor. The result of this process is the so-called T-cell killing. A tumor stroma, however, can present different inhibitory signals that inhibit the function of cytotoxic T cells. The article presents the different ways of influencing the cancer-immunity cycle and the readily-approved immunotherapy drugs. Začetni poskusi zdravljena z imunoterapijo segajo v leto 1893. Skoraj stoletje kasneje pa so bila odobrena prva zdravila, ki jih štejemo med imunoterapijo, kot sta interferon in interlevkin. Vlogo imunskega sistema pri raku znanstveniki v zadnjih letih razlagajo s pomočjo protitumorskega imunskega cikla. V prvem koraku tega cikla pride do sproščanja tumorskih antigenov (neoantigenov), ki jih antigen predstavitvene celice spoznajo in ujamejo. V drugem koraku jih v bezgavkah predstavijo celicam T. Za aktivacijo in determinacijo celic T v smeri citotoksičnih T-celic je potreben še dodaten stimulatorni signal. Morebitni inhibitorni signali pa aktivacijo celic T zavirajo oz. preprečujejo čezmerno aktivacijo in avtoimunost. Aktivirane celice T po krvi potujejo v tumor. Tumorsko celico prepoznajo prek T-ce ličnega receptorja. S tem preide do uničenja tumorske celice. V sami tumorski stromi pa so lahko različni inhibitorni signali, ki delovanje citotoksičnih celic T onemogočijo. V prispevku so predstavljeni možni načini vplivanja na protitumorski imunski cikel in trenutno registrirana zdravila s področja imunoterapije. Keywords: immunotherapy, cancer-immunity cycle, co-sti mulatory signals, co-inhibitory signals. Ključne besede: imunoterapija, protitumorski imunski cikel, stimulatorni signali, inhibitorni signali. Grašič Kuhar Cvetka Korespondenca: doc.dr. Cvetka Grašič Kuhar, Onkološki inštitut, Zaloška cesta 2, 1000 Ljubljana, E-mail: cgrasic@onko-i.si Poslano / Recieved: 05.11.2017 Sprejeto / Accepted: 23.11.2017 Korespondenca: doc.dr. Cvetka Grašič Kuhar, Onkološki inštitut, Zaloška cesta 2, 1000 Ljubljana, E-mail: cgrasic@onko-i.si Poslano / Recieved: 05.11.2017 Sprejeto / Accepted: 23.11.2017 6 \| ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 6 \| ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 6 ZGODOVINA IMUNOTERAPIJE tudi kemoterapijo, ki je doživela pravi razcvet šele po letu 1970. Ves ta čas je bila imunoterapija v zatonu (2). Leta 1953 je Coleyeva hči (Helen Coley Nauts) objavila rezultate zdravljenja očetovih bolnikov, ki so bili zavidljivi (1200 bolnikov, spremlja nje do 45 let, 270 kompletna remisija po zdravljenju z mešano bakterijsko vakcino), vendar vseeno ni uspela vzbuditi zanimanja za imunoterapijo pri zdravnikih (1). Leta 1953 je v New Yorku ustanovila neprofitno organizacijo Cancer Research Institute, ki deluje še danes in podpira financiranje raziskav z imunoterapijo. Raziskovanje imunosti pri raku je bilo dolga desetletja prepove dano področje. Kljub blokadi se je zanimanje za imunoterapijo vrnilo leta 1957, ko sta Thomas in Burnet prvič predstavila teorijo imunskega nadzora, vendar tehnično znanje takrat ni moglo ma nipulirati z limfociti in vitro ter ponuditi dokazov o obstoju tumor specifičnih antigenov in zato je razvoj kar nekaj let zastal (3). Uporaba imunoterapije pri zdravljenju raka je dosegla razmah v zadnjih letih. Leto 2013 so razglasili kot prodorno leto za imunoterapijo. Prvi poskusi zdravljenja z imunoterapijo pa segajo daleč nazaj v leto 1893. Takrat je ameriški kirurg dr. William B. Coley pri bolniku z inoperabilnim recidivnim rakom na vratu opazil popoln regres tumorja po lokalni okužbi (erizipelu). Po retrospektivni analizi primerov raka v svoji ustanovi je našel še več takšnih bolnikov. Na podlagi tega je pripravil mešanico mrtvih toksinov iz Streptoccoccus pyogenesa in Serratia marcescensa (Coleyev toksin ali mešana bakterijska vakcina) ter ga uporabljal za zdravljenje različnih inoperabilnih rakov (predvsem rakov glave in vratu ter sarkomov) (1). To zdravljenje se je nadaljevalo v različnih centrih v Evropi in Ameriki, vendar so bili uspehi nekonsistentni. Z razvojem radioterapije so leta 1915 ukinili uporabo Coleyevega toksina pri zdravljenju raka, ker rezultati zdravljenja niso bili vedno ponovljivi, poleg tega pa niso poznali mehanizmov delovanja imunoterapije. Med drugo svetovno vojno (1943) so za zdravljenje raka začeli uporabljati Leta 1976 so odobrili uporabo liofiliziranega bacila BCG (Myco bacterium bovis) pri raku mehurja in se še danes uporablja za karcinom in situ ter stadij T1 po transuretralni resekciji (intra vezikalna aplikacija) (4). Leta 1986 je bil odobren interferon alfa ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 \| 7 7 TEMELJNE ZNAČILNOSTI RAKA za zdravljenje dlakastocelične levkemije, kasneje pa še za druge indikacije (maligni melanom, folikularni limfom, Kapošijev sarkom pri AIDS-u, kronični hepatitis B in C …). Rak nastane v večstopenjskem procesu, vendar je vsem vrstam raka skupno, da je to zelo kompleksna bolezen, ki ima določene značilnosti. Znanje o tem, katere so temeljne značilnosti raka, je z leti naraščalo. Tako Hanahan in sod. navajajo, da ima rak 10 temeljnih značilnosti (slika 2) in ena od njih je tudi, da se rak izogne uničenju z imunskimi celicami (9). Nato so poročali o vlogi imunskega sistema pri raku ledvic in malignem melanomu (5, 6). Leta 1992 in leta 1998 je bilo za navedena raka odobreno zdravljenje z interlevkinom-2, ki je sicer precej toksično, vendar v 20 odstotkih lahko inducira trajne remisije. V letu 2010 je bila odobrena prva vakcina z dendri tičnimi celicami za zdravljenje hormonsko rezistentnega raka prostate, leta 2011 pa prvi inhibitor kontrolnih točk (ipilimumab); slika 1 (7, 8). Genomska nestabilnost in mutacije Izogibanje uničenju z imunskimi celicami Indukcija angiogeneze Omogočenje neskončne delitve Izogibanje rastnim zaviralcem Aktivacija invazije in metastaziranja Celica se upira smrti Deregulacija celične energije Vnetje, ki pospešuje rast tumorja Selektivni antiinflamatorni agensi CDK inhibitorji EGFR inhibitorji Anti CTL-A4, anti PD-1 Inhibitorji aerobne glikolize Inhibitorji telomerase PARP inhibitorji Proapoptotični BH3 mimetiki Stalni signali za proliferacijo Inhibitorji HGF/c- Met Inhibitorji VEGF signalov Slika 2. Novejši koncept potrebnih mehanizmov nastanka raka (oglati okvirčki) in možna tarčna terapija (ovalni okvirčki). Prirejeno po Hanahan in sod. (9). Šele z razjasnitvijo delovanja imunskega sistema in posameznih imunskih celic pri zdravem človeku je bilo možno razumevanje njegove vloge pri raku. Pri raku so pomembne celice naravne imunosti (dendritične celice) in celice pridobljene imunosti (celice T). Pomembno je, da se morajo celice T potem, ko zaključijo imunski odziv, izklopiti, da preprečijo avtoimunost. To dosežejo tako, da izražajo večje število inhibitorjev kontro lnih točk in inducirajo nastanek regulatornih T-celic (Treg), ki izklopijo proliferativno fazo aktiviranega T-celičnega odgovora. Vnetje, ki pospešuje rast tumorja Indukcija angiogeneze Slika 1. Mejniki v razvoju imunoterapije pri raku. Povzeto po Ventola in sod. (2). Slika 2. Novejši koncept potrebnih mehanizmov nastanka raka (oglati okvirčki) in možna tarčna terapija (ovalni okvirčki). Prirejeno po Hanahan in sod. (9). Slika 2. Novejši koncept potrebnih mehanizmov nastanka raka (oglati okvirčki) in možna tarčna terapija (ovalni okvirčki). Prirejeno po Hanahan in sod. (9). PROTITUMORSKI IMUNSKI CIKEL Vlogo in razumevanje imunskega sistema pri nastanku raka lahko ponazorimo s t. i. protitumorskim imunskim ciklom (slika 3). Pri raku pride do akumulacije genetskih sprememb in izgube normalnih regulatornih procesov. To povzroči izražanje neoanti genov ali diferenciacijskih genov, ki se izrazijo kot peptidi, vezani na molekule glavnega histokompatibilnega sistema I (MHC I), na površini rakavih celic. S tem se ločijo od normalnih celic in jih prepoznajo CD8+ celice T, ki nastajajo spontano pri bolnikih z rakom. Desetletja so raziskovalci skušali doseči, da bi povzročili nastanek celic T, specifičnih proti rakavemu antigenu. Šele nekaj let je znano, kako imunski sistem učinkovito prepozna in uniči rakave celice. Vse mora potekati učinkovito in natančno reguli rano v sosledju, kot je predstavljeno v protitumorskem imunskem ciklu (10, 11). 8 \| ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 8 Slika 3. Protitumorski imunski cikel se dogaja na poti med tumorjem, bezgavko in krvožiljem. Pri vsaki točki je opisano, katere celice so udeležene. Cikel se ponavlja. Povzeto po Chen in sod. (11). Slika 3. Protitumorski imunski cikel se dogaja na poti med tumorjem, bezgavko in krvožiljem. Pri vsaki točki je opisano, katere celice so udeležene. Cikel se ponavlja. Povzeto po Chen in sod. (11). Slika 4. Spodbujevalni (stimulatory) ali zaviralni (suppressive) dejavni ki (co-signals), ki soodločajo o nadaljnji aktivaciji ali zavori na nivoju antigen predstavitvena celica (APC) - celica T (T-cell), ki poteka v bezgavki oz. na nivoju celica T-tumorska celica (target cell), ki se odvija v tumorskem tkivu. Vir: Slike (12). Neoantigene iz odmirajočih rakavih celic ali iz črevesnih mikroorganizmov ujamejo dendritične celice in jih preko MHC I in MHC II v bezgavkah predstavijo celicam T. Pri tej prestavitvi sodelujejo še različni spodbujevalni ali zaviralni dejavniki (slika 4). Za aktivacijo celic T sta potrebna dva aktivirajoča signala. Če je aktivacija celic T uspešna, nastanejo specifične efektorske (citotoksične) celice T, ki po krvi potujejo do tumorja in infiltri rajo tumorsko stromo. Prek T-celičnega receptorja in antigena se vežejo na MHC I in nato ubijejo rakavo celico. Tudi tu sodelujejo številni modulatorji, večina je inhibitornih, ki preprečujejo efek torsko delovanje citotoksičnih celic T (slika 4). Če je aktivacija citotoksičnih celic T uspešna, tumorske celice nekrotizirajo in se ob tem sproščajo dodatni tumorski neoantigeni, tako da se lahko poglobi in razširi imunski odgovor na tumor. Pri bolnikih, ki razvijejo klinično zaznaven rak, ta cikel žal ne deluje optimalno. CILJI IMUNOTERAPIJE PRI RAKU nega T-celičnega odgovora in nastanejo proti tumorju specifične celice T, poleg teh pa še specifične autoreaktivne celice T (kar pa povzroča znatne avtoimunske neželene učinke ipilimumaba) (8, 10, 11). Ipilimumab tako odstrani zavoro za nastanek citotoksič nih celic T in pri določenem deležu bolnikov (20–30 %) povzroči trajne klinične odgovore (8). Predvideva se, da tudi pospeši odstranjevanje Treg (ki preprečujejo avtoimunost). Cilj imunoterapije je zagnati ali ponovno zagnati samodejno delujoč protitumorski imunski cikel, vendar v takšni meri, da ne povzroča avtoimunosti. Ker ima cikel več kontrolnih točk in zaviralcev na vsaki od teh, je najučinkovitejši pristop selektivno tarčno delovanje na tisti člen, ki je najpočasnejši. Najpogosteje se je izkazalo, da je ta člen imunosupresija v sami tumorski stromi. Če bi pomnožili delovanje celotnega protitumorskega cikla, bi to povzročilo resne neželene učinke na normalna tkiva. Na nivoju prepoznavanja tumorskih celic v tumorski stromi je pomemben T-celični receptor. Za tumorske celice je značilno, da znižajo ekspresijo MHC peptidov na svoji površini (proces se imenuje tudi 'immune editing') in s tem uidejo napadu imunskih celic. V septembru 2017 je bila odobrena prva CAR (chimeric antigen receptor) T-celična terapija. Gre za imunoterapijo z gensko modificiranimi T-celicami (vektor za vnos gena je lenti virus), ki je usmerjena proti tarčnim celicam, ki izražajo antigen, povezan z rakom. Pri B-celičnih levkemijah je takšen antigen CD19. Zunanji del CAR-proteina je enoverižni fragment mišjega protitelesa, znotrajcelični del CAR-proteina pa ima domeno za signaliziranje T-celic (CD3-z) in kostimulatorni domeni (CB28, 4-1BB). Posledica je, da pride do ekspanzije CAR T-celic in do sproščanja citokinov. Terapija je zelo toksična (sindrom sprošča nja citokinov, odpoved ledvic, nevrotoksičnost, koagulopatije, možganski edem). Bolniki potrebujejo intenzivno nego, pogosto umetno ventilacijo in hemodializo. Tisagenlecleucel je prva takšna terapija za ponovljeno ali refraktarno B-celično akutno limfatično levkemijo pri otrocih med tretjim in 25. letom starosti (14, 15). Zaradi inkorporacije lentivirusa (ali retrovirusa pri drugih CAR celicah T) v genom, obstaja tveganje, da bo vstavljen virus pridobil sposobnost replikacije, da bo povzročil insercijsko mutagenost (sekundarni raki) in genotoksičnost (povzročitev genskih okvar in prenos le teh na potomce). PROTITUMORSKI IMUNSKI CIKEL Vzroki so lahko: imunske celice ne zaznajo tumorskih antigenov (tumorske celice postanejo neimunogene), dendritične celice in T-celice obravnavajo tumorske gene kot lastne (ne kot tuje), tako da se tvorijo Treg celice, T-celice neustrezno ali sploh ne infiltri rajo tumorske strome ali pa (najpogostejši vzrok) mikrookolje v tumorju inhibira celice T (10, 11). Slika 4. Spodbujevalni (stimulatory) ali zaviralni (suppressive) dejavni ki (co-signals), ki soodločajo o nadaljnji aktivaciji ali zavori na nivoju antigen predstavitvena celica (APC) - celica T (T-cell), ki poteka v bezgavki oz. na nivoju celica T-tumorska celica (target cell), ki se odvija v tumorskem tkivu. Vir: Slike (12). ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 \| 9 MOŽNI NAČINI VPLIVANJA NA PROTITUMORSKI IMUNSKI CIKEL Možni načini vplivanja na imunski cikel na vsaki izmed kontrol nih točk so predstavljeni na sliki 5. Tudi zdravljenje s kemoterapi jo, radioterapijo in tarčno terapijo, ki uniči rakave celice, privede do sproščanja tumorskih antigenov in s tem aktivacije imunskega cikla. Slika 5. Protitumorski imunski cikel in način vplivanja nanj. Vir: Chen in sod. (11). Na nivoju tumorske strome delujejo monoklonska protitelesa proti receptorju za programirano smrt (PD-1, izraženem na celicah T) in ligandu za receptor za programirano smrt (PD-L1). PD-L1 je distalni negativni imunski regulator, ki je izražen v stromi 20 do 50 odstotkov tumorjev in povzroča, da se v citoto ksičnih celicah T ne tvorijo in ne sproščajo citotoksične snovi. Protitumorski cikel je lahko intakten do te zadnje točke ubijanja tumorskih celic. Če to blokado preprečimo, vidimo lahko odgovor tumorja na terapijo v nekaj dneh. Pri zdravljenju z anti PD-L1 in anti PD-1 protitelesi opažamo veliko manj imunsko pogojenih neželenih učinkov kot anti CTLA-4. Pri 20 do 50 odstotkih bolnikov so možni dolgotrajni odgovori na terapijo (8). Slika 5. Protitumorski imunski cikel in način vplivanja nanj. Vir: Chen in sod. (11). Na nivoju antigen predstavitvenih celic delujeta citokin interfe ron alfa (IFN-a) in vakcina iz dendritičnih celic (sipuleucel-T). Na splošno pridobijo tovrstne vakcine tako, da dendritične celice (najmočnejše antigen predstavitvene celice) pridobijo iz človeka (bolnika), centrifugirajo, prenesejo na kulturo, kjer jih namnožijo in aktivirajo s tumorskimi antigeni (prek avtolognega ali alogenega tumorskega lizata, sintetičnih peptidov, mRNK ali celo prek virusnega vektorja) ter nato vrnejo bolniku z vakcino. S tem močno stimulirajo T-celični imunski odgovor. Pri sipuleucelu je imunski sistem stimuliran proti antigenu kisla prostatična fosfataza, ki je močno izražena na večini rakastih celic raka prostate (7). Vakcina kot samostojno zdravljenje po navadi ni dovolj uspešna, saj ji nasprotuje delovanje imunosupresornih molekul, ki nastajajo v tumorski stromi (10–13). KLINIČNA UPORABA IMUNOTERAPIJE PRI RAKU Pri pembrolizumabu je pri zadnji odobreni indikaciji treba poudariti, da se je zgodilo prvič, da je FDA odobrila zdravilo za zdravljenje raka na podlagi značilnosti raka (pomanjkljivi popravljalni mehanizmi (MMR (mismatch repair deficiency)) oz. visokega deleža mikrosatelitne nestabilnosti – (MSI-H: high mi crosatelite instability) in to ne glede na histološki izvor (oz. organ izvora) raka. Gre za rake, ki so hipermutirani in tako izražajo ogromno neoantigenov, zato jih imunske celice prepoznajo in je imunoterapija učinkovita. Tu gre za personalizirano imunotera pijo. Takšnih rakov je okrog 4 odstotke vseh rakov. Kar pa je zelo pomembno, gre za rake, ki so redki raki ali zanje doslej ni bilo uspešne terapije (rak endometrija, materničnega vratu, želodca, tankega črevesja, sarkom uterusa, holangiokarcinom …) (17). KLINIČNA UPORABA IMUNOTERAPIJE PRI RAKU Najdlje v razvoju in klinični uporabi so zdravila, ki vplivajo na aktivacijo celic T v bezgavkah (protitelesa proti CTLA-4). Njim so sledila protitelesa proti receptorjem na celicah T (PD-1) ali ligandom v tumorski stromi, ki preprečujejo delovanje celic T (PD-L1). Paleta odobrenih zdravil se veča in je prikazana v tabeli 1. Nekatera od teh zdravil v Evropi še niso odobrena. Od odobrenih zdravil večina v Sloveniji še ni dostopna ali pa ni dostopna za vse registrirane indikacije (16). Na naslednji kontrolni točki v imunskem ciklu, to je na nivoju aktivacije celic T, delujeta citokin interlevkin 2 in ipilimumab. Ipilimumab je monoklonsko protitelo proti CTLA-4 (common T lymphocyte antigen 4) in ta blokada preprečuje vezavo CTLA-4 z njegovimi ligandi (B7.1 in B7.2). S tem ni več regulatorne vloge CTLA-4, zaradi česar pride do nenadzorovanega in prekomer 10 \| ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 10 Tabela 1. Odobrena nova zdravila s področja imunoterapije (t. i. inhibitorji kontrolnih točk) v Evropi (EMA – Evropska agencija za zdravila) ali ZDA (FDA – Ameriška agencija za zdravila) ter Sloveniji (SLO). MM: maligni melanom; NSCLC: nedrobnocelični pljučni karcinom; SCHNC: ploščatocelični rak glave in vratu; UCC: urotelni karcinom; MMR (mismatch repair) deficientni: s pomanjkljivim popravljalnim mehanizmom za okvare med podvojitvijo DNK; MSI-H: z visoko mikrosatelitno nestabilnostjo. Povzeto po Haanen in sod. (16). Zdravilo Tarča Indikacija Odobritev Ipilimumab CTLA-4 Metastatski MM Adjuvantno MM stadij III FDA+EMA+SLO FDA Nivolumab PD-1 Metastatski MM 2. linija metastatski NSCLC 2. linija metastatski RCC Ponovljeni/metastatski SCHNC Napredovali rak ledvic Lokalno napredovali/metastatski UCC Klasični Hodgkinov limfom FDA+EMA+SLO FDA+EMA+SLO FDA+EMA+SLO FDA+EMA FDA+EMA+SLO FDA+EMA FDA+EMA Pembrolizumab PD-1 Metastatski MM 2. linija metastatski NSCLC (PD-L1 >=1 %) 1. linija metastatski NSCLC (PD-L1 >=50 %) 2. linija NSCLC v komb. pemetr+karboplatin Klasični Hodgkinov limfom Lokalno napredovali/metastatski UCC MSI-H ali MMR deficientni metastatski raki FDA+EMA+SLO FDA+EMA+SLO FDA+EMA+SLO FDA FDA+EMA+SLO FDA FDA Atezolizumab PD-L1 2. linija metastatski NSCLC Lokalno napredovali/metastaski UCC FDA FDA Avelumab PD-L1 Lokalno napredovali/metastatski UCC Metastaski karcinom Merklovih celic FDA FDA Durvalumab PD-L1 Lokalno napredovali/metastatski UCC FDA Ipilimumab + nivolumab CTLA-4, PD-1 Metastatski MM FDA+EMA PD-1 in PD-L1. Po drugi strani pa tumorji, ki niso infiltrirani z imunskimi celicami (t. i. hladni tumorji), ne odgovorijo na inhibitorje PD-1 in PD-L1. Trenutno že vemo, da je kombinacija inhibitorjev PDLA-4 in PD-1 bolj učinkovita kot monoterapija, vendar ima tudi več in huje izražene neželene učinke. NEŽELENI UČINKI IMUNOTERAPIJE Neželeni učinki imunoterapije so drugačni kot pri drugih oblikah sistemske terapije. Ker imunoterapija deluje v smislu ponovnega zagona imunskega cikla in preprečevanja zavor imunskega sistema, so neželeni učinki 'imunsko pogojeni' (13, 17, 18). Gre za različne '–itise' (tabela 2). Najpogostejši neželeni učinki so splošni (utrujenost, gripozno počutje), kožni in gastrointesti nalni (predvsem pri ipilimumabu, kjer lahko pride do življenjsko nevarnega kolitisa) ter motnje v delovanju endokrinih žlez. Predvidevamo lahko, da bi bilo zdravljenje z vakcinami in terapijo CAR bolj učinkovito, če bi ga kombinirali z inhibitorji ONKOLOGIJA \| ISSN 1408-1741 \| PREGLEDNI STROKOVNI ČLANEK \| LETO XXI \| ŠT. 2 \| DECEMBER 2017 11 kontrolnih točk (anti CTLA-4, anti PD-1, anti PD-L1). Organski sistem Vrsta neželenega učinka KOŽA izpuščaj, pruritus, vitiligo, psoriaza, Steven-Johnsonov sindrom, izpuščaj po zdravilih s sistemskimi učinki GASTROINTESTINALNI TRAKT kolitis, ileitis, gastritis, pankreatitis, celiakija MIŠICE, SKELET artritis, miopatije, dermatomiozitis ENDOKRINE ŽLEZE hipotireoza, hipertireoza, insuficienca nadledvične žleze, diabetes, hipofizitis JETRA hepatitis LEDVICE nefritis RESPIRATORNI SISTEM pneumonitis, plevritis, sarkoidozi podobna granulomatoza OČI uveitis, konjunktivitis, skletitis, episkleritis, blefaritis, retinitis, horoiditis, orbitalna miopatija ŽIVČEVJE neuropatija, mielopatija, mielitis, encefalitis, meningitis, Guillain-Barre sindrom, miastenija KARDIOVASKULARNI SISTEM miokarditis, perikarditis, vaskulitis HEMATOPOETSKI SISTEM anemija (aplastična, autoimunska hemolitična), autoimunska trombocitopenija 4. Morales A, Eideger D, Bruce A.W. Intracavitary Bacillus Calmette-Guérin in the treatment of superficial bladder tumor. 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https://openalex.org/W2784968772	https://hal-pasteur.archives-ouvertes.fr/pasteur-01868255/file/2018A-Raimondi-Microbial-Cell.pdf	English	null	Molecular signature of the imprintosome complex at the mating-type locus in fission yeast	Microbial cell	2,018	cc-by	12,970	To cite this version: Célia Raimondi, Bernd Jagla, Caroline Proux, Hervé Waxin, Serge Gangloff, et al.. Molecular signature of the imprintosome complex at the mating-type locus in fission yeast.. Microbial Cell , 2018, 5 (4), pp.169 - 183. 10.15698/mic2018.04.623. pasteur-01868255 Molecular signature of the imprintosome complex at the mating-type locus in fission yeast. Raimondi, Bernd Jagla, Caroline Proux, Hervé Waxin, Serge Ganglof Benoît Arcangioli Célia Raimondi, Bernd Jagla, Caroline Proux, Hervé Waxin, Serge Gangloff, Benoît Arcangioli Distributed under a Creative Commons Attribution 4.0 International License Research Article www.microbialcell.com Abbreviations: Abbreviations: ChiP-seq – chromatin- immunoprecipitation and whole genome sequencing, DSB – double-strand break, HMG – high mobility group, M – minus, MT – mating type, P – plus. INTRODUCTION act as boundary elements [8, 9]. Additionally, a centromer- ic repeated sequence located between mat2P and mat3M, CEN-H, promotes the formation of heterochromatin ([10], see Figure 1). A salient feature of the MT loci is that they are flanked by homologous sequences. The H1 homology box (59 bp) is located on the centromere distal (right) side of the cassettes and the H2 homology box (135 bp) on the centromere proximal (left) side [2]. Both sequences are thought to be essential for base pairing during the initia- tion and resolution steps of the gene conversion process required for MT switching [11, 12]. The H3 box (57 bp) is located to the left of H2 at mat2 and mat3 [2], and is not required for MT switching [13]. HAL Id: pasteur-01868255 https://pasteur.hal.science/pasteur-01868255v1 Submitted on 5 Sep 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 Molecular signature of the imprintosome complex at the mating-type locus in fission yeast Célia Raimondi1, Bernd Jagla2, Caroline Proux3, Hervé Waxin4, Serge Gangloff1, Benoit Arcangioli1,* 1 Genomes and Genetics department, Genome Dynamics Unit, UMR 3525 CNRS, Institut Pasteur, 25-28 rue du docteur Roux, Paris, France. Sorbonne Universités, Université Pierre et Marie Curie, Institut de Formation Doctorale, 75252 Paris Cedex 05, France. 2 Center for Human Immunology, CRT & Hub de Bioinformatique et Biostatistiques, C3BI, Institut Pasteur, 25-28 rue du Docteur Roux Paris, France. 3 Genomes and Genetics department, Plate-forme Transcriptome & Epigenome, Biomics, Centre d’Innovation et Recherche Technologique (Citech), Institut Pasteur, 25-28 rue du Docteur Roux, Paris, France. 4 Enseignement, Institut Pasteur, 25-28 rue du Docteur Roux, Paris, France. * Corresponding Author: Benoit Arcangioli, Genomes and Genetics department, Genome Dynamics Unit, UMR 3525 CNRS, Institut Pasteur, 25-28 rue du docteur Roux, Paris, France. Sorbonne Universités, Université Pierre et Marie Curie, Institut de Formation Doctorale, 75252 Paris Cedex 05, France; Tel: 0033145688454; E-mail: benoit.arcangioli@pasteur.fr doi: 10.15698/mic2018.04.623 Received originally: 05.09.2017; in revised form: 21.12.2017, Accepted 02.01.2018, Published 16.01.2018. ABSTRACT Genetic and molecular studies have indicated that an epigenetic imprint at mat1, the sexual locus of fission yeast, initiates mating type switch- ing. The polar DNA replication of mat1 generates an imprint on the Watson strand. The process by which the imprint is formed and maintained through the cell cycle remains unclear. To understand better the mechanism of im- print formation and stability, we characterized the recruitment of early play- ers of mating type switching at the mat1 region. We found that the switch activating protein 1 (Sap1) is preferentially recruited inside the mat1M allele on a sequence (SS13) that enhances the imprint. The lysine specific demethyl- ases, Lsd1/2, that control the replication fork pause at MPS1 and the for- mation of the imprint are specifically drafted inside of mat1, regardless of the allele. The CENP-B homolog, Abp1, is highly enriched next to mat1 but it is not required in the process. Additionally, we established the computational signa- ture of the imprint. Using this signature, we show that both sides of the im- printed molecule are bound by Lsd1/2 and Sap1, suggesting a nucleoprotein protective structure defined as imprintosome. Keywords: imprint, mating type switching, epigenetics, replication, Lsd1, Lsd2, Sap1. OPEN ACCESS \| www.microbialcell.com INTRODUCTION FIGURE 1: Lsd1, Lsd2, Sap1 and Abp1 are recruited to the mating type region. (A) Distribution of normalized enrichments of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs in a h90 strain (IP RPkM reads per kilobase million/ WCE RPkM). The sequence used for the alignment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. The 3 mat loci are indicated as well as the CEN-H region and the inverted re- peats IR-L and IR-R. Light gray boxes show genes present in this region. (B) Same than in A) except that the sequence used for the alignment is a 44-kb region that contains the MT region with the P allele at mat1: h90 P. is site-specific but sequence-independent [19, 20]. Inter- estingly, the position of the break on the Watson strand at mat1 differs by three nucleotides between the mat1P and mat1M alleles [22], indicating that the position is dictated from within the mat1M and mat1P sequences. The imprint is protected against DNA repair and remains stable throughout the entire length of the cell cycle to be transi- ently converted to a polar DSB during the following S- phase [17, 20]. Repair of this break is not random; mat1P prefers mat3M, and mat1M prefers the nearby mat2P in 90% of switches. This preference is called directionality of switching [23]. In strains in which the donor sequences are deleted [24], the position and level of the imprint are iden- tical to that in wild type, but the DSB is repaired off the sister [24, 25]. Extensive pedigree analysis at the single-cell level demonstrated that two consecutive divisions are required to produce one switched cell among four related cousins. Fission yeast possesses a remarkable genetically pro- grammed system initiated by a site and DNA strand- specific imprint at the mat1 locus for changing its MT (re- viewed in [14-16]). The first division produces the imprint on one of the sister chromatids at mat1, while the second division triggers a double-strand break (DSB) on the im- printed chromatid that initiates MT switching [17]. It was shown that the polarity of DNA replication of mat1 is instrumental in the establishment and the strand specificity of the imprint [18]. INTRODUCTION Haploid Schizosaccharomyces pombe cells exist as two mating types (MTs), P (for plus) and M (for minus), that switch during cell divisions. The MT of the cell is deter- mined by the allele (M or P) expressed at the mat1 locus on the right arm of chromosome II, 500 kb away from the centromere: mat1P in P cells and mat1M in M cells [1, 2]. The mat1 allele can be replaced efficiently by genetic in- formation contained in one of the two silent donor cas- settes mat2P and mat3M [2-4]. mat2P and mat3M are located 17 kb and 29 kb centromere-distal to mat1, respec- tively [3, 5], and are maintained in a silent chromatin state preventing their transcription or recombination [5-7]. Two inverted repeats flank the donor region (IR-L and IR-R) and Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 169 C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 1: Lsd1, Lsd2, Sap1 and Abp1 are recruited to the mating type region. (A) Distribution of normalized enrichments of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs in a h90 strain (IP RPkM reads per kilobase million/ WCE RPkM). The sequence used for the alignment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. The 3 mat loci are indicated as well as the CEN-H region and the inverted re- peats IR-L and IR-R. Light gray boxes show genes present in this region. (B) Same than in A) except that the sequence used for the alignment is a 44-kb region that contains the MT region with the P allele at mat1: h90 P. FIGURE 1: Lsd1, Lsd2, Sap1 and Abp1 are recruited to the mating type region. (A) Distribution of normalized enrichments of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs in a h90 strain (IP RPkM reads per kilobase million/ WCE RPkM). The sequence used for the alignment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. The 3 mat loci are indicated as well as the CEN-H region and the inverted re- peats IR-L and IR-R. Light gray boxes show genes present in this region. (B) Same than in A) except that the sequence used for the alignment is a 44-kb region that contains the MT region with the P allele at mat1: h90 P. OPEN ACCESS \| www.microbialcell.com INTRODUCTION The in- volvement of Lsd1 in DNA replication and DNA damage response is not new [30], and we showed that Lsd1/2 act redundantly to promote the pause through both their HMG (high mobility group) domain and amine oxidase ac- tivity. In addition, the recruitment of Lsd1/2 to mat1 re- quires SS2 [27]. Swi1/3 proteins are associated with the replication fork and stabilize it during the pause [28, 31]. Sap1, the Switch Activating Protein 1 is an essential gene that interacts with SAS1, a sequence covered by the Msmt- 0 deletion [32]. Sap1 was also described to play a role in DNA replication [33-36], retrotransposition targeting [37, 38] and chromosomal organization [39, 40]. Finally, Abp1 (ARS Binding Protein 1), a CENP-B (Centromeric Protein B) homolog that antagonizes Sap1 at LTRs (long terminal re- peat), is enriched next to SAS1, to the right of mat1M [35]. Abp1 was proposed to control the directionality of switch- ing by regulating the alternative expression of the swi2 gene (Switch gene 2) [41-43], important for the spreading of the Swi2/Swi5 recombination-promoting complex on the mat2/3 region [43]. proteins [29]. The complex also associates with topoiso- merase 2 (Top2), replication factor A (Rfa1) and Sap1 [29], essential proteins with a role in DNA replication. The in- volvement of Lsd1 in DNA replication and DNA damage response is not new [30], and we showed that Lsd1/2 act redundantly to promote the pause through both their HMG (high mobility group) domain and amine oxidase ac- tivity. In addition, the recruitment of Lsd1/2 to mat1 re- quires SS2 [27]. Swi1/3 proteins are associated with the replication fork and stabilize it during the pause [28, 31]. Sap1, the Switch Activating Protein 1 is an essential gene that interacts with SAS1, a sequence covered by the Msmt- 0 deletion [32]. Sap1 was also described to play a role in DNA replication [33-36], retrotransposition targeting [37, 38] and chromosomal organization [39, 40]. Finally, Abp1 (ARS Binding Protein 1), a CENP-B (Centromeric Protein B) homolog that antagonizes Sap1 at LTRs (long terminal re- peat), is enriched next to SAS1, to the right of mat1M [35]. Abp1 was proposed to control the directionality of switch- ing by regulating the alternative expression of the swi2 gene (Switch gene 2) [41-43], important for the spreading of the Swi2/Swi5 recombination-promoting complex on the mat2/3 region [43]. INTRODUCTION To advance our understanding of the process of imprint formation and stability, we analyzed by chromatin- immunoprecipitation and whole genome sequencing (ChIP- seq) the recruitment of Abp1, Lsd1, Lsd2, and Sap1 pro- teins to the mat1 region. Because the Lsd1/2 complex in- teracts with a cis-acting element located within the MT cassettes and promotes replication pause at mat1 but not at mat2P and mat3M, we compared the interactions of the four proteins in the wild-type strain and in two strains con- taining a deletion of both the mat2P and mat3M cassettes. Thus, we have established the computational signature of the imprint and propose a model in which Lsd1 and Lsd2 are exclusively recruited to the mat1 locus. FIGURE 2: Abp1 does not control MPS1 but is recruited at swi2 f i f h i ( ) h l i di FIGURE 2: Abp1 does not control MPS1 but is recruited at swi2 as a function of the mating type. (A) The upper panel indicates the probe and restriction enzyme used for the analysis of the replication intermediates. The lower panel represents the 2D gel analysis of the MPS1 site at mat1 in h90 strain and in h90 abp1∆ strain (n=3). (B) Distribution of normalized enrichments of the Abp1 ChIPs at the swi2 locus (IP RPkM / WCE RPkM) in h90 (gray), mat1M stable (green) and mat1P stable (orange). Gray boxes indicate the genes in the region and arrows the transcription direction. Loci where Abp1 is enriched are annotated. ChIP-sequencing mapping of Abp1, Lsd1/2 and Sap1 to the h90 mating type region It has been previously reported that Abp1, Lsd1/2 and Sap1 bind to the mat1 region [27, 35, 44]. However, several limi- tations arose from those studies: The strains that were initially used to map Abp1 and Sap1 by ChIP-seq were rear- ranged in the MT region (h-S), and the length of the reads (30 nucleotides) prevented their unambiguous assignment to mat1, 2 or 3 cassettes [35]. Concerning Lsd1/2, whole genome analyses had been achieved by ChIP-chip [45], but no detailed analysis of the MT region was performed. More recently, we mapped Lsd1/2 to mat1 by ChIP-qPCR [27]. mat1P and mat2P (Figure 1). Additionally, Sap1 was found to be enriched at the IR-L and IR-R boundary elements [8, 46]. Concerning Abp1, we confirmed that it is indeed en- riched at the left border of CEN-H [41, 47]. Interestingly, we found that Abp1 is also enriched at the right border of mat1 in an h90 background. This observation prompted us to investigate the potential involvement of Abp1 in the early steps of MT switching. To extend the mapping of these proteins to the entire MT region, we performed ChIP-seq of Abp1, Lsd1/2 and Sap1 on the wild-type and switching proficient h90 strain (Figure 1). To achieve proper mapping, we constructed two reference genomes carrying either the M or P allele at mat1 (Figure 1). We found that both Lsd1 and Lsd2 are enriched at the mat1, 2 and 3 cassettes, whereas Sap1 was more enriched at the mat1M and mat3M cassettes than OPEN ACCESS \| www.microbialcell.com INTRODUCTION Two sites control the replica- tion polarity at mat1, RTS1 that blocks the fork coming in from the centromere and MPS1 located near the site of the imprint that pauses the fork coming in from the right. The pausing of the replication fork at MPS1 is a prerequisite for the formation of the imprint [14]. Thus, the imprint is made on the newly synthesized lagging strand during the resumption of DNA synthesis at MPS1 [17, 19]. Several cis-acting sequences are required for the for- mation and maintenance of the imprint. The SS2 and SS13 deletions are located within mat1M. SS2 is important for the pause and the imprint, whereas SS13 acts as a spacer element that allows efficient imprinting [19]. To the right of mat1M, the Msmt-0 deletion maintains efficient pausing but abolishes the imprint [18, 26]. The Lsd1/2 (Lysine Spe- cific Demethylases) and the Swi1/Swi3 (Switch gene) com- plexes are necessary for the pause and the subsequent imprint [17, 27, 28]. Lsd1 is part of a complex including Lsd2 as well as Phf1/Phf2, two plant homeodomain finger Molecular studies have suggested that the imprint at mat1 is either a single-strand DNA break [20] or one or two ribonucleotides [21]. The imprint was mapped to the Wat- son strand corresponding to the neo-synthesized lagging strand, at the junction of the mat1 allele and the H1 ho- mology box [22]. Furthermore, the position of the imprint Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 170 C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 2: Abp1 does not control MPS1 but is recruited at swi2 as a function of the mating type. (A) The upper panel indicates the probe and restriction enzyme used for the analysis of the replication intermediates. The lower panel represents the 2D gel analysis of the MPS1 site at mat1 in h90 strain and in h90 abp1∆ strain (n=3). (B) Distribution of normalized enrichments of the Abp1 ChIPs at the swi2 locus (IP RPkM / WCE RPkM) in h90 (gray), mat1M stable (green) and mat1P stable (orange). Gray boxes indicate the genes in the region and arrows the transcription direction. Loci where Abp1 is enriched are annotated. proteins [29]. The complex also associates with topoiso- merase 2 (Top2), replication factor A (Rfa1) and Sap1 [29], essential proteins with a role in DNA replication. Abp1 is recruited to swi2 when M is present at mat1 The sequence used for the alignments is a 10-kb region containing mat1M (for the left panel) or mat1P (for the right panel).( B-C) the imprint site is indicated as well as the H2 (pink) and H1 (blue) ho- mology boxes. FIGURE 3: Abp1, Lsd1/2 and Sap1 are recruited at mat1. (A) Schematic view of the strains used. The stable mat1M ∆2-3 and mat1P ∆2-3 strains as well as the wild type h90 strain are drawn. (B) On the left: distribution of normalized enrichments of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs on the h90 background (IP RPkM / WCE RPkM). The sequence used for the align- ment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. Only mat1M is shown. On the right: same as at the left, with the h90 P sequence used for the alignment. Only mat1P is shown. (C) Same as in (B) with the mat1M ∆2-3 back- ground on the left and the mat1P ∆2-3 background on the right. The sequence used for the alignments is a 10-kb region containing mat1M (for the left panel) or mat1P (for the right panel).( B-C) the imprint site is indicated as well as the H2 (pink) and H1 (blue) ho- mology boxes. shown that Mc, encoded from the mat1M cassette, and Abp1 control the expression of Swi2, a key player in the directionality process [42, 43]. Moreover, the recruitment of Mc to swi2 depends on Abp1 [42]. Reciprocally, using the two stable M and P strains with deleted donor se- quences, we found that the enrichment of Abp1 at swi2 is restricted to M cells, while the enrichment at the neighbor- ing peak is unaffected by the MT of the cells (Figure 2B). The genome wide analysis showed that Abp1 M-specific interaction is unique for the swi2 gene. shown that Mc, encoded from the mat1M cassette, and Abp1 control the expression of Swi2, a key player in the directionality process [42, 43]. Moreover, the recruitment of Mc to swi2 depends on Abp1 [42]. Reciprocally, using the two stable M and P strains with deleted donor se- quences, we found that the enrichment of Abp1 at swi2 is restricted to M cells, while the enrichment at the neighbor- ing peak is unaffected by the MT of the cells (Figure 2B). Abp1 is recruited to swi2 when M is present at mat1 The genome wide analysis showed that Abp1 M-specific interaction is unique for the swi2 gene. richment is independent of the allele present at mat1 (Fig- ure 3B, 3C). Lsd1 and Lsd2 are enriched both in mat1M and mat1P with Lsd2 and Sap1 more enriched in mat1M (Fig- ure 3B, 3C). We next analyzed the reads that can only map to a unique position on the reference genome (unique mapper) in a h90 wild type context (Figure 4A), where the donor se- quences are present (Supplementary Figure 4). As ex- pected, the highly repetitive sequences of IR-L and IR-R are no longer covered in the two h90 genomes (h90 M and h90 P) (Supplementary Figure 4A and 4B). When the M allele is at mat1, we found no unique mapper reads at mat1 and mat3 (Figure 4B). Similarly, when the P allele is at mat1, no unique mapper reads at mat1 and mat2 were found (Fig- ure 4C). Conversely, Abp1 is located outside of the mat1 cassette in a unique region and can thus be used for align- ment. In the case of Lsd1/2, the enriched unique mappers are well maintained at mat1 (both in the M and P context) and strongly diminished to nearly background level at Abp1 is recruited to swi2 when M is present at mat1 Abp1 is recruited to swi2 when M is present at mat1 Using 2D gel analysis, we addressed the role of Abp1 in MPS1 activity. Unlike the situation observed at LTRs, Abp1 is not required for the pausing activity of MPS1 (Figure 2A), consistent with Abp1 being dispensable for the formation and maintenance of the imprint [41]. Earlier work has Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 171 C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 3: Abp1, Lsd1/2 and Sap1 are recruited at mat1. (A) Schematic view of the strains used. The stable mat1M ∆2-3 and mat1P ∆2-3 strains as well as the wild type h90 strain are drawn. (B) On the left: distribution of normalized enrichments of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs on the h90 background (IP RPkM / WCE RPkM). The sequence used for the align- ment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. Only mat1M is shown. On the right: same as at the left, with the h90 P sequence used for the alignment. Only mat1P is shown. (C) Same as in (B) with the mat1M ∆2-3 back- ground on the left and the mat1P ∆2-3 background on the right. The sequence used for the alignments is a 10-kb region containing mat1M (for the left panel) or mat1P (for the right panel).( B-C) the imprint site is indicated as well as the H2 (pink) and H1 (blue) ho- mology boxes. FIGURE 3: Abp1, Lsd1/2 and Sap1 are recruited at mat1. (A) Schematic view of the strains used. The stable mat1M ∆2-3 and mat1P ∆2-3 strains as well as the wild type h90 strain are drawn. (B) On the left: distribution of normalized enrichments of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs on the h90 background (IP RPkM / WCE RPkM). The sequence used for the align- ment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. Only mat1M is shown. On the right: same as at the left, with the h90 P sequence used for the alignment. Only mat1P is shown. (C) Same as in (B) with the mat1M ∆2-3 back- ground on the left and the mat1P ∆2-3 background on the right. Lsd1/2, Sap1 and Abp1 map at mat1 The enrichment positions of the four proteins and the re- peated nature of the MT cassettes introduce an ambiguity that cannot be resolved during alignment. To pinpoint the Lsd1/2 and Sap1 binding sites to mat1, we used the mat1M ∆2-3 and mat1P ∆2-3 strains deleted for the donor se- quences [24] (Figure 3A). Abp1 is enriched outside of the mat1 locus (to the right of the H1 cassette), and its en- Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 172 C. Raimondi et al. (2018) mat2P and mat3M loci, indicating that Lsd1/2 are prefer- entially recruited at mat1. Furthermore, the absence of Lsd1/2 at the two silent loci was confirmed using ChIP- qPCR (Supplementary Figure 4C). The situation for Sap1 is less clear, and its profile suggests that it is recruited to M but poorly to P. This hints that Sap1 is recruited both inside mat1M and to SAS1 outside of mat1. Characterizat We used in vi further deter The gel shift stranded DNA were include mapping insid FIGUR (A) Sch boxes be un lines a lines. U ment w alignm using " covera sequen contain The lin the lin remov unique Same alignm C. Raimondi et al. (2018) The mat1 imprintosome mat2P and mat3M loci, indicating that Lsd1/2 are prefer- entially recruited at mat1. Furthermore, the absence of Lsd1/2 at the two silent loci was confirmed using ChIP- qPCR (Supplementary Figure 4C). The situation for Sap1 is less clear, and its profile suggests that it is recruited to M but poorly to P. This hints that Sap1 is recruited both inside mat1M and to SAS1 outside of mat1. Characterization of the Sap1 binding in the M allele We used in vitro and in vivo complementary approaches to further determine Sap1 binding sequences in the M allele. The gel shift experiments were achieved using 14 double- stranded DNA probes labeled in 5'. Two positive controls were included (SAS1 and Ter1) in addition to 10 regions mapping inside the M allele and 2 mapping outside (Figure FIGURE 4: Lsd1/2 and Abp1 are recruited only at mat1. (A) Schematic representation of the analysis used. Green boxes represent homologous sequences. Reads that can be unambiguously mapped are represented by black lines and multi-mapper reads are represented by green lines. Upper drawing represents the result of an align- ment without filter (1). Lsd1/2, Sap1 and Abp1 map at mat1 Lower drawing represents the alignment after that multi-mapper reads are removed using "samtools" algorithm (2). (B) Distribution of raw coverage of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs. The sequence used for the alignment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. The lines numbered 1 are the coverage without filter and the lines numbered 2 are the coverage obtained after removal of multi-mapper reads. The arrows indicate the unique-mapper reads at the junction of the mat loci. (C) Same as in (B) with the h90 P sequence used for the alignment. The mat1 imprintosome C. Raimondi et al. (2018) FIGURE 4: Lsd1/2 and Abp1 are recruited only at mat1. (A) Schematic representation of the analysis used. Green boxes represent homologous sequences. Reads that can be unambiguously mapped are represented by black lines and multi-mapper reads are represented by green lines. Upper drawing represents the result of an align- ment without filter (1). Lower drawing represents the alignment after that multi-mapper reads are removed using "samtools" algorithm (2). (B) Distribution of raw coverage of the Abp1, Lsd1, Lsd2, and Sap1 ChIPs. The sequence used for the alignment is a 44-kb region that contains the MT region with the M allele at mat1: h90 M. The lines numbered 1 are the coverage without filter and the lines numbered 2 are the coverage obtained after removal of multi-mapper reads. The arrows indicate the unique-mapper reads at the junction of the mat loci. (C) Same as in (B) with the h90 P sequence used for the alignment. mat2P and mat3M loci, indicating that Lsd1/2 are prefer- entially recruited at mat1. Furthermore, the absence of Lsd1/2 at the two silent loci was confirmed using ChIP- qPCR (Supplementary Figure 4C). The situation for Sap1 is less clear, and its profile suggests that it is recruited to M but poorly to P. This hints that Sap1 is recruited both inside mat1M and to SAS1 outside of mat1. Characterization of the Sap1 binding in the M allele We used in vitro and in vivo complementary approaches to further determine Sap1 binding sequences in the M allele. The gel shift experiments were achieved using 14 double- stranded DNA probes labeled in 5'. Lsd1/2, Sap1 and Abp1 map at mat1 Two positive controls were included (SAS1 and Ter1) in addition to 10 regions mapping inside the M allele and 2 mapping outside (Figure Characterization of the Sap1 binding in the M allele Characterization of the Sap1 binding in the M allele We used in vitro and in vivo complementary approaches to further determine Sap1 binding sequences in the M allele. The gel shift experiments were achieved using 14 double- stranded DNA probes labeled in 5'. Two positive controls were included (SAS1 and Ter1) in addition to 10 regions mapping inside the M allele and 2 mapping outside (Figure Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 173 C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 5: Sap1 is recruited in mat1M. (A) Schematic view of right region of the mat1M locus. The probes used for the shift assay as well as the sites of the deletion strains used for the ChIP-seq of Sap1 are indicated. The sequence of the probes that trigger a shift in B and C are indicated. (B) Electrophoretic mobility shift analysis (EMSA) of crude extracts from S. pombe. SAS1 and Ter1 are used as controls. In lanes (-), 1 ng of radio labelled dsDNA is used with no proteins. In lanes (+), 5 µg of total proteins and 1 ng of radio labelled ds DNA are used. (C) EM- SA of Sap1-6xHis protein purified from E. coli. In lanes (-), 1 ng of radio labelled ds DNA is used with no proteins. In lanes (+), 40 ng of Sap1- 6xHis protein and 1 ng of radio labelled ds DNA are used. (D) Distribution of normalized enrichments of Sap1 ChIPs (IP RPkM / WCE RPkM) in the mat1M ∆2-3, mat1M smt-0 ∆2-3, mat1M ∆SS2 ∆2-3 and mat1M ∆SS13 ∆2-3 backgrounds. The sequence used for the alignment is a 10-kb region that contains the mat1M, mat1M smt-0, mat1M ∆SS2 or mat1M ∆SS13, respectively. Only mat1M is shown. The MT switching phenotype of the deletion strains is indicated. FIGURE 5: Sap1 is recruited in mat1M. (A) Schematic view of right region of the mat1M locus. The probes used for the shift assay as well as the sites of the deletion strains used for the ChIP-seq of Sap1 are indicated. The sequence of the probes that trigger a shift in B and C are indicated. Lsd1/2, Sap1 and Abp1 map at mat1 Using crude protein extracts, we observed the ex- pected mobility shift for SAS1 and Ter1 as well as for 3 probes inside the M allele (Figure 5B). We used purified Sap1 protein to confirm that it is responsible for the shift (Figure 5C). Since M4, M5 and M6 are part of a region in- volved in the stability of the imprint (SS13) [19], we ana- lyzed by ChIP-seq the enrichment of Sap1 in several donor less strains with deletions spanning the mat1M locus. The Msmt-0 deletion does not change the enrichment signal within mat1M, whereas the SS13 deletion strongly de- creases the Sap1 signal, supporting the in vitro interaction result (Figure 5D Msmt-0 and SS13). The slight decrease on the right of the mat1M smt-0-containing deletion together with the remaining weak Sap1 signal in SS13 is attributed to the SAS1 interaction located 140 bp away from the im- print. The SS2 deletion abolishes DNA replication pausing and imprinting [19] but does not affect Sap1 enrichment, whose signal is slightly shifted toward the imprint (Figure 5D ∆SS2). These results indicate that Sap1 is preferentially enriched in vivo on two sequences (SS13 and SAS1) that Lsd1/2, Sap1 and Abp1 map at mat1 (B) Electrophoretic mobility shift analysis (EMSA) of crude extracts from S. pombe. SAS1 and Ter1 are used as controls. In lanes (-), 1 ng of radio labelled dsDNA is used with no proteins. In lanes (+), 5 µg of total proteins and 1 ng of radio labelled ds DNA are used. (C) EM- SA of Sap1-6xHis protein purified from E. coli. In lanes (-), 1 ng of radio labelled ds DNA is used with no proteins. In lanes (+), 40 ng of Sap1- 6xHis protein and 1 ng of radio labelled ds DNA are used. (D) Distribution of normalized enrichments of Sap1 ChIPs (IP RPkM / WCE RPkM) in the mat1M ∆2-3, mat1M smt-0 ∆2-3, mat1M ∆SS2 ∆2-3 and mat1M ∆SS13 ∆2-3 backgrounds. The sequence used for the alignment is a 10-kb region that contains the mat1M, mat1M smt-0, mat1M ∆SS2 or mat1M ∆SS13, respectively. Only mat1M is shown. The MT switching phenotype of the deletion strains is indicated. are not necessary for replication fork pausing but required for imprint formation or maintenance, at least at the mat1M locus. 5A). Using crude protein extracts, we observed the ex- pected mobility shift for SAS1 and Ter1 as well as for 3 probes inside the M allele (Figure 5B). We used purified Sap1 protein to confirm that it is responsible for the shift (Figure 5C). Since M4, M5 and M6 are part of a region in- volved in the stability of the imprint (SS13) [19], we ana- lyzed by ChIP-seq the enrichment of Sap1 in several donor less strains with deletions spanning the mat1M locus. The Msmt-0 deletion does not change the enrichment signal within mat1M, whereas the SS13 deletion strongly de- creases the Sap1 signal, supporting the in vitro interaction result (Figure 5D Msmt-0 and SS13). The slight decrease on the right of the mat1M smt-0-containing deletion together with the remaining weak Sap1 signal in SS13 is attributed to the SAS1 interaction located 140 bp away from the im- print. The SS2 deletion abolishes DNA replication pausing and imprinting [19] but does not affect Sap1 enrichment, whose signal is slightly shifted toward the imprint (Figure 5D ∆SS2). These results indicate that Sap1 is preferentially enriched in vivo on two sequences (SS13 and SAS1) that 5A). OPEN ACCESS \| www.microbialcell.com Detection of an imprinted site by high throughput se- quencing p-value is calculated using a theoretical t-student distribution that fits on the distribution of the whole-genome observed 5' count. Regions that have a high 5' count are numerated. expected, both positions are enriched in 5' count in the h90 background that contains a mixture of mat1M and mat1P alleles (Figure 7C). To confirm that the signal results from the imprint, we used the swi1∆ mutant strain that exhibits a low level of imprint. In this genetic background, no nu- cleotide shows any bias in 5' count (Figure 7D). more frequent in the whole chromatin extract even prior to any chromatin immunoprecipitation. Furthermore, it is possible to count for each DNA strand (Watson and Crick) the number of 5' ends at each position in the genome, and we will refer to this count as "5' count" in the rest of the manuscript (see 5’ count analysis in Materials and Meth- ods). A whole genome distribution of the 5’ count was es- tablished on the mat1M ∆2-3 (Figure 6C) and mat1P ∆2-3 and h90 strains (Figure 7B-C) using 130 nucleotide-long reads. 7 enriched regions were detected on the Watson strand (Figure 6C). 6 such regions were also detected on the Crick strand (Figure 6C). They delineate either repeated regions or regions exhibiting homology with the mitochon- drial genome (Supplementary Table 1). The last enriched 5’ count is only found on the Watson strand and corresponds to the imprint site at mat1 (Figure 6C, in red). Because the bias in 5’ count is only visible on the im- printed strand, we conclude that sonication does not pref- erentially break the phosphate bond facing the nick. There- fore, we searched on the Crick strand for a region with a strong 5’ count statistical bias. Using a sliding window with different window sizes, we calculated the p-value in the 10- kb region surrounding mat1 (Figure 8). As expected, the window of one nucleotide at the site of the imprint has the lowest p-value on the Watson strand (Figure 8A and 8B). On the Crick strand, the most significant p-value is con- tained within a window of 17 to 23 nucleotides facing the imprint (Figure 8C and 8D). This corresponds to the molec- ular signature of a confined and persistent single-strand break upon chromatin sonication: A unique nucleotide on one strand and a larger region on the opposite strand (Fig- ure 8E and 8F). Detection of an imprinted site by high throughput se- quencing Lower panel indicates the different types of reads obtained after sequencing. (B) Schematic representation of the blunting of the end that is used during libraries preparation. The Crick and Watson 5' ends are preserved after treatment. The Crick strand can break at 3 positions compared to the imprint site: to the left, across or to the right. FIGURE 6: The imprint site is detectable by high throughput sequencing on the Watson strand. (A) Schematic vi FIGURE 6: The imprint site is detectable by high throughput sequencing on the Watson strand. (A) Schematic view of the result of the sonication of both chromatid types. Red arrows indicate sites of sonication breakage. The black strand is the Watson strand (W) and the gray the Crick (C). 5' of Watson are shown in black and those of Crick in gray. Lower panel indicates the different types of reads obtained after sequencing. (B) Schematic representation of the blunting of the end that is used during libraries preparation. The Crick and Watson 5' ends are preserved after treatment. The Crick strand can break at 3 positions compared to the imprint site: to the left, across or to the right. (C) Distribution of the 5' count per position in the genome on both strands. mat1M ∆2-3 stain was used for the analysis. p-value is calculated using a theoretical t-student distribution that fits on the distribution of the whole-genome observed 5' count. Regions that have a high 5' count are numerated. FIGURE 6: The imprint site is detectable by high throughput sequencing on the Watson strand. (A) Schematic view of the result of the sonication of both chromatid types. Red arrows indicate sites of sonication breakage. The black strand is the Watson strand (W) and the gray the Crick (C). 5' of Watson are shown in black and those of Crick in gray. Lower panel indicates the different types of reads obtained after sequencing. (B) Schematic representation of the blunting of the end that is used during libraries preparation. The Crick and Watson 5' ends are preserved after treatment. The Crick strand can break at 3 positions compared to the imprint site: to the left, across or to the right. (C) Distribution of the 5' count per position in the genome on both strands. mat1M ∆2-3 stain was used for the analysis. OPEN ACCESS \| www.microbialcell.com Detection of an imprinted site by high throughput se- quencing The imprint was described as either a single stranded break [20] or a one/two ribonucleotide(s) [21, 48] that behave(s) as a mechanical fragile site [49]. Because the chromatin preparation and the Illumina sequencing steps will reveal the imprint as a single strand break, we portray the imprint as a single strand break in the remainder of the text. In Figure 6A, we reasoned that upon chromatin sonication, the strand opposite to the nick will preferentially break at its vicinity. Library preparation using the Illumina kit (TruSeq Chip) uses an exonuclease and a DNA polymerase to process the ends to achieve efficient ligation prior to sequencing (Figure 6B). This method conserves the first nucleotide of the 5’ end of all sonicated fragments. Conse- quently, reads starting at the imprint are expected to be Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 174 OPEN ACCESS \| www.microbialcell.com C. Raimondi et al. (2018) The mat1 imprintosome C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 6: The imprint site is detectable by high throughput sequencing on the Watson strand. (A) Schematic view of the result of the sonication of both chromatid types. Red arrows indicate sites of sonication breakage. The black strand is the Watson strand (W) and the gray the Crick (C). 5' of Watson are shown in black and those of Crick in gray. Lower panel indicates the different types of reads obtained after sequencing. (B) Schematic representation of the blunting of the end that is used during libraries preparation. The Crick and Watson 5' ends are preserved after treatment. The Crick strand can break at 3 positions compared to the imprint site: to the left, across or to the right. (C) Distribution of the 5' count per position in the genome on both strands. mat1M ∆2-3 stain was used for the analysis. p-value is calculated using a theoretical t-student distribution that fits on the distribution of the whole-genome observed 5' count. Regions that have a high 5' count are numerated. C. Raimondi et al. (2018) FIGURE 6: The imprint site is detectable by high throughput sequencing on the Watson strand. (A) Schematic view of the result of the sonication of both chromatid types. Red arrows indicate sites of sonication breakage. The black strand is the Watson strand (W) and the gray the Crick (C). 5' of Watson are shown in black and those of Crick in gray. Detection of an imprinted site by high throughput se- quencing We next focused on the 10-kb region surrounding the mat1 locus (Figure 7). The overall distribution of 5’ count is similar on the Watson and Crick strands, except for one nucleotide (in red) significantly over-represented on the Watson strand (Figure 7). The position in mat1M ∆2-3 and mat1P ∆2-3 corresponds to the imprinted position that is 3 nucleotides apart in M and P [22] (Figure 7A and 7B). As Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 175 OPEN ACCESS \| www.microbialcell.com C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 7: 5' count on the Watson strand is biased precisely at the site of the imprint. On the left: distribution of the 5' count of the Wat- son strand (black) and the Crick strand (gray) on the 10-kb region containing mat1. On the right: zoom on the imprint site. Red bars repre- sent the 5' count enriched at the imprint sites. p-value is calculated using a theoretical t-student distribution that fits on the distribution of the whole-genome observed 5' count. (A) The sequencing of mat1M ∆2-3 strain is represented, (B) mat1P ∆2-3 strain, (C) h90 strain and (D) h90 swi1∆ strain. ABCD) The mat1M ∆2-3, mat1P ∆2-3 and h90 P genomes are used, respectively. FIGURE 7: 5' count on the Watson strand is biased precisely at the site of the imprint. On the left: distribution of the 5' count of the Wat- son strand (black) and the Crick strand (gray) on the 10-kb region containing mat1. On the right: zoom on the imprint site. Red bars repre- sent the 5' count enriched at the imprint sites. p-value is calculated using a theoretical t-student distribution that fits on the distribution of the whole-genome observed 5' count. (A) The sequencing of mat1M ∆2-3 strain is represented, (B) mat1P ∆2-3 strain, (C) h90 strain and (D) h90 swi1∆ strain. ABCD) The mat1M ∆2-3, mat1P ∆2-3 and h90 P genomes are used, respectively. FIGURE 7: 5' count on the Watson strand is biased precisely at the site of the imprint. On the left: distribution of the 5' count of the Wat- son strand (black) and the Crick strand (gray) on the 10-kb region containing mat1. On the right: zoom on the imprint site. Red bars repre- sent the 5' count enriched at the imprint sites. OPEN ACCESS \| www.microbialcell.com 176 Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 Detection of an imprinted site by high throughput se- quencing p-value is calculated using a theoretical t-student distribution that fits on the distribution of the whole-genome observed 5' count. (A) The sequencing of mat1M ∆2-3 strain is represented, (B) mat1P ∆2-3 strain, (C) h90 strain and (D) h90 swi1∆ strain. ABCD) The mat1M ∆2-3, mat1P ∆2-3 and h90 P genomes are used, respectively. Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 176 C. Raimondi et al. (2018) The mat1 imprintosome IGURE 8: Signature of sonication across the imprint. Statistical analysis of the 5' count in the mat1 region. -log(p-value) of 5' count as a unction of the window size and genome position is represented. The -log(p-value) scale is indicated on the right of the graphics. The p-value s calculated using theoretical t-student distributions that fit with the distribution of 5' count observe in the 10-kb region that contains mat1. A) A sliding window (+/- 1 kb around the imprint site) with step size one and varying window size (1 to 40 nt) is applied when calculating the og(p-value) of the 5' count of the Watson strand shown. The sequencing of a mat1M ∆2-3 strain is used. (B) Same as in (A) for the Crick trand. (C) Window sizes and window positions compared to imprint site for the top 10 -log(p-value) are indicated. (D, E and F) Same as in A, B and C), respectively for the sequencing of a mat1P ∆2-3 strain. FIGURE 8: Signature of sonication across the imprint. Statistical analysis of the 5' count in the mat1 region. -log(p-value) of 5' count as a function of the window size and genome position is represented. The -log(p-value) scale is indicated on the right of the graphics. The p-value is calculated using theoretical t-student distributions that fit with the distribution of 5' count observe in the 10-kb region that contains mat1. (A) A sliding window (+/- 1 kb around the imprint site) with step size one and varying window size (1 to 40 nt) is applied when calculating the -log(p-value) of the 5' count of the Watson strand shown. The sequencing of a mat1M ∆2-3 strain is used. (B) Same as in (A) for the Crick strand. (C) Window sizes and window positions compared to imprint site for the top 10 -log(p-value) are indicated. (D, E and F) Same as in (A, B and C), respectively for the sequencing of a mat1P ∆2-3 strain. Immunoprecipitation of the imprinted chromatid Result of the 5' count analysis of the ChIP data presented in Figure 3C. (A) Schematic representation of the theoretical 5' count distribution of Lsd1/2 and Sap1. In black, the 5' count of a non-imprinted chromatid and in red that of an imprinted chromatid. (B) Distribution of the 5' count of the Lsd1/2 and Sap1 ChIPs for the Watson strand (black) and Crick strand (gray) in the mat1M ∆2-3 background. The 5' count at the imprint site is indicated. The p-value is calculated using theoretical t-student distributions that fit with the distribution of 5' count observed in the 10-kb region that contains mat1. (C) Analysis of the 5' count bias on the crick strand using a 20 nt window in a mat1M ∆2-3 strains for the IPs of Lsd1-2 and Sap1. A sliding window of 20 nt with set size one is used in a 10 kb region around mat1 is applied. Gray line indicates the bias of the crick across the imprint. (D) Same as in (B) in a mat1P ∆2-3 strain. (E) Same as in (C) in a mat1P ∆2-3 strain. / p p p p y presented in Figure 3C. (A) Schematic representation of the theoretical 5' count distribution of Lsd1/2 and Sap1. In black, the 5' count of a non-imprinted chromatid and in red that of an imprinted chromatid. (B) Distribution of the 5' count of the Lsd1/2 and Sap1 ChIPs for the Watson strand (black) and Crick strand (gray) in the mat1M ∆2-3 background. The 5' count at the imprint site is indicated. The p-value is calculated using theoretical t-student distributions that fit with the distribution of 5' count observed in the 10-kb region that contains mat1. (C) Analysis of the 5' count bias on the crick strand using a 20 nt window in a mat1M ∆2-3 strains for the IPs of Lsd1-2 and Sap1. A sliding window of 20 nt with set size one is used in a 10 kb region around mat1 is applied. Gray line indicates the bias of the crick across the imprint. (D) Same as in (B) in a mat1P ∆2-3 strain. (E) Same as in (C) in a mat1P ∆2-3 strain. cates that Lsd1 also interacts with the chromatid in the absence of imprint (Figure 9B). A similar 5’ count distribu- tion is observed for Lsd1 at mat1P (Figure 9D and 9E). Immunoprecipitation of the imprinted chromatid Lsd2 and Sap1 exhibit a similar pattern at mat1M as well, while their interaction is weaker at mat1P (Figure 9D and 9E). Altogether, our results indicate that Lsd1, Lsd2 and Sap1 interact on both sides of the imprint but also with mat1 regardless of the imprint. Intriguingly, the interaction at mat1P is weaker for Lsd2 and Sap1. Immunoprecipitation of the imprinted chromatid alignment problem by performing the experiment in the donor less strains. For Lsd1, the enrichment of the 5' count at the imprint on the Watson and Crick strands (Figure 9B) at mat1M exhibits the molecular signature defined above (Figure 9C). This result shows that Lsd1 interacts on both sides or directly with the imprinted chromatid. The addi- tional spreading of the 5’ count on both DNA strands indi- To determine whether Lsd1, Lsd2, and Sap1 interact with the imprinted chromatid and on the right or left of the imprint we used the molecular signature defined above. Figure 9A shows the expected distributions of enriched 5’ count upon immunoprecipitation. Since Lsd1/2 and Sap1 bind the repeated mat cassettes, we circumvented the Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 177 C. Raimondi et al. (2018) C. Raimondi et al. (2018) The mat1 imprintosome FIGURE 9: Lsd1/2 and Sap1 immunoprecipitate chromatids on both sides of the imprint. Result of the 5' count analysis of the ChIP data presented in Figure 3C. (A) Schematic representation of the theoretical 5' count distribution of Lsd1/2 and Sap1. In black, the 5' count of a non-imprinted chromatid and in red that of an imprinted chromatid. (B) Distribution of the 5' count of the Lsd1/2 and Sap1 ChIPs for the Watson strand (black) and Crick strand (gray) in the mat1M ∆2-3 background. The 5' count at the imprint site is indicated. The p-value is calculated using theoretical t-student distributions that fit with the distribution of 5' count observed in the 10-kb region that contains mat1. (C) Analysis of the 5' count bias on the crick strand using a 20 nt window in a mat1M ∆2-3 strains for the IPs of Lsd1-2 and Sap1. A sliding window of 20 nt with set size one is used in a 10 kb region around mat1 is applied. Gray line indicates the bias of the crick across the imprint. (D) Same as in (B) in a mat1P ∆2-3 strain. (E) Same as in (C) in a mat1P ∆2-3 strain. The mat1 imprintosome nd Sap1 immunoprecipitate chromatids on both sides of the imprint. Result of the 5' count analysis of the ChIP data 3C. (A) Schematic representation of the theoretical 5' count distribution of Lsd1/2 and Sap1. In black, the 5' count of a FIGURE 9: Lsd1/2 and Sap1 immunoprecipitate chromatids on both sides of the imprint. OPEN ACCESS \| www.microbialcell.com DISCUSSION This result further indicates that the processes required for replication fork pause and imprinting formation/protection are not identi- cal in M and P cells. One possibility would be that Sap1 is either modified and not efficiently recognized during the immunoprecipitation or replaced by another protein in P cells. We also provide data for the chromosomal enrichment at the genome wide level of four proteins, Lsd1, Lsd2, Sap1, and Abp1. Abp1 is strongly interacting with the sequences near mat1, but a function in replication fork pausing and imprinting was ruled out. The M-specific enrichment of Abp1 next to the Mc binding site located in the swi2 gene reinforces the role of Swi2 in directionality [42, 43]. In mammals, Lsd1 plays an important role in gene ex- pression by establishing and maintaining proper epigenetic marks. Several layers control the chromosomal targeting of Lsd1 for positive or negative transcription regulation. CoREST targets the Lsd1 complex to H3K4 [50], whereas the human androgen receptor targets Lsd1 to H3K9 [51]. The plant homeodomain finger protein BHC80 and the HMG protein Braf35 are part of the core complex Lsd1- CoREST (reviewed in [52]). BHC80 participates to the read- ing of histone marks [53] and Braf35 [54] is a structural DNA-binding protein. FIGURE 10: The imprintosome. Schematic view of the model of binding of Lsd1/2 and Sap1 at mat1. Lsd1/2 complex is repre- sented in blue with Phf1/2 in green. Sap1 is represented in pink. The imprint is represented by a red star. Upper panel, Lsd1/2 complex is recruited on the imprint site on the chromatid with no imprint independently of Sap1. Lower panel, when the imprint is formed Sap1 stabilizes the Lsd1/2 complex. In fission yeast, several pieces of evidence support the specific demethylation of H3K9 by Lsd1 at many promoters and boundary elements, but its role on H3K4 was not es- tablished [29, 55]. Lsd1 is part of a complex including Lsd2 and Phf1/Phf2, two plant homeodomain finger proteins [53], and Lsd1 and Lsd2 are both fused to an HMG domain in fungi [56]. Taken together, the protein composition of the Lsd1/2 core complex is quite similar in fission yeast and mammals and the main functional difference resides in the absence of H3K4 demethylation. DISCUSSION In this work, we propose a molecular definition of the im- print upon sonication and genomic sequencing. Our com- putational analysis demonstrates that a site- and strand- Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 178 178 C. Raimondi et al. (2018) The mat1 imprintosome pauses at MPS1 on the chromosome, and sufficient on a plasmid system [19]. Interestingly, when P and M sequenc- es are present at the silent mat2P and mat3M loci, Lsd1 and Lsd2 are not detected (Supplemental Figure S2), con- sistent with the absence of replication fork pause at these loci [19]. In addition, the absence of H3K9 methylation (clr4 mutant) is not sufficient to trigger recruitment of Lsd1/2 at the silent mat2/3 loci (data not shown). We propose that the Phf1/Phf2 histone code readers participate to the tar- geting of Lsd1/2 at mat1 and probably at the other tran- scriptionally active loci [53]. specific persistent imprint/nick can be unambiguously mapped. The computation of the 5’ count reveals a unique accumulation at the site of the imprint on the Watson strand, whereas the opposite Crick strand is mechanically broken by sonication within a window of 20 nucleotides, thus yielding a more diffuse accumulation of 5’ ends. This molecular signature is consistent with the free rotation of the Crick strand that generates a population of molecules with weakened A-T rich Watson-Crick pairing on both sides of the nick and makes the resulting single-strand in this window highly prone to breakage. However, we cannot rule out that the proteins preventing the imprint from be- ing repaired contribute as well to the pattern of shearing of the Crick strand. By using the molecular signature of the imprint, we provide strong evidence that the Lsd1/2 complex is inter- acting at the imprint site, prior and after imprint formation. The same conclusion was reached by showing that Lsd1/2 is working upstream of Swi1/3 and is still enriched at mat1 in the absence of an imprint [27]. The two Sap1 binding sites on either side of the imprint (SS13 and SAS1) overlap with regions that are dispensable for both replication fork pausing and Lsd1/2 initial recruitment, but are essential for imprint formation or maintenance, at least when mat1 contains the M allele. When mat1 contains the P allele, only the SAS1 site remains, since no enrichment for Sap1 could be detected in the P sequences. DISCUSSION Because Sap1 interacts with the Lsd1/2 complex [29] and that Sap1 and Lsd1/2 complex are overlapping at several locations within the genome (see Supplemental Figure 1A) and notably at high- ly expressed genes (see Supplemental Figure 1B-E), we propose that Sap1 participates in the targeting of the Lsd1/2 complex to prevent H3K9 methylation at key pro- moters and therefore define heterochromatin boundaries [29, 57]. OPEN ACCESS \| www.microbialcell.com ACKNOWLEDGEMENTS This work has been supported by the Fondation ARC for Cancer Research and by the Fondation pour la Recherche Médicale (FRM) and the Pasteur Institute. We are dedicat- ing this article to the memory of Amar Klar and Jacob Dal- gaard, two great scientists who have investigated for sev- eral decades the epigenetic aspects of mating-type switch- ing in fission yeast. Amar Klar passed away in March and Jacob Dalgaard in December. Our community has lost two insightful and keen members. The situation is more complicated at the mating type locus, where the methylation state of H3K9 is not involved in the early mating type switching steps. At this locus, the presence or absence of the sole H3K9 methyltransferase Clr4 does not rescue the mating type switching defect ob- served when lsd1 is mutated [27]. Although mat1P and mat1M exhibit no sequence homology, a remarkable find- ing is that Lsd1 and Lsd2 are enriched in both alleles on sequences that are necessary to promote replication fork FIGURE 10: The imprintosome. Schematic view of the model of binding of Lsd1/2 and Sap1 at mat1. Lsd1/2 complex is repre- sented in blue with Phf1/2 in green. Sap1 is represented in pink. The imprint is represented by a red star. Upper panel, Lsd1/2 complex is recruited on the imprint site on the chromatid with no imprint independently of Sap1. Lower panel, when the imprint is formed Sap1 stabilizes the Lsd1/2 complex. FIGURE 10: The imprintosome. Schematic view of the model of binding of Lsd1/2 and Sap1 at mat1. Lsd1/2 complex is repre- sented in blue with Phf1/2 in green. Sap1 is represented in pink. The imprint is represented by a red star. Upper panel, Lsd1/2 complex is recruited on the imprint site on the chromatid with no imprint independently of Sap1. Lower panel, when the imprint is formed Sap1 stabilizes the Lsd1/2 complex. Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com 179 C. Raimondi et al. (2018) The mat1 imprintosome obtained using the ∆∆Cq normalization method using the val- ue of a pair of primers localized 3 kb away from mat1 and the WCE values. The list of primer sequences is in Supplemental Table S4. Experiments were performed 3-times and unpaired t-test were used to calculate the p values. The simplest model accounting for our results is that the Lsd1/2 complex is epigenetically recruited to mat1 independently of Sap1 to promote replication fork pausing at MPS1 (Figure 10, No Imprint). Following replication fork restart and imprinting, Sap1 and Lsd1/2 form a chromatin structure, the imprintosome, that protects the imprint from being repaired (Figure 10, Imprint). We propose that Lsd1/2 controls replication fork pausing at mat1 on both the imprinted and the non-imprinted chromatids, thus allowing the fork to restart in the same environment at each round of DNA replication and produce persistently and efficiently the imprint. Illumina Sequencing of DNA Libraries were prepared using TruSeq ChIP Library Preparation kit following the manufacturer’s instructions (Illumina). Librar- ies are checked for concentration and quality on DNA chips with a Bioanalyser (Agilent). The libraries were quantified by fluorimetric measurements with the Qubit® dsDNA HS Assay Kit (ThermoFisher). 65-bp and 130-bp Single Read sequences were generated on the Hiseq2500 sequencer according to manufacturer’s instructions (Illumina). Library statistics are provided in Supplemental Table S5. Chromatin Immunoprecipitation (ChIP) ChIP-sequencing was performed as previously described [35] with the following modifications: fixation for ChIP of Lsd1 and Lsd2 was carried out at 25°C for 20 min with 1% of paraform- aldehyde (PFA) and fixation for ChIP of Sap1 and Abp1 was performed at 18°C for 15 min with 3% of PFA. For Lsd1 and Lsd2, an Anti-Myc-Tag mAb-magnetic Beads (M047-11 MBL) was used. For Abp1 and Sap1, Dynabeads Protein G 10007D (Invitrogen) coupled with Tap Tag antibody CAB1001 (Pierce Thermo) or with polyclonal serum against the native Sap1[53], respectively, were used. For the ChIP followed by qPCR analy- sis, 3x108 cells were used with one tenth the amount of rea- gents. All scripts used for the analysis and outputs are available upon request. Fission yeast strains and genetic procedures The S. pombe strains used in this study are listed in Supple- mental Table S3. Standard media (YES) and genetic protocols for fission yeast were used [58]. Illumina Read Processing and Alignment of DNA Libraries Illumina reads were aligned to the S. pombe genome assembly ASM294v2.23 using Bowtie v2.1.0 [61] with default parame- ters and multi-mapper reads randomly distributed. Using BamTools [62], read counts were normalized to reads per million per kilo base (RPKM), using the total library size and a bin size of 1 kb. For the analysis of multi-mapper reads, we filtered aligned read files with samtools [63] using the param- eter -q 10. 5' count analysis We used a script to extract the beginning of reads that map on the Watson or Crick strands. Enrichments of 5' count were obtained using the total library size. p-values were obtained using the negative binomial distribution fitting function in R. Gel shift assay d f Production of Sap1-6His by E. coli was induced with 1 mM IPTG as previously described [32]. Sap1-6His was purified us- ing column of Ni-NTA His-tag from Qiagen according to the manufacturer’s instructions. Sequences used are listed in Ta- ble 2. 1.50 ng of a given oligonucleotide was labeled with gamma-32P with T4 polynucleotide Kinase (NEB) and incubated with its cold complementary sequence. 1 ng of the resulting double stranded probe was incubated with 5 µg of total pro- tein extract or with 40 ng of purified Sap1-6His [44]. Gel shift was performed as previously described [60]. ChIP-Sequencing Analysis ChIP peaks versus appropriate inputs (whole-cell extracts) were called using MACS v2.1.0 [64]. Statistic validation of peaks was performed using the IDR method with ENCODE recommendations [65]. Genome browser tracks and meta- analysis were created using enrichment (IP reads per million (RPM) / input RPM) of indicated sequencing. Intersection of lists of peaks were calculated with BEDTools v.2.17.02 [66] (parameters: -u). For the transcriptional analysis 2 groups of 108 genes were defined and intersected with the list of peaks shared by Lsd1/2 and Sap1. The first group contains the 108 most present transcripts and the 108 less present transcripts using the normalized data from [67]. 2D Gel Analysis 2D gel analysis of replication intermediates was carried out as described previously [59]. DNA was prepared and digested in agarose plugs with the indicated restriction enzymes [12]. Enriched fractions for replication intermediates were obtained using BND cellulose columns (SIGMA). Gels were blotted onto Hybond-N+ nylon membrane. The probes were labeled with alpha-32P dCTP and the blot analyzed on a PhosphoImager and quantified with the ImageQuant software (pause/ arc + pause in percentage) (GE Healthcare Life Sciences). Sequences of primers used to produce the probe are indicated in Table 2. REFERENCES REFERENCES 1. Beach D, Nurse P, and Egel R (1982). Molecular rearrangement of mating-type genes in fission yeast. Nature 296(5858): 682–683. PMID: 7070512 14. Dalgaard JZ, and Klar AJ (2001). Does S. pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching? Trends Genet17(3): 153–157. PMID: 11226610 14. Dalgaard JZ, and Klar AJ (2001). Does S. pombe exploit the intrinsic asymmetry of DNA synthesis to imprint daughter cells for mating-type switching? Trends Genet17(3): 153–157. PMID: 11226610 15. Arcangioli B, and Thon G (2004). Mating-Type Cassettes: Structure, Switching and Silencing. In : Egel R. (eds) The Molecular Biology of Schizosaccharomyces pombe. 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Four chromo-domain pro- teins of Schizosaccharomyces pombe differentially repress transcrip- tion at various chromosomal locations. Genetics 155(2): 551–568. PMID: 10835380 19. AUTHOR CONTRIBUTIONS C.R performed experiments. C.P prepared the libraries for sequencing. H.W provided a purified Sap1 protein. C.R and B.J performed the computational analysis. C.R and B.A con- ceived experiments. C.R., S.G. and B.A. wrote the manu- script. © 2018 Raimondi et al. This is an open-access article re- leased under the terms of the Creative Commons Attribu- tion (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged. SUPPLEMENTAL MATERIAL Please cite this article as: Célia Raimondi, Bernd Jagla, Caroline Proux, Hervé Waxin, Serge Gangloff, Benoit Arcangioli (2018). Molecular signature of the imprintosome complex at the mating- type locus in fission yeast. Microbial Cell 5(4): 169-183. doi: 10.15698/mic2018.04.623 All supplemental data for this article are available online at www.microbialcell.com. quantitative PCR (q-PCR) qPCR was performed using a master mix (Eurobio Green qPCR Mix Lo-Rox) on the qTower Analytik Jena AG A6 machine. Each value is the mean of technical triplicates. The enrichment was Microbial Cell \| APRIL 2018 \| Vol. 5 No.4 OPEN ACCESS \| www.microbialcell.com OPEN ACCESS \| www.microbialcell.com 180 The mat1 imprintosome C. Raimondi et al. (2018) REFERENCES Sayrac S, Vengrova S, Godfrey EL, and Dalgaard JZ (2011). Identifi- cation of a novel type of spacer element required for imprinting in fission yeast. PLoS Genet 7(3): e1001328. doi: 10.1371/journal.pgen.1001328 8. Thon G, Bjerling P, Bünner CM, and Verhein-Hansen J (2002). Ex- pression-state boundaries in the mating-type region of fission yeast. Genetics 161(2): 611–622. PMID: 12072458 20. Kaykov A, and Arcangioli B (2004). 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https://openalex.org/W1520829806	https://www.intechopen.com/citation-pdf-url/38577	English	null	Automatic Optical Inspection of Soldering	InTech eBooks	2,013	cc-by	20,655	http://dx.doi.org/10.5772/51699 http://dx.doi.org/10.5772/51699 Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 12.2% 191,000 210M TOP 1% 154 7,200 Chapter 16 Automatic Optical Inspection of Soldering Mihály Janóczki, Ákos Becker, László Jakab, Richárd Gróf and Tibor Takács Mihály Janóczki, Ákos Becker, László Jakab, Richárd Gróf and Tibor Takács Additional information is available at the end of the chapter © 2013 Janóczki et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2. Rise of the AOI systems Manufacturing electronic devices necessitates the constant controlling and inspection of the product. Previously, ICT was the main appliance used for this purpose. It inspected electronic components (e.g. resistor, capacitor etc.), checked for shorts, opens, resistance, capacitance and other basic quantities. Finally, it checked the proper operation of the whole circuit to show whether the assembly had been correctly fabricated. It operated by using a bed of measure- nails type test fixtures, designed for the current PWB and other specialist test equipment. It had the following disadvantages. As the dimensions of components were shrinking and the emplacement density was increasing, the positioning of the test-points became increasingly difficult. Beds of measure-nails are also relatively expensive and they are PWB-specific. This problem was however, partially solved by using flying-probe ICT systems (but at the expense of speed). Another disadvantage of ICT was that only finished product could be examined. It was able to detect failures but not to prevent them. It is also was not suitable for inspecting the quality of various assembling technologies. A further disadvantage was also in the case of functional testing. Extra measurement procedures had to be developed to ensure the enhance‐ ment of the quality of the manufacturing process. Previously, the quality of solder joints had only been verified by manual visual inspection (MVI). The disadvantage of manual inspection, which at best was subjective, was that the tolerance limits were narrower than used in automated machines. A magnifying glass could help for a while, but as the mounting number of components per unit area exceeded the capabilities of manual testing, this option was already proving to be difficult or not even applicable as described in [1]. Because of the rapid development of digital computing, machine vision and image processing, it was obvious that it was becoming necessary to automate the process with the help of various high-resolution cameras, novel lighting devices [2], illumi‐ nation techniques [3]-[5] and efficient image processing algorithms. Such state-of-the-art devices and solutions are described in detail in the following books: [6]-[8]. In cases where the manufacturing of large quantities of precise and high quality products takes place, the capabilities of production appliances can only be used effectively if the inspections, after various technological sequences are automated (in-line), are fast and reliable. As a result, the automatic optical inspection or testing appliances has been developed to replace manual inspection. 1. Introduction Automatic Optical Inspection (AOI) or Automated Visual Inspection (AVI) is a control process. It evaluates the quality of manufactured products with the help of visual information. Amongst its several uses, one is the inspection of PWB (Printed Wiring Boards) after their assembling sequences i.e. paste printing, component placement and soldering. Nowadays, surface mount technology is the main method of assembly. It can be automated with ease. The increasing widespread use of SMT (Surface Mount Technology) in PWB assembly results in down-scaling of component size, increasing of lead count and component density. Parallel to this the latest manufacturing assembly lines have a very high rate of productivity. Not only is productivity required but a high quality also is expected. The quality requirements for electronic devices have already been standardized, e.g. IPC, ANSI-JSTD standards. Modern machines used in manufacturing lines such as paste printers, component placement machines etc. are capable of producing significantly better results than those required in normal standards specifica‐ tions. Nowadays, the capability of modern manufacturing machines now reaches 6 σ as usually applied specification norm and 5 σ for more stringent ones. Even so, manufacturing processes are still kept under constant supervision. There still occasions when even a modern assembly line fails to create fully operational devices. Besides the “classic” electric tests, such as in-circuit-test (ICT) and/or functional tests, there are in-line inspection possibilities: automatic optical inspection and automatic X-ray inspection systems. Because of their capabilities and properties, mostly the AOI systems are used as in- line quality inspection appliances. The main advantage of these systems is their ability to detect failures earlier and not only when the product has been assembled. AOI systems can be used to inspect the quality at each stage of the manufacturing process of the electronic device. Accordingly, there are real financial advantages by using such systems because the sooner a © 2013 Janóczki et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Materials Science - Advanced Topics 388 failure can be detected, the smaller the likelihood of refuse device manufacturing. Because of component down-scaling and increasing in density, optical inspection is now only possible with the help of machine vision as opposed to manual inspection. 3. Sensors, image capturing methods, structure In the early 1970s, CCD (Charge Coupled Device) and CMOS (Complementary Metal–Oxide– Semiconductor) sensors were invented. It presented an opportunity to capture digital images that could be processed and evaluated by a computer. Machine vision was born. The subse‐ quent exponential development resulted in an infinite number of these applications. One such development was the automatic optical inspection. Comparison between these sensors is reported in detailed [30]-[36]. Two kinds of methods exist to capture the images: FOI (Field of Interest) based matrix camera and line scan camera. The first captures several images on an optimized course, the second scans the whole surface of PWB. Both have their advantages and disadvantages. Line-scan is the faster method but the design of a proper source of illumination is more difficult or sometimes not possible at all because the position of components themselves affects the efficiency of illumination. If the component is parallel or perpendicular to the scanning line, the captured image could differ. In case of paste inspection, component positioning is out of question, so line-scan is better choice. For components and meniscus inspection, FOI is better. A new FOI generation method is shown in [37]. Basically AOI systems have three main parts: optical unit (illumination, cameras), positioning mechanism, and control system (Fig. 1). 2. Rise of the AOI systems The words, Automated Optical Inspection imply that when used in the manufac‐ turing and assembly of PWBs, the nature of the inspection process itself, using digital machine vision and image processing, will give objective results. AOI inspects bare and mounted PWBs automatically and uses optical information. It is faster, more accurate and cheaper than manual inspection. In preparing the parameters for such Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 389 inspections, parametric test procedures can be used to evaluate the digital image and on the basis of this they classify the inspected PWB, component or solder joint. Automatic Optical Inspection systems offer a reliable, flexible, fast and cost-effective solution when inspecting each step of the manufacturing process. Using AOI systems also has financial advantages. Detailed calculations show this in [1]. Further works give more reasons why AOI should be used. Several economic, efficiency and suitability studies have been undertaken about these systems [9]-[29]. 4. Identifying PWB AOI appliances identify the PWBs with the help of a separate built-in unit i.e. laser-scanner or by using its inbuilt functionality. On this basis, machines can decide what inspection is necessary. According to data contained in a barcode, the AOI system loads the appropriate inspection program. As barcodes (Fig. 2.a) became more widely used, in some cases the amount of data that could be stored in them was too limited and this became a barrier to its applicability. To solve this problem, the so-called ‘matrix codes’ (Fig. 2.b) were developed. In [30] 22 types of linear barcodes and 48 types of matrix codes are described. Materials Science - Advanced Topics 390 5. Inspection of bare PWBs There are several possibilities, appliances and algorithms when inspecting bare PWB optically. These are able to inspect the copper wire-patterns on a PWB surface with high precision. Optical inspection gives rapid and reliable results regarding the quality of th PWB. Electrical detection methods, (e.g. ICT, Flying Probes) are slower and more expen sive. Bare PWB inspecting AOIs have a special name: Automatic Optical Test (AOT systems. There are several research and survey papers about this topic [31]-[43] and tw manufacturers now have AOT machines [44]-[49]. In Table I a comparison between thes appliances is shown. Figure 1. The three main part of an AOI system (a) (b) Figure 2. Example for: a) linear barcode b) DMC Materials Science - Advanced Topics 90 Figure 1. The three main part of an AOI system Figure 1. The three main part of an AOI system 5. Inspection of bare PWBs (a) (b) Figure 2. Example for: a) linear barcode b) DMC (a) (b) (b) Figure 2. Example for: a) linear barcode b) DMC Figure 2. Example for: a) linear barcode b) DMC 5. Inspection of bare PWBs There are several possibilities, appliances and algorithms when inspecting bare PWBs optically. These are able to inspect the copper wire-patterns on a PWB surface with high precision. Optical inspection gives rapid and reliable results regarding the quality of the PWB. Electrical detection methods, (e.g. ICT, Flying Probes) are slower and more expen‐ sive. Bare PWB inspecting AOIs have a special name: Automatic Optical Test (AOT) systems. There are several research and survey papers about this topic [31]-[43] and two manufacturers now have AOT machines [44]-[49]. In Table I a comparison between these appliances is shown. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 391 Manufacturer Amistar Automation Inc. Model K5L doutech Excalibur phasor redline LD 6000 Board size max. [mm x mm] 510 x 410 610 x 760 610 x 760 760 x 760 (extended: 1000 x 2000) 610 x 760 610 x 760 (extended: 915 x 1525) Board size min. [mm x mm] 50 x 50 n.a. n.a. n.a. n.a. n.a. Board thickness [mm] 0.5 - 3.0 n.a. n.a. n.a. n.a. n.a. Board warp [mm] + 0.5; - 1.0 n.a. n.a. n.a. n.a. n.a. Board clearance top [mm] 50 n.a. n.a. n.a. n.a. n.a. Board clearance bottom [mm] 50 n.a. n.a. n.a. n.a. n.a. Camera type CCD n.a. n.a. n.a. n.a. n.a. Illumination 3-stage LED dome lighting (Upper: IR; Middle: WH; Lower: WH) n.a. n.a. n.a. n.a. n.a. View size [mm] 30.4 x 22.8 500 x 635 500 x 610 500 x 635 (extended: 915 x 1980) 500 x 635 (extended: 1000 x 2000) 500 x 610 (extended: 865 x 1475) Resolution [µm] 19 50 50 60 50 n.a. Inspection time 0.45 sec/screen 20 - 60 sq. m/h 45 sq. m/h n.a. 20 - 60 sq. m/h 15 sec @ 3/3 mil line/space; 27 sec @ 2/2 mil line/space (for 480 x 600 mm board size) Applicability missing components, position shift, rotation error, wrong components, polarity check, bridge, character recognition functional and cosmetic faults functional and cosmetic faults functional and cosmetic faults functional and cosmetic faults functional and cosmetic faults Lloyd Doyle Limited Table 1 Comparison of Automatic Optical Test (AOT) machines Table 1. Comparison of Automatic Optical Test (AOT) machines Table 1. Comparison of Automatic Optical Test (AOT) machines 6. Inspections following SMT sequences In the SMT assembling process there are three main phases where AOI plays an important role; after-paste printing, component placement and soldering. In the case of wave, r selective or partial soldering there are other sequences e.g. glue dispensing, through-hole component insertion etc. But SMT processes are used mainly for inspection in discussions about this method of assembly. Possible locations where AOI can be placed in an SMT line are: the post- paste, post-placement or pre-reflow and post reflow (Fig. 3.). At each location AOI appliances have a special name. These are: Solder Paste Inspection (SPI, also known as Post-Printing Inspection), Automatic Placement Inspection (API. also known as Post-Placement Inspection) and Post-Soldering Inspection (PSI). The AOI systems able to inspect each manufacturing sequence are called: Universal AOI (UAOI). If there is a possibility that the equipment can inspect the finished product optically, it is then called the Automatic Final Inspection (AFI). Materials Science - Advanced Topics 392 6.1. Solder paste inspection According to PWB assemblers, it is very important that the quality of the print solder paste is inspected because it heavily influences the quality of solder joints. In some papers it has been reported that 52%–71% of SMT defects are related to the printing process [50]-[53]. As failures can be detected much earlier, this obviously results in cost savings. According to some other opinions, inspection of the solder paste is not so relevant: “Contrary to the common, frequently quoted opinion that paste faults represent the primary percentage or 70% of all faults in the printed circuit assembly process, this detailed analysis shows that those faults amounted to only 8.3%.” [54]. Figure 3. Possible places of AOI systems Figure 3. Possible places of AOI systems Figure 3. Possible places of AOI systems Figure 3. Possible places of AOI systems 6.1. Solder paste inspection According to PWB assemblers, it is very important that the quality of the print solder paste is inspected because it heavily influences the quality of solder joints. In some papers it has been reported that 52%–71% of SMT defects are related to the printing process [50]-[53]. As failures can be detected much earlier, this obviously results in cost savings. According to some other opinions, inspection of the solder paste is not so relevant: “Contrary to the common, frequently quoted opinion that paste faults represent the primary percentage or 70% of all faults in the printed circuit assembly process, this detailed analysis shows that those faults amounted to only 8.3%.” [54]. Special AOI machines are able to inspect the quality of print of the solder paste. It is an important option because in case of failure, the product can be repaired with minimum cost and with‐ out scrap loss. The size of the print in the 3 dimensions examined (latitude, longitude, alti‐ tude) must fall within the limits specified. To measure these parameters, so-called SPI (Solder Paste Inspection) machines have been developed. These machines are able to inspect only one step i.e. paste printing, but they are cheaper than universal AOI machines. As the control of solder paste presence is one of the easier tasks, then only the width, length and position needs to be inspected and so several failures can be detected such as bridges [55]. This can be solved using image capturing (usually with the help of line scan cameras) and subsequent evaluation. But to measure volume as well the paste thickness is equally as important. Comparison between 2D and 3D solder paste inspections are reported in [56] and [57]. There are several possibilities to enable the measurement of paste volume optically: laser scanner [58]-[63]; projected sinusoidal fringe pattern as in [64]; the development of this technique for solder paste geometry measurement in [65], [66] and some special methods shown in [67]-[69]. Nine manufacturers offer SPI systems [70]-[82]. Several different solutions have been developed in these appliances as can be found in the scientific literature, described above. Comparison between the different methods is shown in Table 2. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 393 Manufacturer Model Board size max. [mm x mm] Board size min. 6.1. Solder paste inspection [mm x mm] Maximum board weight [kg] Maximum inspection area [mm] Maximum pad size in field of view [mm] Typical inspection speed @ high speed [sq. cm/sec] Typical inspection speed @ high resolution [sq. cm/sec] Typical inspection speed @ unload and fiducial find [sec] X and Y pixel size @ high speed [µm] X and Y pixel size @ high resolution [µm] Paste height range [µm] Height resolution [µm] Measurement types CyberOptics SE 300 Ultra 508 x 508 101 x 40 3 508 x 503 5 x 10 29.0 16.0 3 - 4 40 20 50 - 610 0.13 height, area, volume, registration, bridge detection CyberOptics SE 500 510 x 510 50 x 50 3 - 5 508 x 503 15 x 15 80.0 50.0 4 - 5 30 15 50 - 500 0.20 height, area, volume, registration, bridge detection CyberOptics SE 500-X 810 x 610 100 x 100 10 810 x 605 15 x 15 80.0 50.0 4 - 5 30 15 50 - 500 0.20 height, area, volume, registration, bridge detection Koh Young Technology aSPIre n.a. n.a. n.a. n.a. n.a. 41.0 n.a. n.a. 10 20 20 - 400 0.37 height, area, volume, offset, bridge detection, shape deformity Koh Young Technology KY-8030 n.a. n.a. n.a. n.a. n.a. 15.0 19.7 n.a. 20 20 20 - 400 0.37 height, area, volume, offset, bridge detection, shape deformity Marantz Power Spector 510 x 460 50 x 50 n.a. n.a. n.a. 80.0 80.0 n.a. n.a. n.a. max. 600 n.a. height, area, volume, offset, bridge detection, shape deformity Omron VT-RNS-P 510 x 460 50 x 50 n.a. n.a. n.a. n.a. n.a. n.a. 10; 15; 20 10; 15; 20 n.a. n.a. presence of solder, insufficient solder, excessive solder, solder shifting, grazing, bridging, spreading Omron - CKD VP5000L 510 x 460 50 x 50 n.a. n.a. n.a. n.a. n.a. n.a. 12 12 n.a. n.a. average height, volume, excessive deposition, insufficient solder, smearing, misalignment, bridging Orpro Vision Symbian P36 508 x 540 n.a. n.a. n.a. n.a. 60 60 4 - 6 20 20 50 - 300 5.00 n.a. Saki BF-SPIder-M 250 x 330 50 x 60 n.a. n.a. n.a. n.a. n.a. 3 12 12 max. 450 0.40 height, area, volume, shift, shape, spread, bridge Saki BF-SPIder-L 460 x 500 50 x 60 n.a. n.a. n.a. n.a. n.a. 3 - 5 12 12 max. 6.1. Solder paste inspection 450 0.40 height, area, volume, shift, shape, spread, bridge ScanCAD ScanINSPECT SPI 457 x 508 50 x 50 n.a. 419 x 508 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7006/L/LL-20 330 x 280 50 x 50 3 n.a. n.a. 98.8 24.7 n.a. 20 20 40 - 600 n.a. n.a. TRI Innovation TR7006/L/LL-16 510 x 460 50 x 50 5 n.a. n.a. 63.2 15.8 n.a. 16 16 32 - 480 n.a. n.a. TRI Innovation TR7006/L/LL-12 660 x 610 50 x 50 10 n.a. n.a. 35.5 8.8 n.a. 12 12 24 - 360 n.a. n.a. TRI Innovation TR7066-20 510 x 460 50 x 50 3 n.a. n.a. 153.6 23.0 n.a. 20 20 40 - 600 n.a. n.a. TRI Innovation TR7066-16 510 x 460 50 x 50 3 n.a. n.a. 122.9 18.4 n.a. 16 16 32 - 480 n.a. n.a. TRI Innovation TR7066-12 510 x 460 50 x 50 3 n.a. n.a. 92.2 13.8 n.a. 12 12 24 - 360 n.a. n.a. Viscom S3088-II QS 450 x 350 n.a. n.a. n.a. n.a. 110 110 n.a. 22 22 n.a. n.a. n.a. Vi Technology 3D-SPI 510 x 460 n.a. n.a. n.a. n.a. 20.0 20.0 n.a. n.a. n.a. n.a. n.a. height, area, volume, bridge, shape, position Table 2. Materials Science - Advanced Topics 394 Manufacturer Model Conveyor speed [mm/sec] Conveyor height [mm] Board warp Volume repeatability on a certification target Volume repeatability on a circuit board Gage R&R Measurement types CyberOptics SE 300 Ultra 150 - 450 889 - 990 <2% of PCB diagonal or 6.35mm total n.a. n.a. n.a. height, area, volume, registration, bridge detection CyberOptics SE 500 150 - 450 810 - 970 <2% of PCB diagonal or 6.35mm total < 1%; 3 <3%; 3 << 10%; 6 height, area, volume, registration, bridge detection CyberOptics SE 500-X 150 - 450 810 - 970 <2% of PCB diagonal or 6.35mm total < 1%; 3 <3%; 3 << 10%; 6 height, area, volume, registration, bridge detection Koh Young Technology aSPIre n.a. 830 - 970 ± 5.0 mm < 1%; 3 <3%; 3 << 10%; 6 height, area, volume, offset, bridge detection, shape deformity Koh Young Technology KY-8030 n.a. 870 - 970 ± 3.5 mm < 1%; 3 <3%; 3 << 10%; 6 height, area, volume, offset, bridge detection, shape deformity Marantz Power Spector n.a. 830 - 970 ± 5.0 mm n.a. n.a. n.a. Table 3. Comparison of Solder Paste Inspection (SPI) machines Table 3. Comparison of Solder Paste Inspection (SPI) machines 6.2 Automatic placement inspection Inspection of the PWB after component placement is the next possibility. With this method possible placement failures can be detected and some defective paste printing phenomena as well. If there is a sign or mark on the component, it can be read and identified with the help of modern image processing algorithms even if it has more than one different-looking label type. APIs (Table III) are able to measure most parameters of components objectively e.g. X-Y shift, rotation, polarity, labels, size etc. [83]-[90]. Four manufacturers have this special appli‐ ance in stock. [91]-[94]. Manufacturer BeamWorks Landrex Omron Viscom Model Inspector cpv Optima II 7301 Express VT-RNS-Z S3054QV Field of view 12 x 9 mm @ 15 µm; 48 x 36 mm @ 73 µm 10 x 10 mm; 15 x 15 mm n.a. 1280 x 1024 pixel Pixel size [µm] 15; 73 n.a. 10; 15; 20 10; 22 Depth of field (max. component height for inspection) [mm] 10; 15 n.a. n.a. n.a. Number of cameras 1 1 vertical, 4 angled 1 1 Lighting method oblique ring, white LED light n.a. ring-shaped RGB LED n.a. Board size max. @ single board operation [mm x mm] 508 x 406 609 x 558 510 x 460 443 x 406 Board size max. @ dual board operation [mm x mm] none none none 370 x 406 Board size min. [mm] 40 x 28 51 x 76 50 x 50 n.a. Board thickness [mm] 0.8 - 3.2 n.a. n.a. n.a. Conveyor height [mm] n.a. n.a. n.a. 850 - 960 Board edge clearance [mm] 4 n.a. n.a. 3 Board edge clearance top [mm] 25; 37 63 20 35 Board edge clearance bottom [mm] 25; 37 63 50 50 Inspection speed [sq. cm/sec] 2 @ 12 x 9 mm field of view n.a. 250 ms/screen @ 10 sq. mm field of view 20 - 30 Applicability missing component, wrong component, polarity check, offset, skew missing components, misoriented components, extra components, component placement, tombstoned and bill boarded components, lifted leads, insufficient solder and excess solder, wrong part, through hole pins presence of solder, component shifting, polarity error, missing components, wrong components, solder balls, skewing, bridging, foreign objects n.a. Table 4. Comparison of Automatic Placement Inspection (API) machines Table 4. Comparison of Automatic Placement Inspection (API) machines 6.1. Solder paste inspection height, area, volume, offset, bridge detection, shape deformity Omron VT-RNS-P n.a. n.a. n.a. n.a. n.a. n.a. presence of solder, insufficient solder, excessive solder, solder shifting, grazing, bridging, spreading Omron - CKD VP5000L n.a. n.a. n.a. n.a. n.a. n.a. average height, volume, excessive deposition, insufficient solder, smearing, misalignment, bridging Orpro Vision Symbion P36 n.a. 870 - 930 + 3.0 mm; - 6.0 mm n.a. n.a. < 10% n.a. Saki BF-SPIder-M n.a. max. 900 n.a. < 1%; 3 n.a. < 10% height, area, volume, shift, shape, spread, bridge Saki BF-SPIder-L n.a. max. 900 n.a. < 1%; 3 n.a. < 10% height, area, volume, shift, shape, spread, bridge ScanCAD ScanINSPECT SPI n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7006/L/LL-20 n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7006/L/LL-16 n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7006/L/LL-12 n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7066-20 n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7066-16 n.a. n.a. n.a. n.a. n.a. n.a. n.a. TRI Innovation TR7066-12 n.a. n.a. n.a. n.a. n.a. n.a. n.a. Viscom S3088-II QS n.a. 850 - 960 n.a. n.a. n.a. n.a. n.a. Vi Technology 3D-SPI n.a. max. 950 ± 3.5 mm n.a. n.a. < 10% height, area, volume, bridge, shape, position Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 395 6.3 Post soldering inspection Most manufacturers agree from a strategic point of view, that optical inspection after soldering has been completed should not be missed out. At the very least, the defective products must be eliminated because many failures are generated during soldering: “Forty-nine percent of the true faults were detectable only after soldering. These consisted of component and soldering faults. Forty-eight percent of the optically recognizable faults could not be recog‐ nized electrically.” [54]. Materials Science - Advanced Topics 396 In consequence, this is the most important part of the AOI inspection process. Most scientific papers are preoccupied with this subject [95]-[123]. The quality of the solder joint (and the soldering process) can be inspected with the methods described in this section. The quality of the solder joints is determined from geometric and optical properties of the solder meniscus. These parameters determine the reflection properties of the meniscus which is formed from the liquid alloy during the soldering process. After cooling, the meniscus becomes solid and reflects illumination which means that we can evaluate it (Fig.5, Fig. 6). From these reflection patterns and with the help of image processing algorithms we are able to determine the quality of the solder joints. Figure 4. Schematic of the meniscus Figure 5. Reflection pattern on meniscus model with white ring illumination Figure 4. Schematic of the meniscus Figure 4. Schematic of the meniscus Figure 4. Schematic of the meniscus Figure 5. Reflection pattern on meniscus model with white ring illumination Figure 5. Reflection pattern on meniscus model with white ring illumination Figure 6. Reflection pattern on meniscus model with RGB ring illumination Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 3 Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 397 Figure 6. Reflection pattern on meniscus model with RGB ring illumination Figure 6. Reflection pattern on meniscus model with RGB ring illumination Using reflection patterns is the basis of all papers that have been published in this field of study. There are two solutions: gray-scale or colour inspection. Supplier and appliances are shown in Table IV [124]-[126]. One interesting area is wave soldering. It needs different types of algorithms because of the circular solder shape and the pin. Some solutions for this kind of inspection are reported in [127]-[129]. A summary of possible failures and appliances that can detect them are shown in Table V. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 399 Table 6. Possible failures and appliances 6.4. Combined appliances Possible failures and appliances Materials Science - Advanced Topics 400 Manufacturer Model Camera movement Board movement Component inspection Printing/paste inspection Distinction principles Distinction parameters Camera type Camera field of view/resolution Lens Illumination Machine Vision Products Supra E n.a. n.a. n.a. n.a. n.a. n.a. 5MP color camera 7 - 17 µm n.a. programmable variable LED strobe lighting, proprietary multi-color illumination Marantz iSpector HDL 350L X + Y direction stationary during inspection presence, polarity, offset, correctness, soldering offset, smearing, bridges, uniformity synthetic imaging, spectral analysis, grayscale limits brightness, hue, saturation via filters UXGA CCD digital camera with CameraLink 32 x 24 mm @ 20 µm; 40 x 30 mm @ 25 µm; 16 x 12 mm @ 10 µm telecentric lens with built in prism for DOAL lighting omnidirectional triple LED rings; side, main, line sourced DOAL (diffused on axis lighting (coaxial)) Marantz iSpector HDL 650L X + Y direction stationary during inspection presence, polarity, offset, correctness, soldering offset, smearing, bridges, uniformity synthetic imaging, spectral analysis, grayscale limits brightness, hue, saturation via filters UXGA CCD digital camera with CameraLink 32 x 24 mm @ 20 µm; 40 x 30 mm @ 25 µm; 16 x 12 mm @ 10 µm telecentric lens with built in prism for DOAL lighting omnidirectional triple LED rings; side, main, line sourced DOAL (diffused on axis lighting (coaxial)) Marantz iSpector HML 350L X + Y direction stationary during inspection presence, polarity, offset, correctness, soldering offset, smearing, bridges, uniformity synthetic imaging, spectral analysis, grayscale limits brightness, hue, saturation via filters UXGA CCD digital camera with CameraLink 32 x 24 mm @ 20 µm; 40 x 30 mm @ 25 µm; 16 x 12 mm @ 10 µm high resolution telecentric omnidirectional 4- angle LED: RGB- DOAL (coaxial) Marantz iSpector HML 650L X + Y direction stationary during inspection presence, polarity, offset, correctness, soldering offset, smearing, bridges, uniformity synthetic imaging, spectral analysis, grayscale limits brightness, hue, saturation via filters UXGA CCD digital camera with CameraLink 32 x 24 mm @ 20 µm; 40 x 30 mm @ 25 µm; 16 x 12 mm @ 10 µm high resolution telecentric omnidirectional 4- angle LED: RGB- DOAL (coaxial) Orpro Vision Symbion S36 stationary during inspection X + Y + Z direction presence and absence of components, placement accuracy and polarity, optical character recognition insufficient solder, tombstone, billboard, coplanarity, lifted leads, shorts n.a. n.a. 6.4. Combined appliances There are systems that are able to inspect more sequences. These are combined systems, namely API&PSI [140]-[146] (Table VI). And the all-in-one machines are the UAOI systems, detailed: SPI&API&PSI [147]-[163] (Table VII). Materials Science - Advanced Topics 398 Manufacturer TRI Innovation Model TR7530 S3016 S3054QC Field of view (orthogonal camera) [pixel] 1024 x 768 2752 x 2048 @ 55 x 43 mm 672 x 512 Pixel size (orthogonal camera) [µm] 10; 15; 20 22; 10 22; 10 Number of cameras (orthogonal) 1 4 1, 2 or 4 Resolution (angled view camera) [µm/pixel] n.a. 15 n.a. Number of cameras (angled view) n.a. 4 n.a. Illumination ultra-low angle, multi- segment, RGB LED lighting n.a. n.a. Inspection speed 72 sq. cm/sec @ 20 µm; 40 sq. cm/sec @ 10 µm; 18 sq. cm/sec @ 10 µm typical connector with 100 pins 15 sec typical connector with 100 pins 15 sec Board size max. [mm] 400 x 300 430 x 406 443 x 406 Board size min. [mm] 50 x 50 n.a. n.a. Board weight max. [kg] 3 n.a. n.a. Conveyor height [mm] n.a. 850 - 960 850 - 960 Board edge clearance [mm] 3 3 3 Board clearance top [mm] 25 50 15 Board clearance bottom [mm] 40 35 50 Applicability missing component, tombstone, billboard, polarity, skew, marking, defective, insufficient and excess solder, bridge, trough hole pin, lifted lead, golden finger, scratch, blur selective and special solder joints selective and special solder joints Viscom Table 5. Comparison of Post Soldering Inspection (PSI) machines Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 399 SPI API PSI API PSI pre ref low pre wave post wave component in past e component in adhesive missing past e whet her it is not covered by component misalignment past e bridge whet her it is not covered by component whet her it is not covered by component missing component component posit ion (x-y shif t , rot at ion, f ace lif t ) polarit y damaged component unsoldered component insuf f icient solder j oint solder bridge lif t ed lead t ombst one missing adhesive smeared adhesive on pad missing pin end insuf f icient pin solder dewet t ing PWB regist rat ion UAOI post ref low adhesive inspect ion past e inspect ion smeared solder past e cont aminat ion complet eness or/ and volume of solder past e Table 6. 6.4. Combined appliances 4MP XGA high resolution top and 4 side cameras with symmetric image acquisition and color capability n.a. n.a. axial, direct, diffuse and RGB multi-color illumination TRI Innovation 7500 n.a. n.a. missing, tombstone, billboard, polarity, shift insufficient solder, excess solder, bridge n.a. n.a. 1 top view XGA 3CCD camera @ 1024 x 768 pixel, 4 angle view XGA mono camera & 1024 x 768 pixel 10 µm; 15 µm; 20 µm; 25 µm n.a. multi segment, multi angle LED, RGB+W TRI Innovation 7500L n.a. n.a. missing, tombstone, billboard, polarity, shift insufficient solder, excess solder, bridge n.a. n.a. 1 top view XGA 3CCD camera @ 1024 x 768 pixel, 4 angle view XGA mono camera & 1024 x 768 pixel 10 µm; 15 µm; 20 µm; 25 µm n.a. multi segment, multi angle LED, RGB+W TRI Innovation 7550 n.a. n.a. missing, tombstone, billboard, polarity, skew, marking, defective insufficient solder, excess solder, bridge, trough hole pins, lifted lead, golden finger scratch, blur n.a. n.a. 1 top view XGA 3CCD camera @ 1024 x 768 pixel, 4 angle view XGA mono camera & 1024 x 768 pixel 10 µm; 15 µm; 20 µm; 25 µm n.a. multi segment, multi angle LED, RGB (coaxial lighting optional) Sony SI-V200 n.a. n.a. inaccurate mounting, reversed, polarity missing, solder, bridging, solder quantity n.a. n.a. 2MP color CCD camera 24.8 x 18.6 mm @ 15.5 µm; 17.6 x 13.2 mm @ 11 µm n.a. high intensity white LED Saki BF-Tristar n.a. n.a. presence and absence of components, misalignment, tombstone, reverse, polarity, bridge, foreign material, lifted lead, lifted chip absence, insufficient solder, fillet defect n.a. n.a. line color CCD camera 10 µm n.a. LED lighting system Table 7. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 401 Manufacturer Model Minimum inspection component size Position accuracy Board clearance top [mm] Board clearance bottom [mm] Board size max. [mm x mm] Board size min. [mm x mm] Movement speed [mm/sec] Inspection capacity typical Conveyor speed [mm/sec] Conveyor height [mm] Board clamping Board thickness [mm] Machine Vision Products Supra E 0201 and 01005 (0.4 x 0.2 mm) 0.5 µm n.a. n.a. 508 x 508 n.a. n.a. 90 sq. cm/sec n.a. n.a. n.a. n.a. Table 8. Comparison of combined (API&PSI) machines Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 401 6.4. Combined appliances Marantz iSpector HDL 350L 01005 (0.4 x 0.2 mm @ 10 µm) pixel related feedback loop 40 35, 55 350 x 250 50 x 50 720 1500 cps/min 10 - 500 830 - 950 ruler blade, top & edge clamping, sensor stopper 0.6 - 4.0 Marantz iSpector HDL 650L 01005 (0.4 x 0.2 mm @ 10 µm) pixel related feedback loop 40 35, 55 650 x 550 50 x 50 720 1500 cps/min 10 - 500 830 - 950 ruler blade, top & edge clamping, sensor stopper 0.6 - 4.0 Marantz iSpector HML 350L 01005 (0.4 x 0.2 mm @ 10 µm) pixel related feedback loop 30 35, 55 350 x 250 50 x 50 720 1500 cps/min 10 - 500 n.a. ruler blade, top & edge clamping, sensor stopper 0.6 - 4.0 Marantz iSpector HML 650L 01005 (0.4 x 0.2 mm @ 10 µm) pixel related feedback loop 30 35, 55 650 x 550 50 x 50 720 1500 cps/min 10 - 500 n.a. ruler blade, top & edge clamping, sensor stopper 0.6 - 4.0 Orpro Vision Symbion S36 01005 (0.4 x 0.2 mm) down to 0.3 mm pitch n.a. 90 90 550 x 470 n.a. n.a. 40 sq. cm/sec n.a. n.a. n.a. n.a. TRI Innovation 7500 n.a. n.a. 50 50 510 x 460 n.a. n.a. 110 sq. cm/sec @ 25 µm; 72 sq. cm/sec @ 20 µm; 40 sq. cm/sec @ 15 µm; 18 sq. cm/sec @ 10 µm; n.a. n.a. n.a. max. 4.0 TRI Innovation 7500L n.a. n.a. 50 50 660 x 610 n.a. n.a. 110 sq. cm/sec @ 25 µm; 72 sq. cm/sec @ 20 µm; 40 sq. cm/sec @ 15 µm; 18 sq. cm/sec @ 10 µm; n.a. n.a. n.a. max. 4.0 TRI Innovation 7550 n.a. n.a. 40 40 540 x 460 50 x 50 n.a. 110 sq. cm/sec @ 25 µm; 72 sq. cm/sec @ 20 µm; 40 sq. cm/sec @ 15 µm; 18 sq. cm/sec @ 10 µm; n.a. n.a. n.a. 0.6 - 4.0 Sony SI-V200 0402 @ high resolution, 0603 @ normal resolution n.a. 20 40 460 x 510 40 x 50 n.a. 0.27 sec/frame n.a. n.a. n.a. 0.4 - 2.0 Saki BF-Tristar n.a. n.a. 30 30 250 x 330 50 x 70 n.a. 20 sec / 250 x 350 mm n.a. 900 n.a. 0.6 - 2.5 Table 8. 6.4. Combined appliances Comparison of combined (API&PSI) machines Materials Science - Advanced Topics 402 Manufacturer Model Pre-reflow inspection Post-reflow inspection Camera type Orthogonal camera field of view [mm] Orthogonal camera resolution [µm] Number of orthogonal cameras Angular camera resolution [µm] Number of angular cameras Illumination Agilent Technologies Medalist SJ50 Series 3 missing, offset, 2D paste, skewed, polarity, bridging, wrong part, traceability missing, offset, skewed, polarity, billboard, tombstone, lifted/bent leads, excess/insufficient solder, bridging, wrong part, traceability 4 megapixel digital camera 44.7 x 32.8 19; 12 n.a. n.a. n.a. multiple color, multiple angle, multiple segment ED lighting head, auto-calibration Agilent Technologies Medalist SJ50 Series 3 XL missing, offset, 2D paste, skewed, polarity, bridging, wrong part, traceability missing, offset, skewed, polarity, billboard, tombstone, lifted/bent leads, excess/insufficient solder, bridging, wrong part, traceability 4 megapixel digital camera 44.7 x 32.8 19; 12 n.a. n.a. n.a. multiple color, multiple angle, multiple segment ED lighting head, auto-calibration Agilent Technologies Medalist sj5000 missing, offset, 2D paste, skewed, polarity, bridging, billboard, wrong part , extra part, traceability missing, offset, skewed, polarity, billboard, tombstone, lifted/bent leads, excess/insufficient solder, bridging, wrong part, traceability 4 megapixel digital camera 44.7 x 32.8 21; 12 n.a. n.a. n.a. multiple color, multiple angle, multiple segment ED lighting head, auto-calibration Amistar Automation Inc. K2 position shift, blur, solder area, bridge missing components, position shift, rotation error, wrong components, polarity check, bridge, character recognition, solder quantity, lifted leads CCD 30.4 x 22.8 19 n.a. n.a. n.a. auto-adjust 3-stage LED dome lighting (Upper: IR; Middle: WH; Lower: WH) Amistar Automation Inc. K2L position shift, blur, solder area, bridge missing components, position shift, rotation error, wrong components, polarity check, bridge, character recognition, solder quantity, lifted leads CCD 30.4 x 22.8 19 n.a. n.a. n.a. auto-adjust 3-stage LED dome lighting (Upper: IR; Middle: WH; Lower: WH) CyberOptics Flex HR 8 missing, polarity, billboard, flipped, wrong part, gross body and lead damage, gold-finger contamination, missing, polarity, billboard, flipped, wrong part, gross body and lead damage, gold-finger contamination, tombstone, solder bridge, opens, lifted leads, wettability, excess/insufficient solder, debris 5 megapixel color CMOS camera n.a. 17 5 n.a. n.a. fluorescent white light CyberOptics Flex HR 12 missing, polarity, billboard, flipped, wrong part, gross body and lead damage, gold-finger contamination, missing, polarity, billboard, flipped, wrong part, gross body and lead damage, gold-finger contamination, tombstone, solder bridge, opens, lifted leads, wettability, excess/insufficient solder, debris 5 megapixel color CMOS camera n.a. 17 8 n.a. n.a. 6.4. Combined appliances fluorescent white light CyberOptics Flex HR 11 missing, polarity, billboard, flipped, wrong part, gross body and lead damage, gold-finger contamination, missing, polarity, billboard, flipped, wrong part, gross body and lead damage, gold-finger contamination, tombstone, solder bridge, opens, lifted leads, wettability, excess/insufficient solder, debris 5 megapixel color CMOS camera n.a. 17 11 n.a. n.a. fluorescent white light Machine Vision Products Ultra IV n.a. n.a. n.a. n.a. 8 - 17 n.a. n.a. n.a. programmable variable LED strobe lighting, proprietary multi-color illumination Mirtec MV-7 n.a. n.a. 1.3, 2 or 4 megapixel digital color camera 14.0 x 10.5 to 37.2 x 37.2 9.8 - 18.2 n.a. n.a. 4 n.a. Mirtec MV-7L n.a. n.a. 1.3, 2 or 4 megapixel digital color camera 14.0 x 10.5 to 37.2 x 37.2 9.8 - 18.2 n.a. n.a. 4 n.a. Mirtec MV-7xi n.a. n.a. 1.3, 2 or 4 megapixel digital color camera 14.0 x 10.5 to 37.2 x 37.2 9.8 - 18.2 n.a. n.a. 4 n.a. Mirtec MV-7U n.a. n.a. 1.3, 2 or 4 megapixel digital color camera 14.0 x 10.5 to 37.2 x 37.2 9.8 - 18.2 n.a. n.a. 4 n.a. Omron VT-RNS-S presence of solder, wrong components, missing components, bridging, component shifting, lead bending presence of solder, wrong components, missing components, bridging, tombstone, component shifting, fillet, wettability, lead bending, adhesive, solder balls 3-CCD camera n.a. 10; 15; 20 n.a. n.a. n.a. ring-shaped LED (red, green, blue) Omron VT-WIN II presence/absence, skewed, shifted, wrong component, un-inserted, upside-down/backward, polarity, lead bent presence/absence of solder, excessive solder, insufficient solder, blow holes, wettability, bridges, solder balls, skewed, shifted, wrong component, polarity, lead bent triple element CCD camera n.a. 10; 13; 15; 20; 25; 30; 35; 50 n.a. n.a. n.a. 3 ring-shaped LED arrays with automatic brightness control Saki BF-Frontier presence/absence, misalignment, polarity, bridge presence/absence, tombstone, reverse, polarity, bridge, foreign material, absence of solder, insufficient solder, lifted lead, lifted chip, fillet line color CCD camera n.a. 18 n.a. n.a. n.a. LED lighting system Saki BF-Planet-X presence/absence, misalignment, polarity, bridge presence/absence, tombstone, reverse, polarity, bridge, foreign material, absence of solder, insufficient solder, lifted lead, lifted chip, fillet line color CCD camera 10 n.a. n.a. n.a. LED lighting system Viscom S3088-III n.a. n.a. megapixel 57.6 x 43.5 23.4; 11.7 4 16.1; 8.05 4; 8 n.a. Viscom S3088-II n.a. n.a. megapixel 57.6 x 43.5 23.4; 11.7 4 16.1; 8.05 4; 8 n.a. Viscom S6056-ST1 n.a. n.a. 6.4. Combined appliances megapixel 57.6 x 43.5 23.4; 11.7 4 16.1; 8.05 4; 8 n.a. Viscom S6056-DS1W n.a. n.a. megapixel 57.6 x 43.5 23.4; 11.7 4 16.1; 8.05 4; 8 n.a. Viscom S6056-DS2W n.a. n.a. megapixel 57.6 x 43.5 23.4; 11.7 4 16.1; 8.05 4; 8 n.a. Vi Technology 3K Series n.a. n.a. 1620 x 1220 pixel; 2352 x 1728 pixel 42.1 x 31.7; 61.1 x 44.9 8 - 12 n.a. n.a. n.a. i-LITE (red, green, blue); axial and peripheral Vi Technology 5K Series n.a. n.a. 1620 x 1220 pixel; 2352 x 1728 pixel 42.1 x 31.7; 61.1 x 44.9 8 - 12 n.a. n.a. n.a. i-LITE (red, green, blue); axial and peripheral Vi Technology 7K Series n.a. n.a. 1620 x 1220 pixel; 2352 x 1728 pixel 42.1 x 31.7; 61.1 x 44.9 8 - 12 n.a. n.a. n.a. i-LITE (red, green, blue); axial and peripheral Vi Technology Vi-5000 n.a. n.a. 1360 x 1040 pixel 44.5 x 33.6 12 n.a. n.a. n.a. amber, green, blue Vi Technology Vi-5000-2 n.a. n.a. 1600 x 1152 pixel 41.6 x 29.9 8 n.a. n.a. n.a. green, white, blue, axial and peripheral Vi Technology Vi-5000-3 n.a. n.a. 2048 x 2048 pixel 53.2 x 53.2 8 n.a. n.a. n.a. green, white, blue YES Tech YTV-F1 position, missing, wrong, polarity, skew polarity, skew, tombstone, bent lead, lifted, bridging, open solder, insufficient, short, solder balls Multiple Thin Camera megapixel color top- down viewing camera @ 1280 x 1024 pixel n.a. 25; 12 n.a. n.a. n.a. LED top light, proprietary bi-color multiangle LED lighting YES Tech YTV-F1S position, missing, wrong, polarity, skew polarity, skew, tombstone, bent lead, lifted, bridging, open solder, insufficient, short, solder balls Multiple Thin Camera megapixel color top- down and 4 side viewing camera @ 1280 x 1024 pixel n.a. 25; 12 n.a. n.a. n.a. LED top light, proprietary bi-color multiangle LED lighting YES Tech YTV-M1 position, missing, wrong, polarity, skew polarity, skew, tombstone, bent lead, lifted, bridging, open solder, insufficient, short, solder balls YESTech 3 Megapixel Thin Camera top-down viewing camera and telecentric lens @ 2048 x 1536 pixel n.a. 25; 12 n.a. n.a. n.a. proprietary Fusion Lighting Table 9. Table 9. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 403 Manufacturer Model Inspection capacity typical Board size max. [mm x mm] Board size min. [mm x mm] Board warp [mm] Board clearance top [mm] Board clearance bottom [mm] Board thickness [mm] Board weight max. 6.4. Combined appliances [kg] Dual lane capable Conveyor height [mm] Agilent Technologies Medalist SJ50 Series 3 41 sq. cm/sec @ pre-reflow; 32 sq. cm/sec @ post reflow 510 x 510 50 x 50 n.a. n.a. n.a. 0.5 - 4.0 3 Yes n.a. Agilent Technologies Medalist SJ50 Series 3 XL 41 sq. cm/sec @ pre-reflow; 32 sq. cm/sec @ post reflow 620 x 620 75 x 50 n.a. n.a. n.a. 1.5 - 15 13 n.a. n.a. Agilent Technologies Medalist sj5000 41 sq. cm/sec @ pre-reflow; 32 sq. cm/sec @ post reflow 510 x 510 50 x 50 n.a. n.a. n.a. 0.5 - 4.0 3 Yes n.a. Amistar Automation Inc. K2 0.25 sec/screen 330 x 250 50 x 50 + 0.5; - 1.0 28 25 0.5 - 2.0 n.a. n.a. n.a. Amistar Automation Inc. K2L 0.25 sec/screen 485 x 410 50 x 50 + 0.5; - 1.0 28 25 0.5 - 2.0 n.a. n.a. n.a. CyberOptics Flex HR 8 50 sq. cm/sec 203 x 508 110 x 63 ± 0.7 32 3 n.a. n.a. n.a. 813 - 965 CyberOptics Flex HR 12 50 sq. cm/sec 305 x 508 110 x 63 ± 0.7 32 3 n.a. n.a. n.a. 813 - 965 CyberOptics Flex HR 11 50 sq. cm/sec 457 x 508 110 x 63 ± 0.7 32 3 n.a. n.a. n.a. 813 - 965 Machine Vision Products Ultra IV 90 sq. cm/sec 500 x 546 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. Mirtec MV-7 4.94 sq. mm/sec 350 x 250 50 x 50 n.a. 25 - 45 50.8 n.a. n.a. n.a. n.a. Mirtec MV-7L 4.94 sq. mm/sec 500 x 400 50 x 50 n.a. 25 - 45 50.8 n.a. n.a. n.a. n.a. Mirtec MV-7xi 4.94 sq. mm/sec 510 x 460 50 x 50 n.a. 25 - 45 50.8 n.a. n.a. n.a. n.a. Mirtec MV-7U 4.94 sq. mm/sec 660 x 510 50 x 50 n.a. 25 - 45 50.8 n.a. n.a. n.a. n.a. Omron VT-RNS-S 0.25 sec/screen @ 10 sq. mm field of view 510 x 460 50 x 50 n.a. 20 - 40 40 - 50 n.a. n.a. n.a. n.a. Omron VT-WIN II 0.40 sec/screen 460 x 510 50 x 50 n.a. 50 50 0.3 - 4.0 n.a. n.a. n.a. Saki BF-Frontier 24 sec/screen 460 x 500 50 x 60 ± 0.2 40 40 0.6 - 2.5 n.a. n.a. max. 900 Saki BF-Planet-X 23 sec/screen 250 x 330 50 x 60 n.a. 6.4. Combined appliances 20 30 0.6 - 2.5 n.a. n.a. max. 900 Viscom S3088-III 20 - 40 sq. cm/sec 508 x 508 n.a. n.a. 35 40 n.a. n.a. n.a. 850 - 960 Viscom S3088-II 20 - 40 sq. cm/sec 450 x 350 n.a. n.a. 35 40 n.a. n.a. n.a. 850 - 960 Viscom S6056-ST1 20 - 40 sq. cm/sec 457 x 356 n.a. n.a. 35 60 n.a. n.a. n.a. 830 - 960 Viscom S6056-DS1W 20 - 40 sq. cm/sec 457 x 356 n.a. n.a. 35 60 n.a. n.a. n.a. 830 - 960 Viscom S6056-DS2W 40 - 80 sq. cm/sec 457 x 356 n.a. n.a. 35 60 n.a. n.a. n.a. 830 - 960 Vi Technology 3K Series 4 - 20 ms 458 x 406 50 x 50 n.a. 34 34 0.7 - 4.0 3 Yes n.a. Vi Technology 5K Series 4 - 20 ms 533 x 533 50 x 50 n.a. 34 60 0.5 - 4.0 3 n.a. n.a. Vi Technology 7K Series 4 - 20 ms 533 x 610 50 x 50 n.a. 34 60 0.5 - 4.0 3 Yes n.a. Vi Technology Vi-5000 4 - 20 ms 508 x 458 50 x 50 n.a. 34 40 0.7 - 5.0 7 n.a. n.a. Vi Technology Vi-5000-2 4 - 20 ms 508 x 458 50 x 50 n.a. 34 40 0.7 - 5.0 7 n.a. n.a. Vi Technology Vi-5000-3 4 - 20 ms 508 x 458 50 x 50 n.a. 34 40 0.7 - 5.0 7 n.a. n.a. YES Tech YTV-F1 35 sq. cm/sec 560 x 510 n.a. n.a. 50 50 n.a. n.a. n.a. n.a. YES Tech YTV-F1S 35 sq. cm/sec 560 x 510 n.a. n.a. 50 50 n.a. n.a. n.a. n.a. YES Tech YTV-M1 35 sq. cm/sec 350 x 250 50 x 50 n.a. 25 50 n.a. n.a. n.a. max. 950 Table 10. Comparison of Universal Automatic Optical Inspection (UAOI) machines Assuming that the component is fully operational, these systems practically are able to prove that the whole circuit board is working correctly thus replacing the ICT. However, because they are usually connected to SPC (Statistical Process Control) servers, they can also provide much information about the SMT process itself and provides help as to how to improve it. But of course there are disadvantages to using AOI systems. 7. Special AOI solutions — Inspection of lead-free solder joints, flexible substrates, wire bonding and semiconductors According to RoHS and WEEE directives, lead-free solder alloys have to be used in commercial electronics. This has presented a new challenge for AOI systems because of the differing optical properties of lead-free alloy. Some solutions are shown in the following studies [166]-[173]. AOI has several further application possibilities in electronic device manufacturing e.g. semiconductor and wire-bonding inspection. These appliances need extremely high-resolu‐ tion cameras to detect defects in the μm scale. Another interesting area is flexible substrate inspection. Some of these special inspections are described in [174]-[179]. 6.4. Combined appliances They are not able to inspect hidden failures such as soldered BGA (Ball Grid Array) bumps and usually the parameters of inspection algorithms cannot be adjusted perfectly. So from time to time they do not detect real failures which are called ‘slip-through failures’. These are the most significant malfunc‐ tions during the operation of AOI systems because in these cases, they fail to do what they were programmed for. So the number of slip-throughs must be zero and - if they arise - close investigation is necessary to prevent and eliminate them. However if this occurs repeatedly, Materials Science - Advanced Topics 404 then the appropriate parts would seem to be defective. These are the pseudo-failures which can reduce productivity so their number should as close to zero as possible. [164] ALSO indicates some other image processing problems. The problems of AOI systems will be described in more detail later in the chapter. Another disadvantage is that they are usually in the ‘bottle-neck’ of the manufacturing production line because they are not able to inspect the whole circuit board as fast as the line can produce them. Therefore, the practice is usually to place more machines behind each other to enable inspections to take place in parallel. Of course, this also has financial implications which should be taken into consideration. 7. Special AOI solutions — Inspection of lead-free solder joints, flexible substrates, wire bonding and semiconductors 7.1. Differences between lead-based and lead-free solder alloys Solders that contain lead are available with a tin content of between 5% and 70%. The compo‐ sition of the most commonly used lead solder is 63/37 Sn/Pb; this was the main type used in electronics manufacturing until strict controls were imposed on its use for environmental reasons. The homogeneity of the solder meniscus that formed was beneficial in that the melting point of eutectic solder really is manifested as a single point on the phase diagram; in other words the molten alloy solidifies at a specific temperature, rather than within a broader temperature range. The solidified alloy can be broken down into tiny lead and tin phases of almost 100% purity, without intermetallic layers. In the case of non-eutectic solders, the crystallisation begins around cores of differing compo‐ sition and crystal structure, and at differing temperatures, so that during the accretion of the individual cores the composition of the residual melt also changes. Due to this, in the case of lead-free, non-eutectic solder alloys, certain phases solidify earlier, and these solid cores do not form a completely mirror-like, smooth surface on the face of the solder meniscus (and naturally, they also cause differences in the volume of the material). Lead-free solders usually contain tin, silver and copper. Compared to lead-based solders they have several negative properties: they are more expensive, their melting point is higher, and they give rise to problems that do not occur when soldering with lead (the phenomenon of whisker formation has still not been fully explored). Because their surface differs from that of Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 405 http://dx.doi.org/10.5772/51699 lead-based solder alloys – which are much more even and mirror-like – they reflect light differently, so different procedures may be used to verify the presence and quality of the solder menisci. In the case of tin-lead solders, the solidification of the melt begins around the cores that are Figure 7. Tin-lead phase diagram http://dx.doi.org/10.5772/51699 Figure 7. Tin-lead phase diagram lead-based solder alloys – which are much more even and mirror-like – they reflect light differently, so different procedures may be used to verify the presence and quality of the solder menisci. lead-based solder alloys – which are much more even and mirror-like – they reflect light differently, so different procedures may be used to verify the presence and quality of the solder menisci. 7.1. Differences between lead-based and lead-free solder alloys In the case of tin-lead solders, the solidification of the melt begins around the cores that are solid at melting point, and the individual solid phases grow at a virtually consistent rate as the two elements separate from the melt. This is how the volumes that are rich in lead and tin become a smooth-surfaced alloy consisting of lead and tin patches, typical of eutectic solder, that can easily differentiated on the cross-section. Lead-free solders do not usually form eutectic alloys, and exist in many variants with different compositions. Tin is usually alloyed with copper and silver, but there are also alloys containing, for example, bismuth and indium. In the case of the non-eutectic alloys (the vast majority of lead-free alloys), however, one of the phases begins to solidify earlier, and the alloying metal concentration of this phase will be smaller than that of the melt. This means that the composition of the remaining part of the alloy, which is still in a liquid state, continues to change until the eutectic composition is achieved, when it cools and solidifies. As a result of this, the microstructure that is created has a greater surface roughness than in the previous case: as the eutectic melt ebbs away, the intermetallic crystals that were the first to solidify create a more irregular surface. This surface scatters light much more than the smoother, more even surface of the tin-lead solder; in other words the proportion of diffuse reflection will be greater than that of specular reflection. Figure 8. Tin-copper-silver phase diagram Materials Science - Advanced Topics Materials Science - Advanced Topics 406 Figure 8. Tin-copper-silver phase diagram achieved, when it cools and solidifies. As a result of this, the microstructure that is created has a greater surface roughness than in the previous case: as the eutectic melt ebbs away, the intermetallic crystals that were the first to solidify create a more irregular surface. This surface scatters light much more than the smoother, more even surface of the tin-lead solder; in other words the proportion of diffuse reflection will be greater than that of specular reflection. achieved, when it cools and solidifies. As a result of this, the microstructure that is created has a greater surface roughness than in the previous case: as the eutectic melt ebbs away, the intermetallic crystals that were the first to solidify create a more irregular surface. 7.1. Differences between lead-based and lead-free solder alloys This surface scatters light much more than the smoother, more even surface of the tin-lead solder; in other words the proportion of diffuse reflection will be greater than that of specular reflection. An attempt to measure the two solders with AOI equipment using the same settings will probably result in several errors, because after the necessary image conversion procedures the images made by the equipment will differ. For this reason, it would clearly be useful to calibrate the AOI equipment specifically for the different solders. In what follows we present a series of images of tin-lead eutectic and lead-free Sn-Ag-Cu solder alloys made using a scanning electron microscope (SEM). This instrument is not suitable for measuring the surface roughness, but it does provide an accurate, high-resolution image of the examined surfaces and of the two solder alloys with differing composition and surface roughness, showing the differences in height and material with spectacular contrast. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 407 Figure 10. SEM image of the surface of a lead-based solder meniscus Figure 9. Electron microscope image of the surface of a tin-lead solder meniscus Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 Figure 9. Electron microscope image of the surface of a tin-lead solder meniscus Figure 9. Electron microscope image of the surface of a tin-lead solder meniscus Figure 10. SEM image of the surface of a lead-based solder meniscus Figure 10. SEM image of the surface of a lead-based solder meniscus Materials Science - Advanced Topics 408 An important question is precisely what the roughness of the pattern formed on the surface of lead-free solder alloys depends on, and how “reliably” predictable the process of its formation is. Figure 11. SEM image of the surface of a lead-free solder meniscus Figure 11. SEM image of the surface of a lead-free solder meniscus In the above image the two types of solder can be clearly differentiated due to the rougher surface that is typical of the lead-free alloy. In places the surface looks quite similar to the one assumed by the microfacet model, which simplifies reality for the purpose of mathematical manageability; in other words, small semi-spherical formations can be observed side by side with each other. 7.1. Differences between lead-based and lead-free solder alloys The fracture in the solder meniscus seen in the above image is probably due to a contaminant, but within the fracture is a particularly clear example of just how uneven a surface can be formed by the lead-free solder alloy. The surface roughness of the lead-free solder meniscus is visibly greater than that of the tin- lead solder. Taking the scale bar as a guide we can also estimate that the size of the uneven protrusions that increase the surface roughness, in terms of both their breadth and height, is in the order of 10 μm. It is also worth noting that the simplification of the microfacet model described by the Cook-Torrance model is clearly visible, as a visual inspection reveals that the surface is not closely similar to the surface made up of tiny flat plates that is assumed by the microfacet model. This simplification, however, is more than made up for by the model’s simplicity and general ease of use. 7.1. Differences between lead-based and lead-free solder alloys On other parts of the picture, however, areas with no unevenness are also visible; and we have taken the electron microscope image of an area that gives a good illustration of a particular property of lead-free, non-eutectic solder alloys (in this case a tin- copper-silver alloy), namely that due to the unevenness of the surface it reflects the light more diffusely (in other words, it scatters the light more) than the smoother surface of a eutectic solder. In the applied Cook-Torrance model, the roughness of the surface is described by a single parameter, which describes the surface in average terms. The above picture shows an SEM image of a cross-section in which the tin (light) and lead (dark) phases of the eutectic alloy are clearly differentiated. The above image was made at a lower magnification (400x rather than 1500x), but the phase boundaries can still be made out, and the smooth meniscus surface typical of lead-based solder Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 409 Figure 12. SEM image of a cross-section of a lead-based solder meniscus Figure 13. SEM image of a cross-section of a lead-based solder meniscus Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 Figure 12. SEM image of a cross-section of a lead-based solder meniscus Figure 12. SEM image of a cross-section of a lead-based solder meniscus Figure 13. SEM image of a cross-section of a lead-based solder meniscus Figure 13. SEM image of a cross-section of a lead-based solder meniscus Materials Science - Advanced Topics 410 alloys is even more visible. In the following SEM images the rough surface typical of lead-free solder alloys can be observed. Figure 14. SEM image of a cross-section of a lead-free solder meniscus The fracture in the solder meniscus seen in the above image is probably due to a contaminant, but within the fracture is a particularly clear example of just how uneven a surface can be formed by the lead-free solder alloy. Figure 14. SEM image of a cross-section of a lead-free solder meniscus The fracture in the solder meniscus seen in the above image is probably due to a contaminant, but within the fracture is a particularly clear example of just how uneven a surface can be formed by the lead-free solder alloy. Type of measurement result Lead-based solder paste Lead-free solder paste Measured surface roughness (RMS) 0.03092 0.0986 Distribution of measured RMS value 0.004451 0.054626 Table 11. Measured surface roughness values Figure 15. SEM image of a cross-section of a lead-free solder meniscus Figure 15. SEM image of a cross-section of a lead-free solder meniscus At first glance the measured surface roughness values appear realistic; the surface roughness of the lead-free solder turned out to be approximately three times that of the lead-based solder. The distribution of the roughness values for the lead-based solder was below 1%, which is satisfactory because the divergence between the shape of the actual solder and that modelled by the computer showed a greater error (a few percent), and because greater fluctuation than this can be expected to result from the differing heat profiles, printed circuit boards or solder- handling requirements of real production lines. The distribution of the surface roughness values for the lead-free solder was over 5%, which is due to diversity of the size and shape of the surface protrusions that appear with this type of solder, which the Cook-Torrance model handles using statistical simplification, by assuming the surface to be of a consistent roughness. 7.2. Measuring the surface roughness To measure the surface roughness we used a Tencor Alpha Step 500 surface profilometer. Based on the 10 measurements of each solder, made on the lead-based (Heraeus F816 Sn63-90 B30) and lead-free (Senju Ecosolder M705-GRN360-K1-V) joints, the two solders yielded the following values: Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 411 7.3. Simulation created with the computer model We checked the measured surface roughness values by comparing the images made using optical microscopes with the computer-generated graphic representations. The Surface Materials Science - Advanced Topics 412 Evolver software uses finite element analysis to calculate the surface profile at certain points on the surface. In areas with a greater radius of curvature, where the energies are closer to each other in terms of magnitude (in other words none are dominant in comparison to any others) the software uses more measurement points, that is a denser grid, for displaying the graphic representation. Evolver software uses finite element analysis to calculate the surface profile at certain points on the surface. In areas with a greater radius of curvature, where the energies are closer to each other in terms of magnitude (in other words none are dominant in comparison to any others) the software uses more measurement points, that is a denser grid, for displaying the graphic representation. Figure 16. Example of a graphic representation generated using the Surface Evolver software Figure 16. Example of a graphic representation generated using the Surface Evolver software Of the models that use a formula based on the Bi-directional Reflectance Distribution Function (BRDF), which is based on a physical approach, the most widely used is the Cook-Torrance model, which has surface roughness as one of its input parameters and is also capable of handling Fresnel distribution. During our simulation we used this, in a Direct3D environment, so when generating the rendered graphics we were able to use the measured roughness values as input parameters. The majority of optical microscopes – including the Olympus BX51 microscope used by me at the department – are capable of operating in bright field (BF) and dark field (DF) imaging mode. In bright field imaging, both the incident and reflected light fall almost perpendicularly onto the sample, naturally through a focusing lens. Dark field microscopes, on the other hand, collect beams of light that arrive not perpendicularly but from the side, from below a given angle, through a lens, in the direction of the observer; in other words the beams of light travel in the opposite direction but along the same path as would the beams of light that enter perpendicularly but are diffracted, not reflected. 7.3. Simulation created with the computer model Dark field microscopy gives a good resolution and microscopes with this capability are usually more expensive, but they are eminently suitable for the detection of phase boundaries or the examination of surface irregularities highlighted by the side illumination. In the case of metals, in which the proportion of diffuse components is smaller and the incident light is reflected much more in accordance with the principle of optical reflection, bright field microscopy Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 413 results in a darker image and in the case of observation along the z axis (from above), as is typical of microscopes, only the surfaces that are parallel to the horizontal are illuminated. We also modelled both of these different types of illumination using the Direct3D software. What follows is a comparison of the images made using the optical microscope and the graphic representations rendered with Direct3D that most closely replicated the actual light and surface conditions. Where not indicated separately, the soldered joint (at the SMT resistors) is illuminated with scattered light. Figure 17. Photograph and graphic representation of empty solder pad covered in lead-free solder (BF imaging) Figure 17. Photograph and graphic representation of empty solder pad covered in lead-free solder (BF imagi Figure 18. Photograph and graphic representation of empty solder pad covered in lead-free solder (DF imaging) gure 18. Photograph and graphic representation of empty solder pad covered in lead-free solder (DF imaging) Materials Science - Advanced Topics 414 Figure 19. Photograph and graphic representation of SMT joint made with lead-free solder Figure 19. Photograph and graphic representation of SMT joint made with lead-free solder Figure 20. Photograph and graphic representation of empty solder pad covered in lead-based solder (BF imaging) Figure 20. Photograph and graphic representation of empty solder pad covered in lead-based solder (BF imaging) Figure 20. Photograph and graphic representation of empty solder pad covered in lead-based solder (BF imaging) Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 415 Figure 21. Photograph and graphic representation of empty solder pad covered in lead-based solder (DF imaging) Figure 21. Photograph and graphic representation of empty solder pad covered in lead-based solder (DF imaging) Figure 22. Photographs (above: BF, below: DF) and graphic representation of SMT joint made with lead-based solder Figure 22. Photographs (above: BF, below: DF) and graphic representation of SMT joint made with lead-based solder Materials Science - Advanced Topics Figure 23. 7.3. Simulation created with the computer model Photograph and graphic representation of SMT joint made with lead-free (left) and lead-based (right) sol‐ der Figure 23. Photograph and graphic representation of SMT joint made with lead-free (left) and lead-based (right) sol‐ der 8. Detailed analysis of AOI systems When there are even more cameras, each with its separate illumination (matrix arrangement), then the PWB should lie on a positioning table. Two big groups of drive systems are commonly used for this purpose. The first is the conven‐ tional electromechanical drive. It is used for some 2D paste inspection machines. Here the velocity of the camera can be constant, while in most cases it contains line-CCD sensor. The other type of motion system is the linear drive which is more accurate and faster and therefore in more frequent use. The directional route of the moving part highly depends on a second factor, that of speed and the properties of image acquisition system. Here also, three main parts can be singled out: • optics / lenses • camera / sensor type • illumination module / lighting source The system has to get the necessary amount of information and resolution from even the smallest components. In the SMT field this means zooming down to a 10μm pixel resolution. To ensure the constant magnification at all points of the entire Field of View (FOV) the use of telecentric optics is essential. This criterion enables the system to make the required size- measurements. On an image seen through traditional lenses, the apparent shape of compo‐ nents changes with the distance from the centre of the FOV, therefore sometimes making shape recognition a hard task. But it is not just the permanency of magnification that is important, so too is the need to select the correct level. On one hand, the larger detection area of the image sensor can help solve this task, but it also increases the computational resources needed. On the other hand, higher magnification levels give a better resolution but at the expense of reducing the field of view. The best scenario is if the system is capable of optional magnification. Generally, a relative large FOV, between 10-25 cm2, could be used and only in certain cases should dedicated Field of Interests (FOI) should be zoomed out. In most AOI applications, the LED based lighting is used for illumination purposes. But independent of the type of illumination source used, the amount of illumination should be only as much as is required. The optimum depends on the application. For example, a 2D paste or a through-hole-technology (THT) components solder-joint inspection system needs only just a small amount of illumination. 8. Detailed analysis of AOI systems As can be seen in the previous section, AOI machines handle several tasks. Much literature is dedicated to the intelligence of these systems, but from a technical point of view we can also examine other aspects. A large number of these AOIs work on high-mix-high-volume SMT lines where the most important key factors are the inspection duration and quality. The attributes of this system relate to the following sections: 1. actuating parts (drives and axes) 2. image acquisition system (sensors, optics, illumination) 3. software processing part They work in close relationship to each other, so the speed of each has to be in sync. There are three well-defined mechanical constructions for an AOI system: They work in close relationship to each other, so the speed of each has to be in sync. There are three well-defined mechanical constructions for an AOI system: • without special moving parts / drives inside • with PWB positioning table • with camera-module actuating unit The simplest case is when the working-process of the system does not include special posi‐ tioning steps. The PWB is positioned/placed in “one step” into the field of camera system, an image is acquired and the PWB is then taken out for the next process. This could be of great benefit because the machine does not need to synchronize any movements during the image acquisition process. The speed affecting factor can thus be ignored. This is used typically in Automatic Final Inspection (AFI) systems. This does not mean that the system has to only contain one camera. For more complex applications the number of cameras can be increased. More cameras mean more complex image transformation and manipulation tasks so it follows that these systems are only capable of use when looking at pre-defined areas. More cameras mean more complex image transformation and manipulation tasks so it follows that these systems are only capable of use when looking at pre-defined areas. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 417 In case of larger inspection areas, the systems are mounted with special drives which can move the camera system or the inspected part. The movements of these drives have to be synchron‐ ized with the process of illumination and image acquisition. When the system contains small number of cameras and the illumination devices are also built-in, then the module itself should be the moving section. 8. Detailed analysis of AOI systems As the number of failure types / inspection tasks increase so too the number of illumination modes also increase. The programmable illumination module is a good tool to develop lighting requirements for dedicated purposes, but it also carries the risk of inhomogeneous and reduced FOV. Materials Science - Advanced Topics 8 Ring illumination around 4 cameras Grey-level distribution of 1 section Ring illumination around 1 camera Grey-level distribution of 1 camera 23 11 4 -1 -3 -3 0 21 10 3 -2 -5 -5 -2 21 10 3 -1 -2 -2 0 26 15 8 4 2 3 6 34 24 16 13 11 12 15 45 31 21 19 21 34 47 38 22 9 6 9 22 40 36 18 4 2 5 19 37 32 16 2 -3 3 16 33 44 28 16 14 18 44 30 Figure 24. Problem of inhomogeneous grey-level by ring-illumination types Ring illumination around 4 cameras Ring illumination around 1 camera Grey-level distribution of 1 section Grey-level distribution of 1 camera 23 11 4 -1 -3 -3 0 21 10 3 -2 -5 -5 -2 21 10 3 -1 -2 -2 0 26 15 8 4 2 3 6 34 24 16 13 11 12 15 45 31 21 19 21 34 47 38 22 9 6 9 22 40 36 18 4 2 5 19 37 32 16 2 -3 3 16 33 44 28 16 14 18 44 30 Ring illumination around 4 cameras Ring illumination around 1 camera Ring illumination around 1 camera Grey-level distribution of 1 camera Figure 24. Problem of inhomogeneous grey-level by ring-illumination types Fig. 7 illustrates two types of camera-illumination systems. The first system contains 4 cameras, the second only 1 camera. Both have LED-ring illumination modules. The grey-level distribu‐ tion maps shown above have been measured with the same type of illumination and grey- reference flat. The green areas indicate the valuable field of the camera. This example clearly points out the importance of the homogeneity. Of course this phenomenon is also present when the illumination system is multi-coloured. Most optical inspection / control appliance decisions are based on image-processing methods that have been set experientially. The stress is on the word “experientially”. Most of the AOI machines make some kind of template matching. These sample-templates can be colour or greyscale, stand from parts/windows or form a complete pattern. 8. Detailed analysis of AOI systems The machine can be ‘self- learning or directed by means of an “external trainer”. Due to the fact that the overall reliability of these machines is not 100%, the defined limits between good and bad classified patterns are not strict. In some cases it could be that just two pixels differ between the data provided. If the phenomenon which the system needs to detect is not so unambiguous, then it should search for another method to make the gap wider between the 2 classes. 9. Software questions One of the most wide-spread criticisms against the principles and methods of automated optical inspection systems stems from a very interesting paradox. As we have mentioned earlier, the introduction of AOI devices in the manufacturing lines was a result of the growth in manufacturing process complexity. These inspection and control devices have to fulfill certain reliability criteria which need to be validated. But unfortunately, these validation Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 419 processes can only be used to a limited degree because of the high-complex manufacturing process and the equally complex and highly varied appearance of devices under test (DUTs). This contradiction invokes the conclusion that accuracy and reliability of AOI system depends very much on the competence and working quality of the engineers and operators, the correct management of the setting up and controlling the inspection devices. In reality, this sets out several very serious challenges to experts. The quality inspection algorithms have many parameters – in some cases several hundred – (image processing, region of interest, threshold parameters etc.). Their setup requires experience, intuition and inspiration from the process engineers themselves. In addition, during parameter tuning, the engineers need to solve the following contradiction, where the difference between images showing correct and faulty components is often only a few pixels which need to be detected by the AOI devices (Fig. 8). In the case of incorrect parameter settings these small signals can disappear and the system classify a bad component as good (“slip-through”). Certainly this false classification is totally intolerable in quality inspection processes; therefore it is necessary to aim for the complete elimination of this possibility by fine tuning the algorithm’s parameters. Unfortunately because of this, engineers can easily set the algorithm to be too strict, meaning also that some correct components will be dropped out during the inspection process. Although these “false calls” (also known as pseudo failure) do not cause catastrophic consequences nevertheless they are the source of a very serious problem. Namely, in this instance, the human operators performing the re- inspection of components considered “faulty” can easily get used to the repeated mistakes of the AOI system. Therefore they can eventually take the inspection device’s decisions out of consideration even where there is a cases of real errors. 9. Software questions Therefore it can happen that whilst a parameter optimization process reduces the number of bad classifications in the first part of the factory, it increases them on other manu‐ facturing lines. This paradox is one of the reasons why the AOI macro optimization process is a very long and “Sisyphean” task of AOI process engineers. Figure 26. Differences between the appearances of similar components (capacitor C0805) A very interesting and important question is the optimization of classification thresholds. One of the most important requirements of an inspection system is high-level robustness, but this condition can hardly be guaranteed if the classification decision (namely whether a component In addition, AOI engineers need to cope with several other difficulties a major one of which is that the production process changes continuously e.g. the settings of devices on the manufac‐ turing line have to be modified, and this needs to be followed also by modifications to the AOI devices. Therefore the need to monitor the inspection algorithms and adapt to different parameters is a serious challenge to the process engineers. Furthermore, it is necessary to satisfy some practical requirements when selecting and adjusting the inspection algorithms. Usually, electronic factories manufacture more products in parallel in which several similar or identical components can be located. If all the compo‐ nents were to be inspected with a separate AOI algorithm, the code management, version tracking and fixing etc. would be impossible. Therefore, engineers often use only one inspec‐ tion method for similar mountings to achieve simpler AOI algorithm version management. Unfortunately, this strategy cannot always be used successfully because of the very heteroge‐ neous appearance of the same components. Fig.9. shows an image sequence of the C0805 capacitor which illustrates the enormous differences between images taken of similar compo‐ nents. In this varied environment it is very hard to develop an inspection method which results in highly reliable classification of each type of image for the same component. In addition, a parameter setting process that reduces the number of bad classifications in case of one component influences not only the selected manufacturing line but has an effect on the whole factory. Therefore it can happen that whilst a parameter optimization process reduces the number of bad classifications in the first part of the factory, it increases them on other manu‐ facturing lines. 9. Software questions This implies that the reliability of the inspection device itself would be in doubt; the fact of which would result in one of the biggest catastrophic effects on AOI systems. In addition, it seems insignificant but it is important to note that many bad classifications slow the manufacturing process, decrease productivity and increase the product overall production costs. To avoid false calls, process-engineers need to reduce the strictness of the inspection parameters which – as we have mentioned earlier – is inconsistent with principle used by the parameter settings preventing the slip-through. Figure 25. An example for the tiny differences between the images containing correct and faulty components Figure 25. An example for the tiny differences between the images containing correct and faulty componen Materials Science - Advanced Topics 420 In addition, AOI engineers need to cope with several other difficulties a major one of which is that the production process changes continuously e.g. the settings of devices on the manufac‐ turing line have to be modified, and this needs to be followed also by modifications to the AOI devices. Therefore the need to monitor the inspection algorithms and adapt to different parameters is a serious challenge to the process engineers. Furthermore, it is necessary to satisfy some practical requirements when selecting and adjusting the inspection algorithms. Usually, electronic factories manufacture more products in parallel in which several similar or identical components can be located. If all the compo‐ nents were to be inspected with a separate AOI algorithm, the code management, version tracking and fixing etc. would be impossible. Therefore, engineers often use only one inspec‐ tion method for similar mountings to achieve simpler AOI algorithm version management. Unfortunately, this strategy cannot always be used successfully because of the very heteroge‐ neous appearance of the same components. Fig.9. shows an image sequence of the C0805 capacitor which illustrates the enormous differences between images taken of similar compo‐ nents. In this varied environment it is very hard to develop an inspection method which results in highly reliable classification of each type of image for the same component. In addition, a parameter setting process that reduces the number of bad classifications in case of one component influences not only the selected manufacturing line but has an effect on the whole factory. 9. Software questions This paradox is one of the reasons why the AOI macro optimization process is a very long and “Sisyphean” task of AOI process engineers. Figure 26. Differences between the appearances of similar components (capacitor C0805) Figure 26. Differences between the appearances of similar components (capacitor C0805) A very interesting and important question is the optimization of classification thresholds. One of the most important requirements of an inspection system is high-level robustness, but this condition can hardly be guaranteed if the classification decision (namely whether a component gets “faulty” or “good” label) is dependent on only one pixel. Therefore the quality results close to the decision threshold need to be classified in a separate group (“limit error”) and it is necessary to apply a different strategy to them. It follows that AOI experts – apart from the fact that they need to solve the optimization paradox mentioned earlier – have to strive to find such an algorithm parameter setting where during the classification, the number of compo‐ nents classified near the decision threshold are as few as possible. Efficiency of AOI appliances Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 421 can be significantly improved with the help of macro optimization. In the first task, the pseudo rate was reduced while slip-throughs remains zero (Table VIII, Fig. 10) Real failures [pieces] Pseudo failures [pieces] 4 4 672 Real failures [pieces] Pseudo failures [pieces] 2 50 After optimization (30 days testing period) Inspected components (solder joints) [pieces] Detected failures [pieces] Pseudo rate [ppm] 223 006 (446 012) 52 224 Before optimization (30 days testing period) 4 676 Detected failures [pieces] Pseudo rate [ppm] 13 459 Inspected components (solder joints) [pieces] 347 130 (694 260) Table 12. Results of macro optimization Figure 27. Pseudo reduction Table 12. Results of macro optimization Figure 27. Pseudo reduction Materials Science - Advanced Topics 422 Secondly parallel pseudo and slip-through reduction were carried out (Table IX, Fig. 11, Fig. 12). 9. Software questions Real failures (Quasi-tombstone ) [pieces] Pseudo failures [pieces] 364 (0 ) 67 841 Real failures (Quasi-tombstone ) [pieces] Pseudo failures [pieces] 627 (62 ) 58 027 After optimization (30 days testing period) Inspected components (solder joints) [pieces] Detected failures [pieces] Pseudo rate [ppm] 4 655 392 (9 310 784) 58 654 12 560 Before optimization (30 days testing period) 68 205 Detected failures [pieces] Pseudo rate [ppm] Inspected components (solder joints) [pieces] 2 423 334 (4 846 668) 27 995 Table 13. Results of macro optimization Table 13. Results of macro optimization Another very serious question is about the parameter optimization process, namely how can the AOI engineers validate the new parameter values determined by the optimization process? Certainly a correction of a bad classification cannot be validated only by examination of the specified image, but it is necessary to check several other instances. Therefore, to execute a reliable validation process, the engineers have to collect a large image database (“image base”) covering all cases as they occur in the best possible way. Unfortunately, creating a good and usable image base is a long and sometimes impossible task because of several – often contra‐ dicting – criteria. A manual image collection by the engineers is very time-consuming and in case of automatic systems (like AOIs) there is only a limited possibility because of the high number and varied type of data. Automatic methods are faster but during the collection, some falsely classified images can be put in the image base which makes the parameter optimization impossible. For example, if an image containing a faulty component is placed into the “good” part of the image base, the optimization process will try to adjust to the parameters that the AOI algorithm has classified the image as “good”. As a result, the optimized macro cannot recognize this specified error which can indicate slippages causing the greatest type of inspection catastrophe. The number of stored images is also a very important factor. If the image base contains too many images, the resources (processor, hard drive, network etc.) become overloaded and the optimization process can only be executed slowly. On the other hand in case of a small image base the algorithm validation is neither reliable nor accurate enough. Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 423 Figure 28. Pseudo and slip-through reduction Figure 28. 9. Software questions Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 425 Certainly the automatic optimization methods also need to collect the relevant images autonomously to create the reference image base. This work sets serious challenges for optimization processes because of the problems and difficulties mentioned earlier. Certainly the automatic optimization methods also need to collect the relevant images autonomously to create the reference image base. This work sets serious challenges for optimization processes because of the problems and difficulties mentioned earlier. As a summary, we can establish that AOI systems offer a powerful solution for a complex problem by means of simple principles, but the analysis of details can reveal several problems, difficulties and contradictions. Finding a solution for them is an essential condition for the automated optical inspection systems in the future. 9. Software questions Pseudo and slip-through reduction Materials Science - Advanced Topics 424 Figure 29. Quasi-tombstone Figure 29. Quasi-tombstone Figure 29. Quasi-tombstone If we suppose that the optimal size of image base is determined (and which cannot be exceeded) and relevant images are collected resulting in a reasonably good image base. At this point another question arises: how can the engineers update the database with new images? It is very hard to determine which images from the new image-set need to be stored and which images need to be deleted from the current image base. There are several criteria – such as the date created, the number of similar images etc. – which can be used as the basis of the updating decision but a precise numerical factor which shows the usefulness of pictures in the database is much more difficult to determine. The aspects and concepts mentioned in this section have shown that the usage and perfect operation of automated optical inspection system requires human control and supervision. Although the devices’ algorithms are able to execute fast, accurate, efficient, reliable, “assid‐ uous” and continuous inspection (they appear to be much more suitable than human operators as a consequence!) without being fed sufficient intelligence they cannot adapt immediately and independently to changes in manufacturing. Therefore the quality inspection process can hinder the increased spreading of autonomous electronic manufacture. Several researches and developments are focusing on the problem to redeem the status of the human operators’ work and to provide help for AOI engineers. Very interesting research directions are in automatic algorithm parameter optimization methods. The AOI devices on the manufacturing line monitor the quality of the algorithms (number of false calls and slip- through, if possible) and on occasions they adjust the parameters using the image base to create a better, higher quality algorithm. The engineers only need to take care of special cases like changing the lighting or creating new inspection methods. Although the automatic parameter optimization methods do not have to satisfy high real-time criteria, it is important to determine the optimized parameter values in a relatively short time. It is easy to verify that even in the case of having some dozen parameters; the analysis of all parameter-combinations takes a very long time (years) therefore heuristic search methods have to be used to solve the optimization problem. 10. 3D Inspection But the analyze-development is just one route for improving the AOI process. The other is the “extended” optical inspection system with measuring capability. The pioneers of this property are 3D SPI machines. In the last few years, a wide variety of these machines have been developed. The inspection in this application - checking the SMT printing process - means the 3 dimensional measurements of solder paste bumps. These bumps are shaped like cylinders or cubes so that the geometries and surfaces are relative simple. This fact makes the 3D optical techniques a viable option. Several measurement techniques are used for this process, some of these are shown in Fig. 13. Laser triangulation Fringe-pattern analysis Stereovision Shape-from-shading Phase measuring Moiré topography Figure 30. optical measurement techniques Laser triangulation Stereovision Phase measuring Figure 30. optical measurement techniques Laser triangulation Fringe-pattern analysis Shape-from-shading Moiré topography Fringe-pattern analysis Laser triangulation Fringe-pattern analysis Stereovision Shape-from-shading Shape-from-shading Shape-from-shading Stereovision Moiré topography Phase measuring Moiré topography Phase measuring Figure 30. optical measurement techniques Materials Science - Advanced Topics 426 The same is true for inspecting component presence, but for solder-joint detection these technologies are in their infancy at present. The shape of different components’ solder-joints is complex and the specular surface also makes the task even more difficult. There have been a number of research efforts, optical 3D shape measurement technologies, based on several technologies as shown in Fig.2. Some of these researches can be found in the following studies [181-198]. Also some companies are in the development phase such as Koh-Young Technology [180]. So the evaluation of these geometries is as yet more difficult, but with the development of optical metrology there will be more AOI machines with measuring capability. 11. Further developments, the future of AOI AOI systems are following the worldwide trends i.e. multi-task integration, adaptivity, speed, etc. There are already appliances that integrate optical inspection with repair functions: Ersa’s AOI+R solution or optical and X-ray inspection together. Some suppliers have AOI+AXI or Viscom’s AOXI (simultaneous inspection). Another possible area of development is the inspection speed. Faster image capturing (with larger FOV, faster camera positioning etc), parallel inspection of two PWBs are some possible ways for this to be done. The other important area is adaptivity. Mainly adaptive illumination is the future of AOI systems. It would help to drastically reduce pseudo-failures rates and eliminate slip-through failures. A third area is image processing. 3D inspection, neural networks, fuzzy systems, intelligent algorithms which will help to increase the efficiency and reliability of these systems. 12. Conclusion Inspection systems are widely used to determine the quality of electronics modules after assembly sequences. Nowadays this is usually the automatic, non-contact and non-destructive process, such as automatic optical inspection (AOI), supplemented with automatic X-ray inspection (AXI) if necessary. These appliances inspect the ready or the incomplete printed wiring boards to determine the quality of it's given property in any technological sequence, such as paste printing, component placement or soldering. The rapid development of elec‐ tronics module assembly manufacturing requiring parallel development of test procedures. The automatic optical inspection is potential multi-disciplinary research area, because from image acquiring, (illumination, the detection of the reflected light etc.) through image proc‐ essing, to the evaluation each area can be optimized to reach to goal, that the qualification of the inspected object in the field of interest (FOI) by the used appliance, matches the specifica‐ tions as stated. Most manufacturers agree that, from a strategic point of view, the optical inspection after soldering should not be ignored. As a consequence, this is the most important part of an AOI inspection. The quality of solder joints is determined from geometric and optical properties of the solder meniscus. These parameters determine the reflection properties of the Automatic Optical Inspection of Soldering http://dx.doi.org/10.5772/51699 427 meniscus. The meniscus forms from the liquid alloy during the soldering process. After cooling, the meniscus becomes solid and reflects illumination which means that we can classify them. From these reflection patterns and with the help of image processing algorithms we are able to determine the quality of the solder joints. As described above, the correct source of illumination is essential. There are several different kinds of approach: white or RGB; directed or diffuse; ring or hemisphere. This survey gives state of the art review of current automated optical inspection systems in the electronic device manufacturing industry. The aim of the chapter is to give an overview about the development phases, operating mechanisms, advantages and disadvantages of AOI appliances, their technical parameters, field of usage, capabilities and possible trends for further developments. Author details Mihály Janóczki1, Ákos Becker2, László Jakab3, Richárd Gróf4 and Tibor Takács5 Address all correspondence to: mihaly.janoczki@eu.agc.com 1 AGC Glass Hungary Ltd., H-2851 Környe, Hungary 2 DENSION Audio Systems Ltd., Budapest, Hungary Mihály Janóczki1*, Ákos Becker2, László Jakab3, Richárd Gróf4 and Tibor Takács5 3 Department of Electronics Technology, Budapest University of Technology and Econom‐ ics, Budapest, Hungary 3 Department of Electronics Technology, Budapest University of Technology and Econom‐ ics, Budapest, Hungary 4 Epcos AG, Heidenheim, Deutschland 5 Department of Control Engineering and Information Technology, Budapest University of Technology and Economics, Budapest, Hungary [2] Miran Burmen, Franjo Pernuš and Boštjan Likar, LED Light Sources: A Survey of Quality-Affecting Factors and Methods for Their Assessment, Measurement and Sci‐ ence Technology Vol. 19, No.12, 2008, 122002 (15pp) References [1] Matthew T. 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https://openalex.org/W2945741530	https://www.duo.uio.no/bitstream/10852/77352/1/Validation%2bof%2bSHACL.pdf	English	null	Validation of SHACL Constraints over KGs with OWL 2 QL Ontologies via Rewriting	Lecture notes in computer science	2,019	cc-by	9,402	Validation of SHACL Constraints over KGs with OWL 2 QL Ontologies via Rewriting Ognjen Savković1(B), Evgeny Kharlamov2,3, and Steﬀen Lamparter4 1 Free University of Bozen-Bolzano, Bolzano, Italy ognjen.savkovic@unibz.it 2 University of Oslo, Oslo, Norway 3 Bosch Centre for Artiﬁcial Intelligence, Robert Bosch GmbH, Renningen, Germany 4 Siemens CT, Siemens AG, Munich, Germany 1 Free University of Bozen-Bolzano, Bolzano, Italy ognjen.savkovic@unibz.it 2 University of Oslo, Oslo, Norway 3 Bosch Centre for Artiﬁcial Intelligence, Robert Bosch GmbH, Renningen, Germany 4 Siemens CT, Siemens AG, Munich, Germany Abstract. Constraints have traditionally been used to ensure data qual- ity. Recently, several constraint languages such as SHACL, as well as mechanisms for constraint validation, have been proposed for Knowl- edge Graphs (KGs). KGs are often enhanced with ontologies that deﬁne relevant background knowledge in a formal language such as OWL 2 QL. However, existing systems for constraint validation either ignore these ontologies, or compile ontologies and constraints into rules that should be executed by some rule engine. In the latter case, one has to rely on diﬀerent systems when validating constrains over KGs and over ontology-enhanced KGs. In this work, we address this problem by deﬁn- ing rewriting techniques that allow to compile an OWL 2 QL ontology and a set of SHACL constraints into another set of SHACL constraints. We show that in the general case the rewriting may not exists, but it always exists for the positive fragment of SHACL. Our rewriting tech- niques allow to validate constraints over KGs with and without ontologies using the same SHACL validation engines. 1 https://www.w3.org/2001/sw/wiki/ShEx. 2 https://www.stardog.com/. 3 https://www.topquadrant.com/technology/shacl/. c ⃝Springer Nature Switzerland AG 2019 P. Hitzler et al. (Eds.): ESWC 2019, LNCS 11503, pp. 314–329, 2019. https://doi.org/10.1007/978-3-030-21348-0_21 SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting KGs are often enhanced with ontologies, expressed in, e.g., the OWL 2 ontol- ogy language [3]. Ontologies capture the relevant background knowledge with axioms over the terms from the KG’s vocabulary e.g., by assigning attributes to classes, by deﬁning relationships between classes, composed classes, and class hierarchies. We refer to ontology enhanced KGs asKnowledge Bases (KBs). ( ) Ontologies signiﬁcantly impact constraint validation over KGs. Indeed, con- straints over KGs have Closed-World semantics, or Assumption (CWA) in the sense that their validation over a KG boils down to checking whether sub struc- tures of the KG comply with the patterns encoded in the constraints [8,12,15]. On the other hand, KBs have open-world semantics (OWA) in the sense that ontologies allow to derive information from a KG that is not explicitly there. g y As a result, constraint validation over KGs in the presence of ontologies requires to bridge the CWA of constraints and OWA of ontologies [20,21,35,42]. A promising semantics that oﬀers the bridge was proposed in [35]: given a set of constraints C, ontology O, and KG G, validating the KB ⟨O, G⟩against C requires to validate all ﬁrst-order logic models of O and G that are set-inclusion minimal against C. This can be done via a rewriting mechanism: in order to validate ⟨O, G⟩ against C, one can compile O and C into a (possibly disjunctive) logic program and then evaluate the program over G [20,35]. A disadvantage of this approach is that constraint validation in the presence of ontologies requires a diﬀerent evaluation engine than in their absence: it requires an engine for disjunctive logic programs, rather than an engine for validating graph constraints. However, from practical point of view it is desirable to have a mechanism that allows to evaluate constraints over KBs using the same engine as over KGs. In this work we address this issue. We ﬁrst formalise the problem of con- straints rewriting over ontologies: we require that the result of rewriting is again a set of constraints C′ in the same formalism as the original C. We then study the existence of such a rewriting function for the constraint language SHACL and the ontology language OWL 2 QL which is commonly used proﬁle of OWL 2. SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting Our results show that rewriting may not exist in the general case unless co-NP = NP, since constraint validation in presence of ontologies is co-NP-complete, while in absence it is NP-complete. We next consider the restriction of SHACL to positive constraints, that we call SHACL+, and show that in this case the rewriting always exists and provide an algorithm for such rewriting. 1 Introduction Constraints have traditionally been used to ensure quality of data in relational [5] and semi-structured DBs [4]. Recently constraints have attracted considerable attention in the context of graph data [16,17], and in particular for Knowledge Graphs (KGs) (e.g, [35,36,42]), i.e., large collections of interconnected entities that are annotated with data values and types [7]. KGs have become powerful assets for enhancing search and data integration and they are now widely used in both academia and industry [1,2,6,19,22–25,28,40,41]. Prominent examples of constraint languages for KGs include SHACL [31], ShEx1; and of constraint validation systems Stardog2 and TopBraid3. 1 https://www.w3.org/2001/sw/wiki/ShEx. 2 https://www.stardog.com/. 3 https://www.topquadrant.com/technology/shacl/. c ⃝Springer Nature Switzerland AG 2019 P. Hitzler et al. (Eds.): ESWC 2019, LNCS 11503, pp. 314–329, 2019. https://doi.org/10.1007/978-3-030-21348-0_21 1 https://www.w3.org/2001/sw/wiki/ShEx. 2 https://www.stardog.com/. 3 https://www.topquadrant.com/technology/shacl/. c ⃝Springer Nature Switzerland AG 2019 P. Hitzler et al. (Eds.): ESWC 2019, LNCS 11503, pp. 314–329, 2019. https://doi.org/10.1007/978-3-030-21348-0_21 315 2.1 Knowledge Graph A Knowledge Graph (KG) G is a possibly inﬁnite directed labeled graph that consists of triples of the form (s, p, o) over Σ, where s is a constant, p – property, and o – constant or class (in this case p is the special predicate “a”). Example 1. Consider the following fragment of the Siemens KG GSIEM from [23], which describes Siemens industrial assets including two turbines with the iden- tiﬁers :t177 and :t852 and one power plant (PPlant) with the identiﬁer :p063, as well as information about equipment (turbine) categories (hasTuCat, hasCat), their deployment sites (deplAt), and enumeration of turbines at plants (hasTurb): {(:p063, a, :PPlant), (:p063, :hasTurb, :t852), (:t852, a, :Turbine), (:t852, :deplAt, :p063), (:t852, :hasCat, :SGT-800), (:t177, :deplAt, :p063), (:t177, :hasTuCat, :SGT-800)}. {(:p063, a, :PPlant), (:p063, :hasTurb, :t852), (:t852, a, :Turbine), (:t852, :deplAt, :p063), (:t852, :hasCat, :SGT-800), (:t177, :deplAt, :p063), (:t177, :hasTuCat, :SGT-800)}. (:t177, :deplAt, :p063), (:t177, :hasTuCat, :SGT-800)}. ⊓⊔ 2 Preliminaries and Running Example In this section we recall required deﬁnitions. We assume a signature Σ of three inﬁnite countable sets of constants, that correspond to entities, classes of unary predicates, that correspond to types, and properties or binary predicates, that correspond to object properties or a special predicate “a” that labels entities with classes. Note that do not consider datatypes and data properties, and leave them for the future study. We assume an inﬁnite countable domain Δ of entities. 316 O. Savković et al. 2.2 SHACL Syntax We next brieﬂy recall relevant notions of SHACL using a compact syntax of [12] which is equivalent to SHACL’s “Core Constraint Components” [12]. SHACL stands for Shapes Constraint Language. Each SHACL constraint in a set of con- straints C, usually referred to as shape, is deﬁned as a triple: ⟨s, τs, φs⟩, where s is the name, – s is the name, – s is the name, – τs is the target deﬁnition, a SPARQL query with one output variable whose purpose is to retrieve target entities of s from G, i.e., entities (nodes) occurring in G for which the following constraint of the shape should be veriﬁed, – τs is the target deﬁnition, a SPARQL query with one output variable whose purpose is to retrieve target entities of s from G, i.e., entities (nodes) occurring in G for which the following constraint of the shape should be veriﬁed, g p , – and φs is the constraint, an expression deﬁned according to the following grammar: g p , – and φs is the constraint, an expression deﬁned according to the following grammar: φ :: = ⊤\| s′ \| c \| φ1 ∧φ2 \| φ1 ∨φ2 \| ¬φ \| ≥n R.φ \| ≤n R.φ \| EQ(r1, r2), (1) φ :: = ⊤\| s′ \| c \| φ1 ∧φ2 \| φ1 ∨φ2 \| ¬φ \| ≥n R.φ \| ≤n R.φ \| EQ(r1, r2), (1) where ⊤stands for the Boolean truth values, s′ is a shape name occurring in C, c is a constant, R is a property, and n ∈N; moreover, ∧denotes the conjunction, ¬ – negation, “≥n R.φ” – “must have at least n-successors in G verifying φ”, r1 and r2 are SPARQL property paths and “EQ(r1, r2)” means that “r1 and r2 successors of a node must coincide”. where ⊤stands for the Boolean truth values, s′ is a shape name occurring in C, c is a constant, R is a property, and n ∈N; moreover, ∧denotes the conjunction, ¬ – negation, “≥n R.φ” – “must have at least n-successors in G verifying φ”, r1 and r2 are SPARQL property paths and “EQ(r1, r2)” means that “r1 and r2 successors of a node must coincide”. 4 https://www.w3.org/TR/shacl/. 2.3 SHACL Semantics Given a shape s, a KG G, and an entity e occurring G, we say that e veriﬁes s in G if the constraint φs applied to e is valid in G. Finally, G is valid against C if for each s ∈C, each target entity retrieved by τs from G veriﬁes s in G. Since a constraint φs may refer to a shape s′, the deﬁnition of validity for KGs is non-trivial. Indeed, the SHACL speciﬁcation leaves the diﬃcult case of recursion up to the concrete implementation4 and a formal semantics via so-called shape assignments has only recently been proposed [12]. Intuitively, G is valid against C if one can label its entities with shape names, while respecting targets and constraints. A shape assignment σ is a function mapping each entity of G to a set of shape names in C. We call an assignment target-compliant if it assigns (at least) each shape to each of its targets, constraint-compliant if it complies with the constraints, and valid if it complies with both targets and constraints. Then, G is valid against C if there exists a valid assignment for G and C. Example 3. Observe that GSIEM is not valid against CSIEM. Shape s1 has targets :t852 and :t177, since both are deployed. :t852 satisﬁes the constraint for s1, since it has a category, but :t177 violates it. Shape s2 has the target :t177 only, which violates it, since it is not declared to be a turbine. Shape s3 has no target in GSIEM. The case of shape s4 is more involved. It has only :t852 as the target, and one may assign s4 to :t852 and s3 to :p063, in order to satisfy the recursive constraint. But since :t177 violates s1 and s2, there is no “global” valid shape assignment for G and S, i.e. which would satisfy all targets and constraints simultaneously. ⊓⊔ ⊓⊔ Example 2. Consider CSIEM = {⟨si, τsi, φsi⟩\| i = 1, 4}, where: φs1 = (≥1 :hasCat.⊤), φs2 = (≥1 a.:Turbine), φs3 = (≥1 :hasTurb.s4), φs4 = (≥1 :deplAt.s3). τs1 = ∃y(:deplAt(x, y)), φs1 = (≥1 :hasCat.⊤), τs2 = ∃y(:hasTuCat(x, y)), φs2 = (≥1 a.:Turbine), τs3 = :PPlant(?x), φs3 = (≥1 :hasTurb.s4), τs4 = :Turbine(?x), φs4 = (≥1 :deplAt.s3). τs1 = ∃y(:deplAt(x, y)), Here s1 essentially says that any deployed artifact should have a category, and s2 says that only turbines can have a turbine category. The last two shapes s3 and s4 are mutually recursive, and they respectively say that each power plant should have at least one turbine and each turbine should be deployed in at least one location. ⊓⊔ 2.2 SHACL Syntax where ⊤stands for the Boolean truth values, s′ is a shape name occurring in C, c is a constant, R is a property, and n ∈N; moreover, ∧denotes the conjunction, ¬ – negation, “≥n R.φ” – “must have at least n-successors in G verifying φ”, r1 and r2 are SPARQL property paths and “EQ(r1, r2)” means that “r1 and r2 successors of a node must coincide”. With a slight abuse of notation we identify the shape with its name. We note that the syntax for constraints allows for shapes to reference each other. A set of constraints is recursive if it contains a shape that reference itself, either directly or via a reference cycle. SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting 317 Example 2. Consider CSIEM = {⟨si, τsi, φsi⟩\| i = 1, 4}, where: Example 2. Consider CSIEM = {⟨si, τsi, φsi⟩\| i = 1, 4}, where: 2.5 Constraint Validation over KGs Enhanced with Ontologies Consider the semantics of [35], that naturally extends constraint validation from KGs to ontology-enhanced KGs and has been adopted in, e.g., [20]. Given a KG G, ontology O, and a set of constraints C, the idea of this semantics is to validate C over all set inclusion minimal models of G and O. Formally, G enhanced with O is valid against C if for each minimal model M of G with O, the KG skol(M) is valid against C, where skol(M) is the Skolemization of models. Example 5. Observe that ⟨O, G, ⟩is valid against CSIEM. Indeed, shape s1 is still satisﬁed by :t852, since no new information can be entailed about :t852 from ⟨OSIEM, GSIEM⟩. Moreover, s1 is now not violated by :t177: ⟨OSIEM, GSIEM⟩ entails that :t177 has a category. Similarly, s2 is now not violated by :t177: one can can infer that it is a turbine. The shape s4 now has an additional target (:t177), and it is veriﬁed by both its targets, thanks to the following assignment: {s1 →{:t852, :t177}, s2 →{:t177}, s3 →{:p063}, s4 →{:t852, :t177}}. ⊓⊔ 3 The Problem of Constraint Rewriting We now formalise and discuss the problem of constraint rewriting over ontologies. 2.4 OWL 2 QL We now recall the syntax and semantics of OWL 2 QL relying on the the Descrip- tion Logics DL-LiteR [9] that is behind this proﬁle. (Complex) classes and prop- erties in OWL 2 QL are recursively deﬁned as follows: B:: = A \| ∃R, C:: = B \| ¬B, R:: = P \| P −, and E:: = R \| ¬R, B:: = A \| ∃R, C:: = B \| ¬B, R:: = P \| P −, and E:: = R \| ¬R, O. Savković et al. 318 where A is a class from Σ, P a property from Σ, and P −the inverse of P. Expression A we call also an atomic class or concepts and B a basic class or concepts. A DL-LiteR ontology is a ﬁnite set of axioms of the form B ⊑C or R ⊑E. A Knowledge Base (KB) is a pair ⟨O, G⟩of an ontology and a KG. The formal semantics of DL-LiteR is given in terms of ﬁrst-order logic interpretations I = (Δ, ·I) over Δ in the standard way. where A is a class from Σ, P a property from Σ, and P −the inverse of P. Expression A we call also an atomic class or concepts and B a basic class or concepts. A DL-LiteR ontology is a ﬁnite set of axioms of the form B ⊑C or R ⊑E. A Knowledge Base (KB) is a pair ⟨O, G⟩of an ontology and a KG. The formal semantics of DL-LiteR is given in terms of ﬁrst-order logic interpretations I = (Δ, ·I) over Δ in the standard way. Example 4. Consider the following OWL 2 QL ontology OSIEM: Example 4. Consider the following OWL 2 QL ontology OSIEM: {:hasTuCat ⊑:hasCat, ∃:hasTuCat.⊤⊑:Turbine}, that says that if x has y as a turbine category, then x has y as a category, and also x can be inferred to be a turbine. ⊓⊔ A useful property of DL-LiteR exploited in Sect. 4, is the existence, for any satis- ﬁable KB ⟨O, G⟩, of a so-called canonical model, which can be homomorphically mapped to any model of ⟨O, G⟩. ⟨O, G⟩is valid against C iﬀG is valid against C′. 5 Recall that for query rewriting the input is a query q and ontology O and the output is another query q′ such that for any database D so-called certain answers of q over ⟨O, D⟩coincide with the answers of q′ over D alone [9]. 3.1 SHACL-Rewriting In order to deﬁne SHACL rewriting we adapt the notion of rewriting (or refor- mulation) of queries over ontologies from [9,18]. In order to deﬁne SHACL rewriting we adapt the notion of rewriting (or refor- mulation) of queries over ontologies from [9,18]. Deﬁnition 1. Let C be a set of constraints and O an ontology. A set of con- straints C′ is a constraint-rewriting of C over O if for any KG G it holds that: Deﬁnition 1. Let C be a set of constraints and O an ontology. A set of con- straints C′ is a constraint-rewriting of C over O if for any KG G it holds that: ⟨O, G⟩is valid against C iﬀG is valid against C′. ⟨O, G⟩is valid against C iﬀG is valid against C′. SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting 319 We now illustrate this notion on the following example. Example 6. Consider a set of SHACL constraints and an OWL 2 QL ontology: C = {⟨s, τs, φs⟩}, where τs = :MechDevice(x) and φs = (≥1 :hasCat.⊤), O = {:Turbine ⊑:MechDevice, ∃:hasTuCat ⊑∃:hasCat}. One can show that a rewiring of C over O is S′ = {⟨s, τ ′ s, φ′ s⟩}, where One can show that a rewiring of C over O is S′ = {⟨s, τ ′ s, φ′ s⟩}, where τ ′ s = :MechDevice(x) ∨:Turbine(x) and φ′ s = (≥1 :hasCat.⊤) ∨(≥1 :hasTuCat.⊤). ⊓⊔ τ ′ s = :MechDevice(x) ∨:Turbine(x) and φ′ s = (≥1 :hasCat.⊤) ∨(≥1 :hasTuCat.⊤). ⊓⊔ Observe that in the example both the target deﬁnition τs and the constraint deﬁnition φs were rewritten over O in order to guarantee that the ontology O can be safely ignored. In particular, the rewriting of τs guarantees that in any graph G, each instance of :Turbine should also be veriﬁed against s, whereas the rewriting of φs guarantees that any entity in G with a :hasTuCat-successor validates s, even if it has no :hasCat-successor. Thus, despite the similarity of query and constraint rewriting overt ontologies there are signiﬁcant diﬀerences5. The ﬁrst diﬀerence as illustrated above is that a shape contains a target deﬁnition and a constraint that in the general case should be rewritten independently. But more importantly, as opposed to queries, SHACL constraints can be recursive which makes the rewriting signiﬁcantly more involved (see Sect. 4 for details). We now show that rewritings may not exist. 3.2 Non-existence of SHACL-Rewritings We start with the hardness of SHACL validation that can be shown by reduction from the 3-coloring co-problem. Theorem 1. There exists a DL-LiteR ontology, a set of SHACL constraints C, and a KG G such that deciding whether ⟨O, G⟩is valid against C is co-NP-hard in the size of G. Proof. [Sketch] The proof is based on an encoding of the 3-coloring co-problem into the validity problem. For a given undirected graph F = ⟨V, E⟩, where V is a set of vertices and E of edges, we construct the following KG GF: {(vi, a, V ) \| vi ∈V } ∪{(vi, E, vj) \| (vi, vj) ∈E} ∪{(v′, U, vi) \| vi ∈V } ∪{(v′, a, T)}, and T are needed for technical reasons as will be explained below. 5 Recall that for query rewriting the input is a query q and ontology O and the output is another query q′ such that for any database D so-called certain answers of q over ⟨O, D⟩coincide with the answers of q′ over D alone [9]. 320 O. Savković et al. Then, we deﬁne O = {V ⊑∃R.C, Cred ⊑C, Cblue ⊑C, Cred ⊑¬Cblue}, where the axiom V ⊑∃R.C enforces that in each minimal model M of ⟨O, GF⟩, each vertex vi has an R-successor ai, which intuitively stands for the color of vertex vi in F6. The two other axioms intuitively enforce that either (ai, a, Cred) ∈M or (ai, a, Cblue) ∈M, or none of the two. Intuitively, vi is either red, or blue or none of the two (i.e. green). Now we introduce a singleton set of constraints C = {⟨s, τs, φs⟩} that requires that at least one pair of adjacent vertices has the same color: τs = T(x), and φs = (≥1 U.(φ1 ∨φ2 ∨φ3)), where φ1 = (≥1 R. ≥1 a.Cred) ∧(≥1 E. ≥1 R. ≥1 a.Cred) φ2 = (≥1 R. ≥1 a.Cblue) ∧(≥1 E. ≥1 R. ≥1 a.Cblue) φ3 = (≥1 R. ≥1 a.(¬Cred ∧¬Cblue)) ∧(≥1 E. ≥1 R. ≥1 a.(¬Cred ∧¬Cblue)) φ1 = (≥1 R. ≥1 a.Cred) ∧(≥1 E. ≥1 R. ≥1 a.Cred) φ2 = (≥1 R. ≥1 a.Cblue) ∧(≥1 E. ≥1 R. ≥1 a.Cblue) 2 = (≥1 R. ≥1 a.Cblue) ∧(≥1 E. ≥1 R. ≥1 a.Cblue) φ3 = (≥1 R. ≥1 a.(¬Cred ∧¬Cblue)) ∧(≥1 E. ≥1 R. ≥1 a.(¬Cred ∧¬Cblue)). 6 The axiom of the kind V ⊑∃R.C in syntactically not in DL-LiteR but it can be expressed using a “fresh” role R1 and three axioms: V ⊑∃R1, R1 ⊑R and ∃R− 1 ⊑C. 3.2 Non-existence of SHACL-Rewritings Intuitively, the formula φ1 evaluates to true at the node vi if vi is colored as red and has a red neighbour. The formulas φ2 and φ3 evaluate similarly, but for blue and green. Finally, the shape s has the node v′ as the unique target, and v has every other node in GF as a U-successor, ensuring that GF is valid against C iﬀthere is no 3-colouring for F. ⊓⊔ ⊓⊔ In [13] it has been shown that validation of SHACL constraints over KGs without ontologies is NP-complete in the size of the graph. Thus, we can imme- diately conclude the following negative result that holds under the assumption that co-NP ̸⊆NP. Corollary 1. There exists an DL-LiteR ontology and a set of SHACL con- straints for which no SHACL-rewriting over this ontology exists. Corollary 1. There exists an DL-LiteR ontology and a set of SHACL con- straints for which no SHACL-rewriting over this ontology exists. In order to overcome the non-existence problem we found a restriction on SHACL as will be presented in the following section. SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting HACL Constraint Validation over Ontology-Enhanced KGs via Rewriting The rest of this section we organize in four parts. (i) First we show why it is suﬃcient to consider only satisﬁable KGs; (ii) Then we show that over such satisﬁable KGs it suﬃcient to focus only on their canonical models (iii) then we show how to rewrite shape targets given an ontology, and (iv) ﬁnally we show how to rewrite ontologies. 4.1 Satisﬁable Knowledge Graphs We observe that in general KGs may contain disjointness and thus they may be unsatisﬁable (that is, they have no model). First we introduce an axillary property that shows that for constraint validation it is suﬃcient to consider only satisﬁable KGs. Lemma 1. Let O be a DL-LiteR ontology and G a graph. If ⟨O, G⟩is unsatis- ﬁable then for any shape s and any node in v in G there is exists no satisfying shape assignment over G and the set of shapes CO that validates s(v). Proof. Assume that ⟨O, G⟩is unsatisﬁable. Wlog we assume that the cause of being unsatisﬁable is the following: ⟨O, G⟩\|= C(a) ∧D(a) holds for some node a in G and some basic concepts C and D, and at same time O \|= C ⊑¬D holds (similarly it can be shown for role disjointness). From ⟨O, G⟩\|= C(a) ∧D(a) we conclude (using the properties on PerfRef in [9]) that G \|= PerfRef(C(a) ∧D(a), O). Then, since we have τsC⊑¬D = PerfRef(C(x) ∧D(x), O) it follows that a is the target of the shape sC⊑¬D. On the other hand φsC⊑¬D = ⊥, thus for every shape assignment σ it must be σ(sC⊑¬D) = ⊥, i.e., there exists no satisfying assignment for sC⊑¬D. Hence, there exist no satisfying assignment over G and CO that also validates s(v). ⊓⊔ 4 Rewriting of SHACL+ Constraints over OWL 2 QL As discussed above, a rewriting may not exist for an arbitrary set of SHACL shapes and a DL-LiteR ontology. Thus, in order to gain rewritability one can restrict the expressivity of SHACL. In the following we do so by restricting SHACL to positive SHACL shapes. For this setting we develop Algorithm 1 that allows to compute constraint rewritings. A SHACL shape is in SHACL+ if it does not contain negation and cardinality restriction of kind “≤n R.φ ”. 321 4.2 Validity over Canonical Models (⇐) The entailment ⟨O, G, C⟩\|= φs(v) holds if ⟨M, C⟩\|= φs(v)7 in each minimal model M, including can(O, G). (⇒) Let M be a minimal model of ⟨O, G⟩. Lemma 2 implies the existence of a homomorphism μ from can(O, G) to M s.t. M, C \|= φs(μ(v)) for μ(v) = v. ⊓⊔ (⇒) Let M be a minimal model of ⟨O, G⟩. Lemma 2 implies the existence of a homomorphism μ from can(O, G) to M s.t. M, C \|= φs(μ(v)) for μ(v) = v. ⊓⊔ 7 In this entailment we consider M as an inﬁnite conjunction of atoms. 4.2 Validity over Canonical Models We also notice that μ has to be surjective; otherwise the μ-image of can(O, G) would be a minimal model “smaller” than M which is a contradiction. Assume now that ⟨can(O, G), C⟩\|= φs(v) for some shape s from C and node v in can(O, G). Let σ be a satisfying assignment for can(O, G), C such that [[φs]]can(O,G),σ,v = true. We deﬁne an assignment σ′ over M, C in the following way: for a shape s1 and node v1 in M we set s1 ∈(v1, σ′) iﬀexists a node v2 in can(O, G) such that μ(v2) = v1 and s1 ∈(v2, σ). Now analyzing diﬀerent cases for s: φs = ⊤, φs = I, φs =≥k R.s1, φs = s1 ∧s2, φs = s1 ∨s2 and φs = EQ(r1, r2), it is not hard to show that if [[φs]]can(O,G),σ,v = true then [[φs]]M,σ′,μ(v) = true. ⊓⊔ Assume now that ⟨can(O, G), C⟩\|= φs(v) for some shape s from C and node v in can(O, G). Let σ be a satisfying assignment for can(O, G), C such that [[φs]]can(O,G),σ,v = true. We deﬁne an assignment σ′ over M, C in the following way: for a shape s1 and node v1 in M we set s1 ∈(v1, σ′) iﬀexists a node v2 in can(O, G) such that μ(v2) = v1 and s1 ∈(v2, σ). Now analyzing diﬀerent cases for s: φs = ⊤, φs = I, φs =≥k R.s1, φs = s1 ∧s2, φs = s1 ∨s2 and φs = EQ(r1, r2), it is not hard to show that if [[φs]]can(O,G),σ,v = true then [[φs]]M,σ′,μ(v) = true. ⊓⊔ Using the lemmas above we show the following property of canonical models. Using the lemmas above we show the following property of canonical models. Lemma 3 (canonical model characterization). For a DL-LiteR ontology O, basic concept C, graph G, node v in G, set C of SHACL+ shapes and shape s deﬁned in C we have that: ⟨O, G, C⟩\|= φs(v) iﬀ⟨can(O, G), C⟩\|= φs(v). Lemma 3 (canonical model characterization). For a DL-LiteR ontology O, basic concept C, graph G, node v in G, set C of SHACL+ shapes and shape s deﬁned in C we have that: ⟨O, G, C⟩\|= φs(v) iﬀ⟨can(O, G), C⟩\|= φs(v). Proof. (⇐) The entailment ⟨O, G, C⟩\|= φs(v) holds if ⟨M, C⟩\|= φs(v)7 in each minimal model M, including can(O, G). Proof. 4.2 Validity over Canonical Models In this part we show that in order to check the validity over all minimal models it is suﬃcient to check the validity over the canonical model for the given KG. In [9], the authors deﬁned a canonical model of a KB as a model that can be homomorphically mapped to any other model of that KB. Now we extend this notion to shapes: Given two graphs G1 and G2 with set of constants Δ1 and Δ2 respectively, and the set of shapes C, a SHACL-homomorphism μ from G to G′ is a mapping μ : Δ1 →Δ2 such that, for each shape s ∈C and each constant v ∈Δ1, if ⟨G1, C⟩\|= φs(v) then ⟨G2, C⟩\|= φs(μ(v)). Lemma 2 (canonical homomorphism for positive shapes). Let O be a DL-LiteR ontology, G a graph, and let M be a minimal model of ⟨O, G⟩. Let C be a set of SHACL+ shapes. Then, there is a SHACL-homomorphism from can(O, G) to M given C. In particular, there exists a SHACL-homomorphism that maps every node from G to itself. Lemma 2 (canonical homomorphism for positive shapes). Let O be a DL-LiteR ontology, G a graph, and let M be a minimal model of ⟨O, G⟩. Let C be a set of SHACL+ shapes. Then, there is a SHACL-homomorphism from can(O, G) to M given C. In particular, there exists a SHACL-homomorphism that maps every node from G to itself. O. Savković et al. 322 Proof. From [9], we have that there exists a homomorphism μ from can(O, G) to M such that for a basic concept C and node v it holds if C(v) ∈can(O, G) (resp. R(v1, v2) ∈can(O, G)) then C(μ(v)) ∈M (resp. R(μ(v1), μ(v2)) ∈M. In particular, it is possible to select μ such that μ(v) = v for v ∈G. We also notice that μ has to be surjective; otherwise the μ-image of can(O, G) would be a minimal model “smaller” than M which is a contradiction. Proof. From [9], we have that there exists a homomorphism μ from can(O, G) to M such that for a basic concept C and node v it holds if C(v) ∈can(O, G) (resp. R(v1, v2) ∈can(O, G)) then C(μ(v)) ∈M (resp. R(μ(v1), μ(v2)) ∈M. In particular, it is possible to select μ such that μ(v) = v for v ∈G. 4.4 Rewriting of the Ontology In this part, we present our rewriting algorithm. In order to make notations more concise, we write G, C \|= φ(v) to denote that the node v satisﬁes the constraint φ in the graph G given a set C of shapes. Similarly, we use ⟨O, G⟩, C \|= φ(v) to denote that v satisﬁes φ in the graph that corresponds to the canonical model of ⟨O, G⟩given C. Then, we assume that the shape constraints in C are normalised, i.e. contain at most one operator. Note that this can always be obtained by introducing nested shape names. Our rewriting procedure is presented in Algorithm 1 and we now guide the reader through it. Our algorithm relies on auxiliary shapes of three kinds: – CO that contains for each concept C in O the corresponding shape sC in CO; this ensures that for every node v the fact C(v) is in the canonical model iﬀ v is valid in the shape sC. – “virtual” shapes Cv O and Cs O that are used to capture the part of the canonical model generated by existential quantiﬁcation. – “virtual” shapes Cv O and Cs O that are used to capture the part of the canonical model generated by existential quantiﬁcation. The shapes CO, Cv O and Cs O will help us to establish rewriting over O of the original shapes from C into C′′. We now deﬁne CO, Cv O and Cs O explain how they are used in Algorithm 1 and show correctness of the algorithm. Rewriting ShapeT for Active Nodes: CO. For every concept of the form A (resp. ∃R) in O, we introduce a shape sA (resp. s∃R), with targets τsA = A (resp. τs∃R = ∃R) and with constraint where R, R′ may be inverse roles: φsA = (≥1 a.A) ∨ O \|= C⊑A sC, φs∃R = (≥1 R.⊤) ∨ O \|= C⊑∃R sC ∨ O \|= R′⊑R s∃R′. Next, we introduce shapes that encode negative assertions. For each GCI of the form (C ⊑¬D) in O, we introduce one shape sC⊑¬D, where targets are all instances of C and D in G, where the constraint is always violated. To this end, Next, we introduce shapes that encode negative assertions. 4.3 Rewriting of Shape Targets In the absence of an ontology, the targets of shapes s are retrieved by evaluating the target deﬁnitions τs over the graph G, written [[τs]]G. In SHACL, a target deﬁnition is a monadic query with a single atom that corresponds to a basic concept in an ontology. In the presence of an ontology, we follow the semantics described in Sect. 2.5, and retrieve targets over all minimal models, or equiva- lently over the canonical model, written as [[τ]]⟨O,G⟩. To achieve this, since τs is a unary conjunctive query, one can apply PerfRef. Lemma 4. For any shape s from SHACL, DL-LiteR ontology O, and graph G it holds that [[τs]]⟨O,G⟩= [[PerfRef(τs, O)]]G. Proof. The authors in [9] established the correspondence between certain answers of conjunctive queries over knowledge graphs and perfect reformula- tion (Lemma 35): For a KG ⟨O, G⟩and conjunctive query q we have that the certain answers of q over the KG correspond to perfect reformulation in the sense that cert(q, ⟨O, G⟩) = [[PerfRef(τs, O)]]G, where, a ∈cert(q, ⟨O, G⟩) iﬀ for every minimal model M of ⟨O, G⟩it holds that a ∈qM. At the same time SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting 323 Algorithm 1. Constraint rewriting Input: ontology O possible with existential rules, set of positive shapes C 1: CO ←ShapeT(O) 2: Cv O ←shapeVirtual(O) 3: Cs O ←successorT(O) 4: C′′ ←{⟨PerfRef(τs, O), RewriteCompl(φs, O)⟩\| s ∈C} 5: return CO ∪Cv O ∪Cs O,C ∪C′′ SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting 323 SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting 323 Algorithm 1. Constraint rewriting Input: ontology O possible with existential rules, set of positive shapes C 1: CO ←ShapeT(O) 2: Cv O ←shapeVirtual(O) 3: Cs O ←successorT(O) 4: C′′ ←{⟨PerfRef(τs, O), RewriteCompl(φs, O)⟩\| s ∈C} 5: return CO ∪Cv O ∪Cs O,C ∪C′′ Algorithm 1. Constraint rewriting the formula [[τs]]⟨O,G⟩deﬁnes a special set of target nodes over the graph and ontology: [[τs]]⟨O,G⟩returns a node v iﬀit does so for every minimal model M of ⟨O, G⟩. ⊓⊔ the formula [[τs]]⟨O,G⟩deﬁnes a special set of target nodes over the graph and ontology: [[τs]]⟨O,G⟩returns a node v iﬀit does so for every minimal model M of ⟨O, G⟩. ⊓⊔ In other words, the targets of s according to the KB ⟨O, G⟩can be retrieved by evaluating the query PerfRef(τs, O) over G alone. 4.4 Rewriting of the Ontology For each GCI of the form (C ⊑¬D) in O, we introduce one shape sC⊑¬D, where targets are all instances of C and D in G, where the constraint is always violated. To this end, O. Savković et al. 324 we exploit results based on PerfRef [9]: τsC⊑¬D = PerfRef(C(x) ∧D(x), O), and φsC⊑¬D = ⊥. Similarly, for negative role inclusions, we use sR1⊑¬R2, with τsR1⊑¬R2 = PerfRef(∃yR1(x, y) ∧R2(x, y), O), and φsR1⊑¬R2 = ⊥. we exploit results based on PerfRef [9]: τsC⊑¬D = PerfRef(C(x) ∧D(x), O), and φsC⊑¬D = ⊥. Similarly, for negative role inclusions, we use sR1⊑¬R2, with τsR1⊑¬R2 = PerfRef(∃yR1(x, y) ∧R2(x, y), O), and φsR1⊑¬R2 = ⊥. ⊑ 2 1⊑ 2 We denote the set of shapes produced above with CO, and the corresponding slation function as ShapeT in Algorithm 1, i.e., ShapeT(O) = CO. 1⊑ 2 1⊑ 2 We denote the set of shapes produced above with CO, and the corresponding translation function as ShapeT in Algorithm 1, i.e., ShapeT(O) = CO. ( ) With cl(O, G) we denote the maximal subset of can(O, G) without fresh nodes. If there were no fresh nodes, i.e., when can(O, G) = cl(O, G), we observe that shapes in C ∪ShapeT(O) would be suﬃcient to validate the facts in can(O, G). Intuitively, this is because dependencies among shapes in ShapeT(O) corresponds to the construction of the “closure” cl(O, G). ( ) With cl(O, G) we denote the maximal subset of can(O, G) without fresh nodes. If there were no fresh nodes, i.e., when can(O, G) = cl(O, G), we observe that shapes in C ∪ShapeT(O) would be suﬃcient to validate the facts in can(O, G). Intuitively, this is because dependencies among shapes in ShapeT(O) corresponds to the construction of the “closure” cl(O, G). Fresh Nodes in Canonical Models. Observe that for DL-LiteR ontologies O the canonical model can(O, G) can be arbitrary large (even inﬁnite) in which case it will contain “fresh” nodes that are not occurring in G. Thus, one should be able to check constraints also on this fresh nodes, as shown next. Example 7. Consider the ontology O = {A ⊑∃U, ∃U −⊑∃P} and graph G = {(v, a, A)}. Then can(O, G) = {(v, a, A), (v, U, a1), (a1, P, a2)} where a1 and a2 are fresh nodes. Now consider shapes ⟨s1, A(x), (≥1 U.s2)⟩and ⟨s2, ⊥(x), (≥1 P.⊤)⟩. 4.4 Rewriting of the Ontology It is not hard to see that C is valid over (O, G). ⊓⊔ Example 7. Consider the ontology O = {A ⊑∃U, ∃U −⊑∃P} and graph G = {(v, a, A)}. Then can(O, G) = {(v, a, A), (v, U, a1), (a1, P, a2)} where a1 and a2 are fresh nodes. Now consider shapes ⟨s1, A(x), (≥1 U.s2)⟩and ⟨s2, ⊥(x), (≥1 P.⊤)⟩. It is not hard to see that C is valid over (O, G). ⊓⊔ These properties of can(O, G) make rewriting technically involved since SHACL constraints cannot express fresh values. We address this with auxil- iary shapes Cv O and Cs O that mimic the construction of the canonical model and validate facts in G′ = can(O, G)\cl(O, G). The graph G′ is a forest (by construc- tion of can(O, G)) where each tree has the root in some assertion of cl(O, G). We call this root the witness of the tree. In example above, (v, a, A) is the only witness. Rewriting shapeVirtual for Fresh Nodes: Cv O. For each concept C appearing in a GCI in O, we introduce a shape svirtual C , such that, for a node v in G, veriﬁes svirtual C (v) iﬀthere is a node v′ in G′ with v as witness such that G′ \|= C(v′). For instance, in Example 8, we introduce shape svirtual ∃R which is veriﬁed by the witness v. Note that v′ is not necessarily an immediate successor of v in G′. More formally, for concept C, the virtual shape svirtual C = ⟨PerfRef(C), φsvirtual C ⟩is created. Then a function similar to RewriteSim is applied to each φsvirtual C , in order to ensure the above property. In our running example, this yields φsvirtual A = sA, i.e. φsvirtual A remains unchanged, but φsvirtual ∃U = s∃U ∨svirtual A ∨svirtual ∃U − . Here, sub-formula svirtual A is added because of the GCI A ⊑U, and s∃U −is added because if ∃U holds at some node a1 in the tree of G′ rooted in v, then ∃U −must hold at some U-successor a2 of a1. Let shapeVirtual be the function which produces (and rewrites) these “virtual” shapes. More formally, for concept C, the virtual shape svirtual C = ⟨PerfRef(C), φsvirtual C ⟩is created. Then a function similar to RewriteSim is applied to each φsvirtual C , in order to ensure the above property. 4.4 Rewriting of the Ontology In our running example, this yields φsvirtual A = sA, i.e. φsvirtual A remains unchanged, but φsvirtual ∃U = s∃U ∨svirtual A ∨svirtual ∃U − . Here, sub-formula svirtual A is added because of the GCI A ⊑U, and s∃U −is added because if ∃U holds at some node a1 in the tree of G′ rooted in v, then ∃U −must hold at some U-successor a2 of a1. Let shapeVirtual be the function which produces (and rewrites) these “virtual” shapes. Rewriting successorT for Fresh Nodes: Cs O. The second kind of shapes is needed in order to check if two roles are concatenated in the same tree in G′. For each pair of roles R1 and R2 in O, we introduce the shape ssucc R1,R2 such that a node v ∈G veriﬁes φssucc R1,R2 iﬀ(a1, R1, a2) and (a2, R2, a3) are on the subtree with SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting 325 the witness v, for some a1, a2, a3 in G′. In our running example, v veriﬁes φssucc R,P , but not φssucc P,R. Formally, for every two roles R1 and R2 in O, τssucc R1,R2 = ⊥(x), and if O \|= ∃R− 1 ⊑∃R2, then φssucc R1,R2 = s∃R1, otherwise φssucc ∃R1,∃R2 = ⊥. The special case R2 = R− 1 , is also covered by the deﬁnition φssucc R1,R− 1 = s∃R1. Let successorT denote the function creating these fresh shapes. the witness v, for some a1, a2, a3 in G′. In our running example, v veriﬁes φssucc R,P , but not φssucc P,R. Formally, for every two roles R1 and R2 in O, τssucc R1,R2 = ⊥(x), and if O \|= ∃R− 1 ⊑∃R2, then φssucc R1,R2 = s∃R1, otherwise φssucc ∃R1,∃R2 = ⊥. The special case R2 = R− 1 , is also covered by the deﬁnition φssucc R1,R− 1 = s∃R1. Let successorT denote the function creating these fresh shapes. Rewriting RewriteComplT for Shapes. Finally, we need to rewrite the shapes in C. To this end, we extend the procedure RewriteSim in the following way. For each shape s in C, we set s′ = RewriteCompl(s) ∨svirtual where RewriteCompl is identical to RewriteSim for operators ∧, ∨and constant I but it changes for φs = (≥k R.s1) as follows: φ′ s = (≥k R.s′ 1) ∨svirtual where φsvirtual = svirtual ∃R ∧svirtual 1 ∧s∃R,s1. 4.4 Rewriting of the Ontology In other words, the witness v veriﬁes svirtual if it veriﬁes both svirtual ∃R and svirtual 1 (that is, both are veriﬁed by some anonymous node with v as the witness), and the range of R can be validated against s1, expressed with the new shape s∃R,s1. Then φs∃R,s1 = s∃R,s2 ∧s∃R,s3 if φs1 = s2 ∧s3 (and similarly for ∨). If φs1 = (≥k P.s2), then φ∃R,s1 = ssucc R,P , that is P has to be the successor of R in G′. Let RewriteComplT denote the corresponding rewriting of C. In other words, the witness v veriﬁes svirtual if it veriﬁes both svirtual ∃R and svirtual 1 (that is, both are veriﬁed by some anonymous node with v as the witness), and the range of R can be validated against s1, expressed with the new shape s∃R,s1. Then φs∃R,s1 = s∃R,s2 ∧s∃R,s3 if φs1 = s2 ∧s3 (and similarly for ∨). If φs1 = (≥k P.s2), then φ∃R,s1 = ssucc R,P , that is P has to be the successor of R in G′. Let RewriteComplT denote the corresponding rewriting of C. Correctness of Rewriting. We now proceed to the correctness of our rewriting procedure and start with the property of possibly inﬁnite canonical models. Correctness of Rewriting. We now proceed to the correctness of our rewriting procedure and start with the property of possibly inﬁnite canonical models. Lemma 5 (Inﬁnite canonical model). Let O be a DL-LiteR ontology, C a concept in O, R and P properties in O, G a graph, v a node in G, and shapes CO, Cv O and Cs O as speciﬁed in Algorithm 1. Then, the following holds: – G, CO ∪Cv O ∪Cs O \|= φsvirtual C (v) iﬀthere is a node a1 in can(O, G) with the witness v such that can(O, G) \|= C(a1). \| ( ) – G, CO ∪Cv O ∪Cs O \|= φsvirtual C (v) iﬀthere is a node a1 in can(O, G) with the witness v such that can(O, G) \|= C(a1). 4.4 Rewriting of the Ontology – G, CO ∪Cv O ∪Cs O \|= φssucc R,P (v) iﬀthere are nodes a1, a2, a3 in can(O, G) with th it h th t (O G) \| R( ) d (O G) \| P( ) ( ) \| ( ) – G, CO ∪Cv O ∪Cs O \|= φssucc R,P (v) iﬀthere are nodes a1, a2, a3 in can(O, G) with the witness v such that can(O, G) \|= R(a1, a2) and can(O, G) \|= P(a2, a3). ( ) \| ( ) – G, CO ∪Cv O ∪Cs O \|= φssucc R,P (v) iﬀthere are nodes a1, a2, a3 in can(O, G) with the witness v such that can(O, G) \|= R(a1, a2) and can(O, G) \|= P(a2, a3). Example 8. We illustrate the rewriting of the running example. Shapes that are not relevant for the reasoning are omitted. The presented shapes are ordered in the way one would reason with them, starting bottom-up (which is possible if C is not recursive). To illustrate the reasoning, we underline in each formula the disjuncts for which one can construct a satisfying shape assignment. φsvirtual A = sA, φsvirtual ∃U = s∃U ∨svirtual A ∨svirtual ∃U − , φs∃U = (≥1 U.⊤) ∨svirtual ∃U , φssucc U,P = s∃U, φs∃U,s2 = ssucc U,P , φsvirtual 1 = svirtual ∃U ∧svirtual 2 ∧s∃U,s2, φs′ 1 =≥1 U.s′ 2 ∨svirtual 1 , φsvirtual 2 = svirtual ∃P , φsvirtual ∃P = s∃P ∨svirtual ∃U − ∨svirtual ∃P − , φsvirtual ∃U−= svirtual ∃U . The only target of s is v, and v veriﬁes φs w.r.t the rewritten set of shapes. ⊓⊔ φsvirtual A = sA, φsvirtual ∃U = s∃U ∨svirtual A ∨svirtual ∃U − , φs∃U = (≥1 U.⊤) ∨svirtual ∃U , φssucc U,P = s∃U, φs∃U,s2 = ssucc U,P , φsvirtual 1 = svirtual ∃U ∧svirtual 2 ∧s∃U,s2, φs′ 1 =≥1 U.s′ 2 ∨svirtual 1 , φsvirtual 2 = svirtual ∃P , φsvirtual ∃P = s∃P ∨svirtual ∃U − ∨svirtual ∃P − , φsvirtual ∃U−= svirtual ∃U . The only target of s is v, and v veriﬁes φs w.r.t the rewritten set of shapes. ⊓⊔ The only target of s is v, and v veriﬁes φs w.r.t the rewritten set of shapes. ⊓⊔ e only target of s is v, and v veriﬁes φs w.r.t the rewritten set of shapes. We are ready to present the main result of this section. 4.4 Rewriting of the Ontology ⊓⊔ We are ready to present the main result of this section. We are ready to present the main result of this section. O. Savković et al. 326 Theorem 2. Let O be a DL-LiteR ontology, C a set of SHACL+ shapes, s ∈C, C′ the shapes returned by Algorithm 1, and s′ the rewriting of s in C′. Then, for any graph G and node v in G it holds that: ⟨O, G⟩, C \|= φs(v) iﬀ G, C′ \|= φ′ s(v). Theorem 2. Let O be a DL-LiteR ontology, C a set of SHACL+ shapes, s ∈C, C′ the shapes returned by Algorithm 1, and s′ the rewriting of s in C′. Then, for any graph G and node v in G it holds that: ⟨O, G⟩, C \|= φs(v) iﬀ G, C′ \|= φ′ s(v). Note the size of the returned rewriting is polynomial in the size of O and C. Note the size of the returned rewriting is polynomial in the size of O and C 5 Related Work As discussed in Sect. 1, constraint validation in the presence of ontologies was studied in [20,35,42]. While these approaches allow for very expressive ontologies (e.g., SHOIN) they require an engine for disjunctive logic programs, which we believe makes these approaches less practically interesting. Rewriting of conjunctive queries over OWL 2 QL ontologies of [9] cannot be applied to SHACL shapes since they may be recursive or contain negation. From a more general prospective our rewriting can be seen a special case of the backward-chaining algorithm [32] over ∀∃-rules (where the body and the head are conjunctions of atoms, and only the variables that occur in the head can be existentially quantiﬁed). However, in such cases the existence of a rewriting is undecidable in general for arbitrary rules, and even for some decidable fragments, diﬀerently from our approach, such algorithms may not terminate. Naturally, one may think of relating SHACL to Datalog programs [14] since they poses recursion and negation. However, Datalog programs can at most have one unique minimal model, and SHACL constraints should be checked for all possible assignments [12] (including also non-minimal ones). If we consider more expressive version of Datalog like Datalog with negation under the stable model semantics (SMS) [14], then relating it to SHACL is more promising, while the actual relation is not obvious as SMS is also based on minimal models. Nevertheless, our preliminary results show that this is not straightforward. 6 Conclusion We have studied the rewriting of constraints over ontologies for constrain vali- dation. We focused on a prominent language for graph constraints SHACL and on ontologies from the widely used OWL 2 QL ontology language. We deﬁned semantics for constraint rewriting, showed the non-existence of such rewritings in the general case, and identiﬁed restrictions on SHACL to positive (but recursive) fragment SHACL+ for which they always exist. For SHACL+ we showed how to rewrite ontologies and SHACL+ constraints into the unique set of SHACL+ constraints. Moreover, SHACL+ validation over OWL 2 QL is tractable. We see this work as an important step towards practical constraint rewrit- ing algorithms and systems. Next, we plan to analyse optimisation techniques in order to obtain more eﬃcient rewritings. For instance, we plan to consider datatypes. They can be used to optimize eliminate unnecessary rewritings, but this need to be done in a controlled way to ensure tractability (e.g., [38]). 327 SHACL Constraint Validation over Ontology-Enhanced KGs via Rewriting Then, we plan to extend this work to account for OWL 2 EL. Moreover, we plan to implement our approach and evaluate it in various industrial settings [11,24–27,33,34,37,39]. An important research direction is also to understand how to repair data that fails to satisfy SHACL constraints and we see the work on ontology evolution as a good source of inspiration [10,29,30,43]. Acknowledgments. This work was partially funded by the SIRIUS Centre, Norwe- gian Research Council project number 237898; by the Free University of Bozen-Bolzano projects QUEST, ROBAST and ADVANCED4KG. References Horrocks, I., Giese, M., Kharlamov, E., Waaler, A.: Using semantic technology to tame the data variety challenge. IEEE Internet Comput. 20(6), 62–66 (2016) 20. 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https://openalex.org/W2565904822	https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-016-1310-4	English	null	DegreeCox – a network-based regularization method for survival analysis	BMC bioinformatics	2,016	cc-by	8,535	© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Modeling survival oncological data has become a major challenge as the increase in the amount of molecular information nowadays available means that the number of features greatly exceeds the number of observations. One possible solution to cope with this dimensionality problem is the use of additional constraints in the cost function optimization. LASSO and other sparsity methods have thus already been successfully applied with such idea. Although this leads to more interpretable models, these methods still do not fully profit from the relations between the features, specially when these can be represented through graphs. We propose DEGREECOX, a method that applies network-based regularizers to infer Cox proportional hazard models, when the features are genes and the outcome is patient survival. In particular, we propose to use network centrality measures to constrain the model in terms of significant genes. Results: We applied DEGREECOX to three datasets of ovarian cancer carcinoma and tested several centrality measures such as weighted degree, betweenness and closeness centrality. The a priori network information was retrieved from Gene Co-Expression Networks and Gene Functional Maps. When compared with RIDGE and LASSO, DEGREECOX shows an improvement in the classification of high and low risk patients in a par with NET-COX. The use of network information is especially relevant with datasets that are not easily separated. In terms of RMSE and C-index, DEGREECOX gives results that are similar to those of the best performing methods, in a few cases slightly better. Conclusions: Network-based regularization seems a promising framework to deal with the dimensionality problem. The centrality metrics proposed can be easily expanded to accommodate other topological properties of different biological networks. Keywords: Regularization, Cox proportional models, Network metrics a largely unsolved problem, especially when the num- ber of features or covariates involved, such as related to molecular data (which can easily reach tens of thou- sands), greatly outnumbers the observations (typically in the hundreds). This fact severely hampers the modeling task, usually leading to a degradation in the classifier accu- racy and a greater difficulty in extracting knowledge from data [2, 3]. Furthermore, datasets suffering from this curse of dimensionality often lead to over-fitted models which, although they represent the training data, exhibit a sig- nificant decrease in their accuracy on new observations [4]. This problem may persist even when feature selection and validation schemes are used. One possible solution to DegreeCox – a network-based regularization method for survival analysis André Veríssimo1,2, Arlindo Limede Oliveira2,3, Marie-France Sagot4,5 and Susana Vinga1* From The 10th International Workshop on Machine Learing in Systems Biology (MLSB) Den Haag, The Netherlands. 3-4 September 2016 From The 10th International Workshop on Machine Learing in Systems Biology (MLSB) Den Haag, The Netherlands. 3-4 September 2016 *Correspondence: susanavinga@tecnico.ulisboa.pt 1IDMEC, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, 1049-001 Portugal Full list of author information is available at the end of the article The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 DOI 10.1186/s12859-016-1310-4 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 DOI 10.1186/s12859-016-1310-4 Background Precision medicine shows the promise of additional effi- cacy by bringing more information into the diagnosis pro- cess. It is, however, highly dependent on rapid advances in science and technology as data analysis and knowl- edge discovery techniques are indeed struggling to keep pace with the challenges related to what computer sci- entists have called big data [1]. In this regard, dealing with the high-dimensionality of patients’ data represents Page 110 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Page 110 of 135 data [14]. Since there is an underlying structure on the gene feature space given by the patterns of co-expression, these correlations can be applied as constraints to the Cox proportional hazards model. Although the results are promising, there are still few studies that fully explore the network properties of the feature space beyond this connectivity. tackle this problem is to impose further constraints on the solution space. This can be accomplished through regular- ization methods, that penalize more complex structures of the solution space. The goal is to penalize the cost func- tion (e.g. quadratic error, log-likelihood) with additional functions in order to impose a structure on the parameter space. In this context, we propose and explore a novel network- degree-constraint Cox proportional hazard model, that we called DEGREECOX, which uses a priori knowledge to leverage the correlation or functional information present in gene expression data. In this survival model, a graph degree constraint is introduced that expresses the impor- tance of a gene by how highly connected it is in the overall network. For linear regression, a regularization method that is widely used is LASSO - Least Absolute Shrinkage and Selection Operator [5], which penalizes the error function with the L1 norm of the regression parameters, leading to a sparse solution. Other possible regularizers include feature or group sparsity, smoothness of the features’ coef- ficients, or a graph representing how the features are connected [5–11]. We applied DEGREECOX to identify gene expression signatures associated with survival of ovarian carcinoma patients. This type of cancer is the fifth-leading cause of cancer death in US women [20]. DEGREECOX was applied to three large-scale ovarian cancer gene expres- sion datasets [20–22] to predict a patient’s risk and to identify genes associated to death events. We compared DEGREECOX with similar methods such as NET-COX [14] and elastic net [6]. Background Our results show that using vertex degree can improve the model in terms of its generaliza- tion capability. These techniques have led to models that are par- tially capable of dealing with the dimensionality prob- lem and, additionally, are able to improve model interpretability [12–14]. In this context, survival analysis in oncology research represents one of the most challenging areas of applica- tion, with the recent development of public databases such as TCGA - The Cancer Genome Atlas [15]. Survival anal- ysis involves modeling the time to an event of interest, by uncovering relationships between the given covariates and time distributions [16], and allowing for censored obser- vations (for which the event does not occur). The Cox proportional hazard model [16] is used to model these relationships and has been widely applied in this con- text. However, it also exhibits problems for datasets with more covariates than observations. For example, using genomic data to determine the relationship of the expres- sion levels of thousands of genes to a death event leads to an under-determined problem that can have multiple solutions. The code to reproduce the results is available at http:// sels.tecnico.ulisboa.pt/gitlab/averissimo/degree-cox. Methods The proposed method DEGREECOX is based on applying network-based regularizers in Cox proportional hazards model estimation. This section will overview several reg- ularizers based on centrality measures of a network and will briefly describe which networks can be applied in the context of gene expression data. Survival models and regularization in the context of Cox regression are then overviewed. Recent efforts to combine Cox modeling with regular- ization techniques have already shown promising results [11, 17, 18]. In particular, sparse models have been devel- oped to identify a small set of gene signatures related to high or low risk patients. Furthermore, the predictability of the model was tested with datasets from five geograph- ically distant populations [17]. Cox regularized models have also been used to predict a patient’s risk of con- version from a mild cognitive impairment to Alzheimer’s disease [18]. Network centrality metrics A biological network is represented as a graph G := (V, E), with V denoting the set of vertices, or nodes, and E the set of edges. In the present context of gene net- works, G represents the co-expression or functional map network where the vertices are P genes, with P := \|V\|, and edges represent a weighted relation between two genes. The graph G may also be represented by a P ×P positively weighted adjacency matrix that we denote by W. Besides these sparsity methods, other techniques tried to embed network-based regularizers, following work on group sparsity [19]. When the features can be connected through a graph, one can further explore this struc- ture in order to improve the models. One example is to impose smoothness on the parameters associated with connected features (in the network). This technique pro- vided good results for modeling survival of ovarian cancer patients where the features correspond to gene expression g j y y The matrix W is further normalized, leading to the matrix S with sij = wij· P n=1 win −1/2 · P n=1 wnj −1/2 , i.e., each normalized value in S is obtained by dividing the weights by the square root of the sum over all rows and columns. The matrix W is further normalized, leading to the matrix S with sij = wij· P n=1 win −1/2 · P n=1 wnj −1/2 , h l d l S b d b d d h j j j i.e., each normalized value in S is obtained by dividing the weights by the square root of the sum over all rows and columns. i.e., each normalized value in S is obtained by dividing the weights by the square root of the sum over all rows and columns. The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Page 111 of 135 Page 111 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Network centrality measures characterize each vertex in a network, creating a ranking of the most relevant ones [23]. Research on this topic emerged in the 1950s on the role of central vertices in social networks [24–26]. Different metrics have been proposed in the literature. These typically use network topology to define a function that determines a measure for vertex yi. Network centrality metrics Among the pro- posed methods to classify important vertices are degree, betweenness and closeness centrality, briefly described below and illustrated in Fig. 1, where the size and color of a vertex reflect the importance of the vertex for each method. Betweenness centrality The betweenness centrality Bi is equal to the frequency of the presence of vertex yi in the shortest paths between every two vertices (yj, yk) in the network, i ̸= j ̸= k. This will rank vertices by their importance on the com- munication flow of the network. It may be used to identify possible bottlenecks or relevant regulators of the network. It is defined by: These typically use network topology to define a function that determines a measure for vertex yi. Among the pro- posed methods to classify important vertices are degree, betweenness and closeness centrality, briefly described below and illustrated in Fig. 1, where the size and color of a vertex reflect the importance of the vertex for each method. Bi = P j=1 P k:k>j gjk(yi) gjk , (3) (3) where gjk is the number of shortest paths between yj and yk and gjk(yi) is the number of shortest paths that include vertex yi. Computation of this metric for dense graphs can be done in (\|V\|3) time and for sparse graphs in O(\|V\|2 · log(\|V\|) + \|V\| · \|E\|) time. In the Results Section, all these measures will be tested on real datasets in order to choose the best ones to be integrated in the proposed regularizer. Degree centrality h d f The degree of a vertex is the number of its adjacent ver- tices. Vertices with a high degree are called hubs and may bridge the path between other low degree vertices in the network keeping the network diameter low. The simplest description of network centrality based on the degree of a vertex was first presented by Nieminem [27] and counts the adjacent edges of vertex yi: Closeness centrality The idea that the centrality of a vertex is related to its con- nectivity in the network was suggested by [24, 25]. This measure, denoted by C, is based on calculating, for each vertex yi, its distance gji to every vertex yj, j ̸= i, in the net- work, defined as the length of the corresponding shortest path, summing all these distances and taking the inverse: di = P j=1 aij, (1) (1) c−1 i = P j̸=i gji. (4) (4) where aij = 1 if vertices yi and yj are connected and aij = 0 otherwise. The rationale is that the more central vertices have lower total distances from all other vertices. This measure requires that the graph is connected, as two disconnected vertices are at an infinite distance from one another. Extensions of this definition to include weighted net- works have been proposed, where the values sij represent the normalized weight of the connecting edge instead of a binary value [28, 29]: di = P j=1 sij. (2) Regularized Cox regression A gene functional map (GFM) describes the functional activity and corresponds to an interaction network that includes information from ∼30, 000 genome-scale exper- iments and ∼25, 000 human genes. It was built using a regularized Bayesian integration system proposed by Huttenhower and colleagues [31] and is available at http:// giant.princeton.edu/. Each edge between two genes is probabilistically weighted based on experimental evidence which integrates many different datasets. The functional map used in the present work includes 7562 genes inferred from experiments using ovarian cells. When the number of gene features P is much larger than the observations n (n ≪P), the estimation procedure exhibits identifiability problems. In fact, applying the stan- dard Cox proportional hazard model to infer parameters will lead to multiple possible solutions with a large num- ber of non-zero parameters, which severely hampers the classification of new observations. Cox proportional hazards model Given D = ((X1, Y1, δ1), · · · , (Xn, Yn, δn)), where Xi, i = 1, . . . , n is the gene expression profile of n patients over P genes, X′ i = (Xi1, · · · , XiP), Y is the response variable that indicates the survival time for patient i and δi is an indicator of whether patient i has observed the event (δi = 1) or not (δi = 0). The hazard function for a patient given his expression profile is given by: lL1L2(β, h0) = n i=1 −exp(X′ iβ)H0(ti)+ δi log(h0(ti)) + X′ iβ −1 2λ α\|β\|1 + (1 −α)\|β\|2 2 , (10) (10) h(t\|Xi) = h0(t) exp(X′ iβ), (5) (5) where β = (β1, · · · , βP) is a vector of regression coef- ficients and h0(t) is the baseline hazard function. The regression coefficients are estimated by maximizing the Cox’s partial log-likelihood: where λ is the parameter controlling the penalizing weight and α the balance between the two norms. In particular, α = 0 leads to the RIDGE regression and when α = 1, LASSO regression is obtained. The R package “glmnet” [11] was used to estimate the coefficients with this type of regularizer. l(β) = n i=1 δi ⎧ ⎨ ⎩X′ iβ −log ⎡ ⎣ n j:yj≥yi exp X′ jβ ⎤ ⎦ ⎫ ⎬ ⎭. (6) (6) Gene networks (2) In order to apply a network-based regularizer, two types of gene networks will be used: 1) Gene Co-expression Networks (GCN); and 2) Gene Functional Maps (GFM). Both networks consider genes as vertices and the weight of each edge corresponds to the association between the connected genes, which can be the correlation between gene expression or functional annotation. Methods to determine the centrality of a vertex are local, since they are functions of the neighborhood of yi, therefore not taking into account global properties. For a comparison of multiple networks, this value should be normalized by the total number of vertices [23]. Fig. 1 Centrality measures Page 112 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 The inference of the optimal coefficients ˆβ is done by maximizing the total log-likelihood in two steps, alternat- ing between maximizing with respect to β and updating the h0(t) estimation (in Eq. 7). A gene co-expression network (GCN) is specific for each dataset and is generated using the ranking of Pearson’s correlation coefficients between gene gi and gj, for all genes in the dataset [30]. The resulting matrix M, is given by M−1 ij = rij · rji, where rij is the position of gene gj in the correlation ranking of gene gi. LASSO and RIDGE regression Strategies that can be used to minimize this problem include the application of L1 and L2 norms, in order to restrict the solution space, in particular imposing sparsity and small coefficients for the parameters [5, 6, 33]. This can be done by penalizing the total log-likelihood with a weighted sum of the L1 and L2 norms, a method called elastic net [6]: DEGREECOX regression The function proposed in DEGREECOX combines the total log-likelihood of Cox regression with degree regulariza- tion. As previously, the total log-likelihood is calcuted using the Breslow estimator (Eq. 8). The novelty is the introduction of a penalizing term that conveys a ver- tex centrality information of the subjacent network. To this purpose, both Gene Co-expression Networks (GCN) and Gene Functional Maps (GFM) are used in order to extract the corresponding vertex centrality information. More specifically, each of the different network centrality measures is tested for each of the two networks. NET-COX regression In the NET-COX model previously proposed [14], a Laplacian matrix constraint is introduced as a smoothness operator among adjacent coefficients in the network. This operator adds a cost, for every pair of genes connected by an edge, which is proportional to the edge weight and the difference between their coefficients. This hypothesis determines that genes that are connected should be cor- related. This implies that the coefficients of the features related to the genes should be similar, i.e., vary smoothly through the network. One of the most used estimators for the baseline hazard is the Breslow estimator [32] given by: ˆh0(ti) = 1 n j:yj≥ti exp(X′β). (7) (7) The partial likelihood and the Breslow estimator are induced by the total log-likelihood: l(β, h0) = n i=1 −exp(X′ iβ)H0(ti)+ δi log(h0(ti)) + X′ iβ , (8) g The Laplacian is then given by: (8) δi log(h0(ti)) + X′ iβ , (β) = 1 2 p i,j=1 Sij βi −βj 2 = β′(I −S)β = β′Lβ, (11) with with (11) H0(ti) = tk≤ti h0(tk). (9) = β′(I −S)β = β′Lβ, (9) The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Page 113 of 135 where L is a positive semidefinite matrix derived from the network. The the full model of NET-COX is based on: When adding the constraint to the Cox model, we get the full likelihood as follows: lDegreeCox(β, h0) = n i=1 −exp(X′ iβ)H0(ti)+ δi log(h0(ti)) + X′ iβ −1 2λ β′Dβ . (14) lNETCOX(β, h0) = n i=1 −exp(X′ iβ)H0(ti)+ δi log(h0(ti)) + X′ iβ −1 2λβ′ ((1 −α) L + αI) β, (12) (14) This model adds a cost for each gene/vertex that increases as its coefficient βi increases, but is also inversely proportional to how well connected that vertex is in the graph, given by its degree. Thus, the objective function drives the assignment of larger coefficients to genes that are highly connected in the network. The ratio- nale behind the application of this regularizer is then to identify a set of genes that not only predicts the survival, but that also has a relevant role in the underlying network. where λ is a parameter that controls the penalizing weight of the regularizer and α is the parameter that weights the two penalizations. Results and discussion In the following experiments, the DEGREECOX, NET-COX, LASSO and RIDGE models were applied to ovarian can- cer gene expression datasets. The experiments ran with multiple parameter values, which were selected using the same cross-validation technique as described in [14]. The selected models were then evaluated by comparing the prognostic risk of each patient in the sample, the obtained clustering in high and low risk groups based on Kaplan-Meier estimators [34] and log-rank tests. Anal- ysis of the deviance residues [35] and the concordance c-index of the selected models [36] is also presented for all combinations of datasets and methods. More formally, we introduce a network degree-based constraint to the Cox model that uses the function ϒ(β) as additional cost function: ϒ(β) = p i=1 β2 i dii = β′Dβ. (13) (13) Datasets and networks The three datasets used in these experiments, here- after named Bonome, TCGA and Tothill, are publicly available from three independent ovarian cancer studies [20–22]. All three contain gene expression data and sur- vival follow-up times for each patient in the study. The where D is a diagonal matrix with D−1 ii = p j=1 sij, i.e., the inverse of the vertex weighted degree. Figure 2 illustrates this measure, that will be used in the DEGREECOX method. Fig. 2 DEGREECOX network regularizer The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Page 114 of 135 Page 114 of 135 datasets were obtained from the HG-U133A platform and the raw files were normalized using the Robust Multichip Average (RMA) preprocessing methodology. Gene Functional Networks have an edge between any pair of genes and, as a consequence, the diameter of the networks is 1, making the centrality metrics based on shortest paths or unweighted degree uninformative. In order to tackle this problem, the original networks were split into sub-networks by ranking the edges on their weight and removing them if sij was below a given thresh- old. By working with both the full network and smaller sub-networks, we can attempt to better understand their structure. g g gy The Bonome dataset comprises the follow-up time, sur- vival status and microarray gene expressions for 185 patients. The microarray data contain 12,442 gene expres- sion levels [21]. The TCGA dataset comprises the follow- up time, survival status and microarray gene expression of 517 patients and the microarray data contain 12,042 gene expression levels [20]. The Tothill dataset also comprises the follow-up time, survival status and microarray gene expression of 278 patients and 19,816 gene expression lev- els [22]. These three datasets have 6,965 genes in common that were therefore adopted for all the experiments using the Gene Co-expression Network. The same number of genes are present in the Gene Functional Network, which will be considered the benchmark to determine and con- firm the weighted degree as the best centrality measure to be used in DEGREECOX. The full network had 28,588,141 edges and was progres- sively reduced using this method, by applying a threshold that varied between 0 (full network) and 1 (fully discon- nected). Each sub-network was then studied in terms of its diameter, power law distribution and, for a ranking of the vertices, according to their degree, weighted degree, betweenness and closeness centrality measures. In Fig. Datasets and networks 3, we show how varying this threshold affects the top ranking genes for the centrality measures described and the total number of edges kept. High edge weights imply a strong connection between the corresponding genes/vertices. This is desirable for centrality measures such as the weighted degree. How- ever, for the betweenness and closeness centrality mea- sures, this would lead to more highly connected vertices having lower betweenness, since they will not be present in the shortest paths. In order to include these strongly connected vertices, the following transformation is applied in these cases: Two criteria for selecting the best centrality measure are evaluated: 1) observing which metric better overlaps the top ranking genes across metrics can help identify a good candidate to test the proposed regularization method; and 2) looking into how rankings change for each metric as the number of edges is reduced should also give insight into the best candidate. For the first criterion, we take the 1,000 top-ranking genes over the studied metrics and analyse their overlap. While the weighted degree and closeness have 90 % of common genes, the betweenness overlaps with less than 45 % of either the closeness or weighted degree. We can assess that the weighted degree and closeness hold similar information as they value vertices that are well connected in the network, locally for the first one and globally for the latter. It is interesting how a local measure such as the degree of a vertex gives similar results as when using a global measure as is the closeness. s′ ij = log 1 sij . (15) (15) Centrality measures evaluation In order to choose the most adequate centrality mea- sure for the regularization, several tests where performed regarding the topological and connectivity properties of each network. The Gene Co-expression Networks and Fig. 3 Comparison of the fraction of top-ranked genes calculated for starting networks for the centrality metrics analized: a weighted degree, b betweenness and c closeness. Sub-network properties obtained by removing edges from the starting network Fig. 3 Comparison of the fraction of top-ranked genes calculated for starting networks for the centrality metrics analized: a weighted degree, b betweenness and c closeness. Sub-network properties obtained by removing edges from the starting network The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Page 115 of 135 Page 115 of 135 The second criterion is studied in Fig. 3, which denotes the percentage of top-ranking genes that are kept with dif- ferent measures as edges are being removed. A ranking of the top 200 genes was calculated for all sub-networks (represented in the x-axis). Each line denotes a differ- ent starting network and shows the fraction of the top- ranked genes that are kept as edges are removed. The data shown in Fig. 3 indicate that the betweenness cen- trality does not perform well with the full graph or big sub-networks as the overlap deteriorates quite fast. On the other hand, weighted degree and closeness show that the top-ranking genes are mostly kept while removing edges, until reaching a critical point near the sub-network with 1,000 edges. between the log-likelihood (Eq. 6) for each individual in the dataset using the global inferred model ( ˆβ) and a saturated, or full model, ( ˙β). The saturated model is a per- fect fit for the data, as the β coefficients are allowed to be different for each individual. This residual is centered in zero and can be regarded as the generalization of the residual sum of squares [37]: resDeviance = −2 log(l( ˆβ)) −log(l( ˙β)). (17) (17) The concordance c-index [38] is a relative measure that will assess all permissible pairs of individuals in the sam- ple and compare if their survival time is in line with the hazard relative risk. Pairs where both individuals are cen- sored or when only one is censored and has a shorter time than the uncensored are not considered valid. Performance evaluation of the Cox models With the best candidate metric selected, experiments were carried out with DEGREECOX using the weighted degree of the network and compared against three exist- ing models: NET-COX, LASSO and RIDGE. The latter two are sub-cases of the elastic net with regularization parameters α = 1 and α = 0, respectively. The other parameters for the models were selected using five-fold cross-validation, following the same procedure previously used [14]. The Kaplan-Meier estimator [34] is a non-parametric method that estimates the survival function, providing information, at any time point in the data, about the frac- tion of individuals where the event did not occur. It allows for right censored data and, when calculated for two dif- ferent groups, we use the log-rank test [39] to compare survival distributions. In the cross-validation procedure, the dataset is parti- tioned in 5 different folds, where four of them are used in model training to find the model’s coefficients ( ˆβ(−i) λα ) and the i-th set is left out. This procedure is performed 5 times for each (λ, α) parameter combination, or (λ), depending on the model. The test itself will determine the parame- ters that best fit the training data and perform best to new unseen data. This is done by maximizing the partial like- lihood (pl) between the full dataset (X) and the pl of the test set (X(−i)). In order to test the predictability of the models the fol- lowing procedure was used: find the best parameters for a training dataset using 5-fold cross validation and then test on the same dataset and 2 others. For example train a model with Bonome to test with the TCGA and Tothill dataset. The results obtained are summarized in Tables 1 and 2 to assess the generalization capability of the methods with new data and how it fits with the training set. We observe that DEGREECOX, NET-COX and RIDGE regression perform very similarly across all three evalua- tion measurements. Regarding the deviances, as measured by RSME, we can conclude that network information improves the results in all the datasets except for TCGA tested on TCGA, where RIDGE achieves lower deviances. For the Bonome and Tothill datasets, DEGREECOX has the best results. When using cross-testing, NET-COX has the best results for the Bonome and Tothill datasets and DEGREECOX for the TCGA dataset. Centrality measures evaluation The algo- rithm increases a concordance count by 1 with every pair that is in one of three cases: (a) individual with higher risk has shorter survival time; (b) hazard risks and survival time are the same; (c) one individual is censored and has a lower risk. Otherwise the count is increased by 0.5. The c-index is calculated by dividing the count by the number of permissible pairs [38]. Combining all information, we decided to choose weighted degree as the network-based regularizer to be used (DEGREECOX). It combines local and global informa- tion on the network due to its similarity with the closeness measure. The degree is more robust and predictable on the impact of edge removal as well as it is cheaper to compute. Performance evaluation of the Cox models NET-COX determines a very good model using the Tothill dataset as train- ing, but then alternates with RIDGE and DEGREECOX CV(λ, α) = 5 i=1 pl X, ˆβ(−i) λα −pl X(−i), ˆβ(−i) λα . (16) (16) Three different analytical methods were used to eval- uate the models: the root mean squared error (RMSE); the concordance index (c-index); and the Kaplan-Meier estimator. The residuals used to calculate the RMSE were the deviance residuals [37], that calculate the difference Page 116 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Table 1 Deviance and C-index results for models chosen by 5-fold cross-validation and tested on all 3 datasets (including 2 that were hidden from the training phase). The LASSO and RIDGE methods do not use network information so the values for GCN and GFM are the same, they are only shown in both networks when they are better than DEGREECOX and NET-COX Train Bonome TCGA Tothill Test Bonome TCGA Tothill Bonome TCGA Tothill Bonome TCGA Tothill Network GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM RMSE DEGREECOX 0.5581 0.7724 1.3189 1.1538 1.2139 1.1027 1.2367 1.3619 0.9201 0.8043 1.0573 1.1083 1.6326 1.2975 1.3749 1.1679 0.5116 0.7013 NET-COX 0.8131 0.8353 1.1438 1.1285 1.0992 1.0886 1.3514 1.3045 0.8361 0.8508 1.1003 1.0802 1.2917 1.2591 1.1612 1.1403 0.7363 0.7606 RIDGE 0.7807 1.1413 1.0986 1.3755 0.7215 1.1769 1.5649 1.3252 0.5432 LASSO 0.7887 1.4619 1.2586 1.7419 0.8105 1.3019 1.9595 1.4208 0.5444 C-Index DEGREECOX 0.9795 0.9401 0.6020 0.6037 0.6455 0.6494 0.6444 0.6427 0.8476 0.9089 0.6711 0.6695 0.6011 0.6088 0.6100 0.6215 0.9834 0.9519 NET-COX 0.9260 0.9202 0.6079 0.6054 0.6483 0.6506 0.6416 0.6439 0.8918 0.8892 0.6633 0.6705 0.6152 0.6106 0.6244 0.6250 0.9389 0.9352 RIDGE 0.9410 0.6177 0.6569 0.6492 0.9394 0.6579 0.6000 0.5926 0.9829 LASSO 0.9309 0.5615 0.6124 0.6405 0.9043 0.6399 0.5075 0.5728 0.9784 Values in bold represent the best performing method for the dataset/network combination (per RMSE and C-Index) Page 117 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Table 2 P-values for log-rank test results for models chosen by 5-fold cross-validation and tested on all 3 datasets (including 2 that were hidden from the training phase). The log-rank tests the separation in two categories of patients, high and low risk based on the expression dataset, using the top and lower 40 % PI groups and the top and lower 50 % PI groups. Performance evaluation of the Cox models The dataset that had the worst separation was the Tothill one, as LASSO and RIDGE perform in a similar way to the other methods up until month 30, which can be seen in Fig. 5c and d. Afterwards, both curves start to con- verge to each other. This observation is coherent with the p-value results of the log-rank test in Table 2. This result in particular shows that enriching the models with network-based information can lead to better predictive models. To further evaluate how these accuracy measures vary, we assessed the distribution of the residuals for the different methods. In Fig. 4, we show a typical result obtained when applying the four studied models on the TCGA/Bonome example. This illustrates that all the resid- uals exhibit a bimodal distribution. However DEGREECOX leads to a smaller variance and LASSO presents the highest dispersion of RMSE values. p The results are slightly different when observing the concordance c-index. The results of RIDGE are consistently better than those of both NET-COX and DEGREECOX. Although the difference is small, at most of 2 %, between the models. LASSO continues to perform worse than the other models with this evaluation measure. Finally, the comparison between the methods involved the evaluation of their potential to correctly classify patients accordingly to their survival risks. This was per- formed by dividing the samples into two groups, high and low risk individuals, based on each individuals’ esti- mated hazard function and using a given (optimal) thresh- old. This value, called prognostic index (PI), is estimated, for each model, by choosing the threshold for PIn = P i=1 Xi,n · βi that leads to the lowest p-value, as assessed by the log-rank test. The results are slightly different when observing the concordance c-index. The results of RIDGE are consistently better than those of both NET-COX and DEGREECOX. Although the difference is small, at most of 2 %, between the models. LASSO continues to perform worse than the other models with this evaluation measure. When measuring the separation between two groups by assessing the p-value of the log-rank test, there is a slight improvement in the results of DEGREECOX for the 50 %- 50 % partition over the top 40 %-lower 40 % case (where 20 % of the observations are excluded), which might indi- cate a better performance in the presence of noisy infor- mation. Performance evaluation of the Cox models The LASSO and RIDGE methods do not use network information so the values for GCN and GFM are the same, they are only shown in both networks when they are better than DEGREECOX and NET-COX. The p-values when the model is tested on the same dataset used in training are always 0 and are ommited from the table Train Bonome TCGA Tothill Test TCGA Tothill Bonome Tothill Bonome TCGA Network GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM GCN GFM 40 % PI Thres. DEGREECOX 2.084E-5 2.124E-5 0.0013 4.390E-4 2.046E-4 3.990E-4 6.547E −6 5.822E-6 9.833E-5 1.757E-4 8.347E-8 3.125E-8 NET-COX 1.082E-4 2.791E-5 7.726E-4 1.514E-4 2.815E-4 1.185E-4 4.241E-5 1.432E-6 1.696E-4 2.545E-4 7.717E-9 4.503E-9 RIDGE 1.594E-6 2.537E-4 4.233E-4 1.765E-5 0.0016 1.864E-5 LASSO 0.0364 0.0048 7.436E-5 0.0036 0.5630 0.0033 50 % PI Thres. DEGREECOX 3.332E-4 5.284E-5 0.0076 0.0084 4.394E-4 0.0090 5.781E-5 1.309E-4 0.0045 4.302E-4 5.264E-7 7.183E-7 NET-COX 2.169E-5 5.086E-5 0.0170 0.0179 0.0036 0.0015 1.247E-4 3.126E-4 0.0026 8.138E-4 1.105E-8 1.632E-7 RIDGE 1.795E-5 0.0013 3.193E-4 0.0029 0.0050 3.499E-5 LASSO 0.0720 0.0048 0.0022 0.0193 0.6464 0.0050 Values in bold represent the best method for the dataset/network combination (per 40 % and 50 % separation) Page 118 of 135 Page 118 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 on the other datasets. Such similar results are expected, as both NET-COX and DEGREECOX use the same addi- tional information, namely the GCN and GFM net- works. The small difference in the results could be explained by how the networks are being used. While NET-COX takes the weighted edges of every two genes, DEGREECOX takes the sum for every vertex losing some detail in the process. However, this does not seem relevant as the difference in the deviance is not significant. high-risk group. The results obtained by using a 50 %-50 % stratification are also reported since they correspond to a less favorable partition of the patients, by including those with intermediate risks. Then, the Kaplan-Meier curves are estimated (Fig. 5) and log-rank tests performed, all available as Additional file 1. The analysis was done for each model and shows that when testing with the Bonome and TCGA datasets, there is a statistically significant difference between the sur- vival functions of the two groups across all models. Performance evaluation of the Cox models This will be further explored in the future. For the 50 %-50 % partition and considering the log-rank tests, RIDGE regression achieves the lowest p-values in half of the tests. Comparing the methods that use network infor- mation in this experimental setting, DEGREECOX achieves better results than NET-COX for the majority of the com- binations (except for Tothill training and testing on in the TCGA). Finally, the comparison between the methods involved the evaluation of their potential to correctly classify patients accordingly to their survival risks. This was per- formed by dividing the samples into two groups, high and low risk individuals, based on each individuals’ esti- mated hazard function and using a given (optimal) thresh- old. This value, called prognostic index (PI), is estimated, for each model, by choosing the threshold for PIn = P i=1 Xi,n · βi that leads to the lowest p-value, as assessed by the log-rank test. The separation of high and low risk patients is sta- tistically significant although it could be improved by adding as variables to the model physiological character- istics, such as tumour stage, age groups, ethnicities or gender. These are not currently included, as proposing a new classifier is out of the scope of the present work, that instead, introduces a new regularization model that requires further research. We stratified the patients as in the NET-COX proposal (Zhang et al. 2013), by assigning those with the lowest 40 % PI to the low-risk group, and the top 40 % PI to the −4 −2 0 2 4 0.0 0.2 0.4 0.6 0.8 Residuals (N = 185) Density DegreeCox rmse = 1.2367083 NetCox rmse = 1.3513747 Ridge rmse = 1.3755335 Lasso rmse = 1.7419194 Fig. 4 Residuals when models are trained with the correlation network and TCGA dataset and tested with the Bonome dataset −4 −2 0 2 4 0.0 0.2 0.4 0.6 0.8 Residuals (N = 185) Density DegreeCox rmse = 1.2367083 NetCox rmse = 1.3513747 Ridge rmse = 1.3755335 Lasso rmse = 1.7419194 Fig. 4 Residuals when models are trained with the correlation network and TCGA dataset and tested with the Bonome dataset The results obtained in this study for the NET- COX model are comparable with those of the origi- nal paper [14] using all the genes (see the Additional file 1). Performance evaluation of the Cox models The obtained p-value results are of the same order of magnitude between both experiments, with the small differences being explained by differences in the pre-processing. Although none of the methods seems to perform better in all situations, we can conclude that including network information does not deteriorate the accuracy and can provide better interpretability of the obtained Cox sur- vival models, which will be further explored in future work. es dua s ( 85) Fig. 4 Residuals when models are trained with the correlation network and TCGA dataset and tested with the Bonome dataset ( ) Fig. 4 Residuals when models are trained with the correlation network and TCGA dataset and tested with the Bonome dataset The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Page 119 of 135 Fig. 5 Kaplan-Meier curves for high vs. low risk groups with the model learnt from the TCGA dataset and tested on Bonome (a and b) and Tothill (c and d). When a death event occurs for an individual, the cumulative survival decreases Fig. 5 Kaplan-Meier curves for high vs. low risk groups with the model learnt from the TCGA dataset and tested on Bonome (a and b) and Tothill (c and d). When a death event occurs for an individual, the cumulative survival decreases ig. 5 Kaplan-Meier curves for high vs. low risk groups with the model learnt from the TCGA dataset and tested on Bonom c and d). When a death event occurs for an individual, the cumulative survival decreases Conclusions We proposed DEGREECOX, a novel method to estimate survival models using network-based regularization. The results show that DEGREECOX consistently performs as well as NET-COX and RIDGE in all scenarios and with better results against LASSO. The evaluation was per- formed using deviance residuals and the log-rank test of the Kaplan-Meier estimator for two different groups, high risk and low risk individuals, and this is somewhat expected as all three methods are based on the same norm. Additional file 1: Kaplan Meier curves and log-rank tests. A PDF file that includes figures of Kaplan-Meier curves and log-rank tests obtained for all the combinations of the three datasets (Bonome, TCGA, Tothill) that are described in the manuscript. (PDF 2682 kb) Declarations This article has been published as part of BMC Bioinformatics Volume 17 Supplement 16, 2016: Proceedings of the Tenth International Workshop on Machine Learning in Systems Biology (MLSB 2016). The full contents of the supplement are available online at http://bmcbioinformatics.biomedcentral. com/articles/supplements/volume-17-supplement-16. These methods show promising results, and possible extensions can include more topological and network measures. 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Funding This work was partially supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 633974 (SOUND project), and FCT, through IDMEC, under LAETA, projects UID/EMS/ 50022/2013 and PERSEIDS (PTDC/EMS-SIS/0642/2014). SV acknowledges support by Program Investigador FCT (IF/00653/2012) from FCT, co-funded by the European Social Fund (ESF) through the Operational Program Human Potential (POPH). AV acknowledges support from FCT (SFRH/BD/97415/2013). Page 120 of 135 Page 120 of 135 The Author(s) BMC Bioinformatics 2016, 17(Suppl 16):449 Published: 13 December 2016 19. Bach F, Jenatton R, Mairal J, Obozinski G. Structured sparsity through convex optimization. Stat Sci. 2012;27(4):450–68. http://projecteuclid.org/ euclid.ss/1356098550. Availability of data and materials 13. Zhang JX, Song W, Chen ZH, Wei JH, Liao YJ, Lei J, Hu M, Chen GZ, Liao B, Lu J, Zhao HW, Chen W, He YL, Wang HY, Xie D, Luo JH. 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https://openalex.org/W2793299928	https://figshare.com/articles/journal_contribution/Circulating_tumour_DNA_analyses_reveal_novel_resistance_mechanisms_to_CDK_inhibition_in_metastatic_breast_cancer/12482324/1/files/23146922.pdf	English	null	Circulating tumour DNA analyses reveal novel resistance mechanisms to CDK inhibition in metastatic breast cancer	Annals of oncology	2,018	cc-by	3,030	Circulating tumour DNA analyses reveal novel resistance mechanisms to CDK inhibition in metastatic breast cancer recommendations, not all patients with HRþ breast cancer respond to CDK inhibition and a signiﬁcant fraction progress within 2 years of initiation of treatment [1–3]. This underscores the need to identify mechanism of resistance to these targeted therapies to anticipate and target novel or subclonal resistance mechanisms driving breast cancer progression in these patients. Cyclin-dependent kinase (CDK) 4/6 inhibition has been demon- strated to improve progression-free survival (PFS) in patients with human epidermal growth factor receptor 2 (HER2), hor- mone receptor positive (HRþ) in advanced breast cancer [1–3]. Palbociclib, ribociclib and abemaciclib are orally bioavailable selective CDK 4/6 inhibitors. These small molecules likely bind the ATP-binding pocket within the CDK4/6 protein kinases thereby inhibiting phosphorylation of retinoblastoma tumour suppressor protein (Rb). In its hypophosphorylated state Rb remains bound to E2F thereby preventing progression through the G1-S-cell cycle checkpoint [4]. The mechanism behind the observed efﬁcacy of CDK inhibition in metastatic breast cancer may relate to a dependence of HRþ breast cancer on CDK4/6 activity to override Rb mediated repression of cell cycle progres- sion (Figure 1) [5]. Circulating tumour DNA (ctDNA) describes molecules of cell-free DNA circulating in plasma that originate from a patient’s tumour. ctDNA analyses by next-generation sequenc- ing are demonstrating translational utility within clinical con- texts ranging from non-invasive screening [8], tracking cancer burden and identifying residual disease in patients undergoing treatment of their disease [9–11] and identifying cancer associ- ated mutations with therapeutic implications [12, 13]. In this edition of Annals of Oncology Condorelli et al. [14] leverage the ability of ctDNA analysis to interrogate the mutational land- scape of progressive metastatic cancer to highlight loss of Rb function as a potential resistance mechanism to CDK4/6 inhibition. They provide a case-series of three patients treated at different institutions, by separate investigators, who developed progressive metastatic breast cancer following treatment with CDK4/6 inhibitors. In each case evidence of somatic alteration involving the RB1 gene was noted through plasma ctDNA analy- ses at the point of disease progression. In the ﬁrst patient a fra- meshift event involving exon 8 of RB1 was observed that was predicted to result in a non-functioning truncated version of the protein. This event was not observed through NGS analysis of a liver biopsy acquired before CDK4/6 inhibition. In the second patient of the case-series four RB1 alterations were noted at progression on palbociclib that were not detectable before initia- tion of therapy. References 1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144(5): 646–674. 10. Galon J, Mlecnik B, Marliot F et al. Validation of the immunoscore (IM) as a prognostic marker in stage I/II/III colon cancer: results of a world- wide consortium-based analysis of 1,336 patients. J Clin Oncol 2016; 34(suppl): 3500. 2. McAllister SS, Weinberg RA. 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The relationship between tumour stroma percentage, the tumour microenvironment and survival in patients with primary operable colorectal cancer. Ann Oncol 2014; 25(3): 644–651. 13. Hynes SO, Coleman HG, Kelly PJ et al. Back to the future: routine morphological assessment of the tumour microenvironment is prognos- tic in stage II/III colon cancer in a large population-based study. Histopathology 2017; 71(1): 12–26. 6. Danielsen HE, Hveem TS, Domingo E et al. Prognostic markers for col- orectal cancer: estimating ploidy and stroma. Ann Oncol 2018; 29(3): 616–623. 14. Le DT, Uram JN, Wang H et al. PD-1 blockade in tumors with mismatch-repair deﬁciency. N Engl J Med 2015; 372(26): 2509–2520. 15. Roseweir AK, McMillan DC, Horgan PG, Edwards J. Colorectal cancer subtypes: translation to routine clinical pathology. Cancer Treat Rev 2017; 57: 1–7. 7. Dekker TJ, van de Velde CJ, van Pelt GW et al. Prognostic signiﬁcance of the tumor-stroma ratio: validation study in node-negative premenopausal breast cancer patients from the EORTC perioperative chemotherapy (POP) trial (10854). Breast Cancer Res Treat 2013; 139(2): 371–379. 8. Courrech Staal EF, Wouters MW, van Sandick JW et al. Annals of Oncology 9. Roxburgh CSD, McMillan DC. The role of the in situ local inﬂammatory response in predicting recurrence and survival in patients with primary operable colorectal cancer. Cancer Treat Rev 2012; 38(5): 451–466. Annals of Oncology Annals of Oncology References The stromal part of adenocarcinomas of the oesophagus: does it conceal targets for ther- apy? Eur J Cancer 2010; 46(4): 720–728. V C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/annonc/article-abstract/29/3/535/4815740 by Francis Crick Institute user on 12 April 2018 V C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// reproduction in any medium, provided the original work is properly cited. Circulating tumour DNA analyses reveal novel resistance mechanisms to CDK inhibition in metastatic breast cancer Transcriptional activation of cyclin-D1 (CCND1) through the estrogen receptor (ESR1), promotes dimerization of CCND1 and CDK4, and CCND1 and CDK6, escaping inhibition by p16. The cyclin-D/CDK complex phosphorylates Rb, releasing E2F to promote cell cycle pro- gression through transcriptional activation of S-phase and G2/M gene sets. Additional transcriptional activation through E2F induction may affect genes involved in DNA methylation and PD-L1 expression. Pharmacological inhibition of ER and CDK4/6 synergistically affects downstream activation of E2F and inhibits cell cycle progression in the context of wild-type Rb. Mutational inactivation of Rb promotes therapeutic resistance. plasma at progression (Chr13(GRCh37): g.48937094G>A) has been previously shown in lung cancer to result in loss of the Rb protein region responsible for the binding of Rb to E2F- transcription factor complexes [15]. The ﬁnal patient was observed to have a p.His483Tyr RB1 variant following ribociclib that is pre- dicted to be deleterious. There are however caveats to consider regarding this case- series. The number of patients described within the manuscript is small and there is no indication as to the frequency by which Rb1 alterations are detected at progression on CDK4/6 inhibition in this patient population. Additionally, patients 1 and 3 in the case-series were treated with two lines of therapy in between the biopsies showing lack of RB1 alterations and ctDNA analyses demonstrating acquired RB1 alterations—patient 1 received everolimus and exemstane before palbociclib and patient 2 received capecitabine and paclitaxel following ribociclib. Therefore, we cannot be certain that the acquisition of Rb1 altera- tions solely associate with selective pressure induced by CDK4/6 inhibition. Advancing the ﬁndings reported in this case-series will require a larger cohort to determine the incidence of Rb1 alterations as resistance mechanisms in patients with metastatic breast cancer on CDK4/6 inhibitors. Furthermore, more frequent ctDNA monitoring is necessary to follow the dynamics by which RB1 alterations emerge and ascertain the association of their emergence with disease progression. This study is of interest for the following reasons. Firstly, it provides observational evidence of deleterious RB1 alterations potentially being selected at disease progression following intervention with CDK4/6 inhibitors in patients with metastatic breast cancer. These observations build on a previous in vivo investigation of CDK4/6 inhibitor resistance using patient- derived tumour xenograft models that suggested Rb1 inactiva- tion as a resistance mechanism to chronic CDK4/6 inhibition [16]. Downloaded from https://academic.oup.com/annonc/article-abstract/29/3/535/4815740 by Francis Crick Institute user on 12 April 2018 Circulating tumour DNA analyses reveal novel resistance mechanisms to CDK inhibition in metastatic breast cancer The variant with the highest allele frequency in CDK4/6 inhibitors have been approved by the US Food and Drug Administration (FDA) for initial endocrine therapy in post- menopausal women with metastatic or advanced HRþ/HER2 breast cancer in combination with an aromatase inhibitor and for the treatment of endocrine therapy-resistant HRþ/HER2 advanced or metastatic breast cancer in combination with Fulvesterant (a selective estrogen receptor degrader) [6]. In December 2017 the National Institute for Health and Care Excellence (NICE) has recommended CDK4/6 inhibitors in com- bination with aromatase inhibition as a ﬁrst-line option for treat- ing locally advanced or metastatic HRþ/HER2 breast cancer [7]. Despite the success of the clinical studies that led to these CDK4/6 Rb CCND1 ESR1 CDK4/6 CCND1 p16 CDKi E2F E2F Rb P P P P M G1 S G2 ERi E2F regulation of DNA methylation & PD-L1 expression? Figure 1. Cell cycle progression through E2F regulation, and the role of CDK and estrogen (ER) inhibitors. Transcriptional activation of cyclin-D1 (CCND1) through the estrogen receptor ESR1), promotes dimerization of CCND1 and CDK4, and CCND1 and CDK6, escaping inhibition by p16. The cyclin-D/CDK complex phosphorylates Rb, releasing E2F to promote cell cycle pro- gression through transcriptional activation of S-phase and G2/M gene sets. Additional transcriptional activation through E2F induction may affect genes involved in DNA methylation and PD-L1 expression. Pharmacological inhibition of ER and CDK4/6 synergistically affects downstream activation of E2F and inhibits cell cycle progression in the context of wild-type Rb. Mutational inactivation of Rb promotes therapeutic resistance. Editorials Annals of Oncology Annals of Oncology Annals of Oncology Figure 1. Cell cycle progression through E2F regulation, and the role of CDK and estrogen (ER) inhibitors. Transcriptional activation of cyclin-D1 (CCND1) through the estrogen receptor (ESR1), promotes dimerization of CCND1 and CDK4, and CCND1 and CDK6, escaping inhibition by p16. The cyclin-D/CDK complex phosphorylates Rb, releasing E2F to promote cell cycle pro- gression through transcriptional activation of S-phase and G2/M gene sets. Additional transcriptional activation through E2F induction may affect genes involved in DNA methylation and PD-L1 expression. Pharmacological inhibition of ER and CDK4/6 synergistically affects downstream activation of E2F and inhibits cell cycle progression in the context of wild-type Rb. Mutational inactivation of Rb promotes therapeutic resistance. Figure 1. Cell cycle progression through E2F regulation, and the role of CDK and estrogen (ER) inhibitors. Circulating tumour DNA analyses reveal novel resistance mechanisms to CDK inhibition in metastatic breast cancer Secondly, this study provides an early glimpse into the potential of ctDNA panels to detect acquisition of actionable alterations in patients who experience disease progression on anticancer therapy. Such a resource could inform mechanisms underlying resistance across a range of systemic therapies. There are advantages to ctDNA analyses as a research tool to understand the biology of heavily treated metastatic disease. The non-invasive nature of ctDNA examination overcomes barriers to tissue acquisition in late stage disease that include poor patient health, increased risk from biopsy procedures and cost. Given this work, it is notable that CDK4/6 inhibition has recently been associated with increasing tumour cell antigen presentation through a mechanism involving downregulation of Rb1-E2F induced DNA methyltransferase 1 (DNMT1) activ- ity, increased expression of endogenous retroviral elements and type III interferon production [17]. This response to CDK4/6 Volume 29 \| Issue 3 \| 2018 References 17. Goel S, DeCristo MJ, Watt AC et al. CDK4/6 inhibition triggers anti- tumour immunity. Nature 2017; 548(7668): 471. 1. Finn RS, Martin M, Rugo HS et al. Palbociclib and letrozole in advanced breast cancer. N Engl J Med 2016; 375(20): 1925–1936. 1. Finn RS, Martin M, Rugo HS et al. Palbociclib and letrozole in advanced breast cancer. N Engl J Med 2016; 375(20): 1925–1936. 1. Finn RS, Martin M, Rugo HS et al. Palbociclib and letrozole in advanced breast cancer. N Engl J Med 2016; 375(20): 1925–1936. 18. Zhang J, Bu X, Wang H et al. Cyclin D–CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance. Nature 2018; 553(7686): 91. 2. Finn RS, Crown JP, Lang I et al. The cyclin-dependent kinase 4/6 inhibi- tor palbociclib in combination with letrozole versus letrozole alone as ﬁrst-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol 2015; 16(1): 25–35. 2. Finn RS, Crown JP, Lang I et al. The cyclin-dependent kinase 4/6 inhibi- tor palbociclib in combination with letrozole versus letrozole alone as ﬁrst-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol 2015; 16(1): 25–35. doi:10.1093/annonc/mdy017 Published online 17 January 2018 doi:10.1093/annonc/mdy017 Published online 17 January 2018 y 3. Cristofanilli M, Turner NC, Bondarenko I et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, 3. Cristofanilli M, Turner NC, Bondarenko I et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, 3. Cristofanilli M, Turner NC, Bondarenko I et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, Disclosure 16. Herrera-Abreu MT, Palafox M, Asghar U et al. Early adaptation and acquired resistance to CDK4/6 inhibition in estrogen receptor–positive breast cancer. Cancer Res 2016; 76(8): 2301–2313. The authors have declared no conﬂicts of interest. Funding This work is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001169, FC001202), the UK Medical Research Council (FC001169, FC001202) and the Wellcome Trust (FC001169, FC001202). CS is funded by Cancer Research UK (TRACERx and CRUK Cancer Immunotherapy Catalyst Network), the CRUK Lung Cancer Centre of Excellence, Stand Up 2 Cancer (SU2C), the Rosetrees Trust, NovoNordisk Foundation (ID 16584), the Prostate Cancer Foundation, the Breast Cancer Research Foundation and the European Research Council (THESEUS), and support was pro- vided to CS by the National Institute for Health Research, the University College London Hospitals Biomedical Research Centre and the Cancer Research UK University College London Experimental Cancer Medicine Centre. 10. Garcia-Murillas I, Schiavon G, Weigelt B et al. Mutation tracking in cir- culating tumor DNA predicts relapse in early breast cancer. Sci Transl Med 2015; 7(302): 302ra133. 11. Tie J, Wang Y, Tomasetti C et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med 2016; 8(346): 346ra92. 12. Zheng D, Ye X, Zhang MZ et al. Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance. Sci Rep. 2016; 6(1): 20913. 13. Thierry AR, El Messaoudi S, Mollevi C et al. Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select meta- static colorectal patients for anti-EGFR treatment. Ann Oncol 2017; 28(9): 2149–2159. 14. Condorelli R, Spring L, O’Shaughnessy J et al. Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer. Ann Oncol 2018; 29(3): 640–645. 15. Liu J, Lee W, Jiang Z et al. Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events. Genome Res 2012; 22(12): 2315–2327. Annals of Oncology HER2-negative metastatic breast cancer that progressed on previous endo- crine therapy (PALOMA-3): ﬁnal analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol 2016; 17(4): 425–439. HER2-negative metastatic breast cancer that progressed on previous endo- crine therapy (PALOMA-3): ﬁnal analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol 2016; 17(4): 425–439. inhibition was ameliorated by silencing of RB1 and therefore could conceivably underlie an immune predatory selection pressure toward selection of Rb1 altered populations whilst undergoing treatment with CDK4/6 inhibitors. The fact that CDK4/6 inhibition has recently been shown to increase PD-L1 expression in mouse models of breast cancer provides a clear rationale for anti-PD1 treatment as a combination therapy with CDK4/6 inhibition before the emergence of Rb1 loss of function [18]. 4. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov 2015; 14(2): 130–146. 5. Thangavel C, Dean JL, Ertel A et al. Therapeutically activating RB: rees- tablishing cell cycle control in endocrine therapy-resistant breast cancer. Endocrine Relat Cancer 2011; 18(3): 333–345. 6. Walker AJ, Wedam S, Amiri-Kordestani L, Bloomquist E, Tang S, Sridhara R et al. FDA approval of palbociclib in combination with fulves- trant for the treatment of hormone receptor-positive, HER2-negative metastatic breast cancer. Clin Cancer Res 2016; 22(20): 4968–4972. C. Abbosh1, C. Swanton1,2 & N. J. Birkbak1,2* 1Cancer Research UK Lung Cancer Centre of Excellence, London and Manchester; University College London Cancer Institute, London; 2Translational Cancer Therapeutics Laboratory, The Francis Crick Institute, London, UK (*E-mail: nicolai.birkbak@crick.ac.uk) 7. National Institute for Health and Care Excellence. Palbociclib with an aro- matase inhibitor for previously untreated, hormone receptor-positive, HER2-negative, locally advanced or metastatic breast cancer. Technology Appraisal Guidance [TA495], December 2017. 8. Aravanis AM, Lee M, Klausner RD. Next-generation sequencing of circulating tumor DNA for early cancer detection. Cell 2017; 168(4): 571–574. 9. Chaudhuri AA, Chabon JJ, Lovejoy AF et al. Early detection of molecular residual disease in localized lung cancer by circulating tumor DNA proﬁling. Cancer Discov 2017; 7(12): 1394–1403. Uncovering the links between systemic hormones and oncogenic signaling in the pathogenesis of meningioma between meningioma risk and sex hormones has been of keen interest for decades, sparked by several observations. The most important of these has been the ﬁnding of estrogen receptor and progesterone receptor (PR) expression in a substantial portion of meningiomas [1, 2]. In addition, a link between meningiomas and hormones has been supported by the skewed gender distribu- tion of meningiomas [3]. Low-grade meningiomas develop two times more often in women than in men, and three times more A number of risk factors have been associated with meningioma development including radiation exposure (radiation-induced meningioma), female gender, germline mutations, high body mass index and hormone exposure (Figure 1). The relationship doi:10.1093/annonc/mdy010 \| 537
https://openalex.org/W4391117450	https://e-journallppmunsa.ac.id/index.php/jeb/article/download/1390/1348	Indonesian	null	ANALISIS PENYERAPAN TENAGA KERJA PADA SEKTOR USAHA MIKRO KECIL DAN MENENGAH DI KABUPATEN SUMBAWA	Jurnal ekonomi dan bisnis/Jurnal Ekonomi dan Bisnis	2,023	cc-by-sa	5,779	\|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jour \|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 Pp. 458 - 469 ANALISIS PENYERAPAN TENAGA KERJA PADA SEKTOR USAHA MIKRO KECIL DAN MENENGAH DI KABUPATEN SUMBAWA Suci Lestari1, Marisa Sutanty2* 12Universitas Samawa, Sumbawa Besar, Indonesia Suci Lestari1, Marisa Sutanty2* 12Universitas Samawa, Sumbawa Besar, Indonesia Penulis Korespondensi: marishasutanty@gmail.com Article Info Article History Received: 13 Desember 2023 Revised: 26 Desember 2023 Published: 31 Desember 2023 Keywords Sales Rate; Wage Rate; Labors Absorption. Abstrak This study aims to analyze labor absorption in the MSME sector in Sumbawa District. The factors thought to influence labor absorption in the MSME sector in Sumbawa District are sales rates and wage rates. The type of this study was associative. The type of data used was quantitative data in the form of sales data, wages and number of labors obtained from documen or archip recording financial reports for MSMEs in Sumbawa District which is the subject of the study. The population that is the subject of this study is all 153 milk candy MSMEs in Penyaring Village, Sumbawa District. Determining the sample in this study used a census sampling technique, which is a sampling technique when all members of the population are used as samples. Thus, the sample in this study was 153 MSMEs in Sumbawa District. All of data was studied using multiple linear regression analysis techniques, partial hypothesis testing (t test), simultaneous hypothesis testing (F test), and determinant coefficient testing (R2). The results of this study showed that the sales rates and wage rates have a positive and significant effect on labor absorption in the MSME sector in Sumbawa District, either partially or simultaneously. The ability of the sales rates and wage rates variables to influence the labor absorption in the MSME sector in Sumbawa District is 74.1%, while the remaining 25.1% is influenced by other variables not examined, such as capital, productivity level and education level of the labors. PENDAHULUAN sebesar perusahaan besar namun kontinuitas dan konsistensi dari sumbangsih UMKM terhadap pendapatan negara mengindikasikan kemampuannya menopang kondisi perekonomian untuk dapat terus berjalan berkelanjutan secara konsisten dan menjadi lapangan pekerjaan bagi tenaga kerja produktif dengan pendidikan mereka yang tidak tinggi sebagai sumber pendapatan, pengurangan pengangguran, bentuk pemerataan distribusi pendapatan (Novitasari, 2022). Berdasarkan pengalaman selama krisis yang telah terjadi Indonesia tersebut, sudah sewajarnya jika pengembangan perekonomian di sektor swasta difokuskan pada pengembangan UMKM. Secara umum, UMKM dalam perekonomian memiliki peran, antara lain sebagai pemeran utama dalam kegiatan perekonomian, penyedia lapangan kerja, pemain penting dalam pengembangan perekonomian lokal dan pemberdayaan masyarakat, pencipta pasar baru dan sumber inovasi, dan kontribusinya terhadap neraca pembayaran (Al-Farisi, et al., 2022). p y Dengan berdirinya usaha di sektor UMKM mampu menyerap jumlah angkatan kerja yang siap bekerja tetapi belum mendapat pekerjaan sehingga dapat mengurangi jumlah pengangguran. Berkembangnya partumbuhan di sektor UMKM artinya semakin terbukanya kesempatan peluang kerja bagi masyarakat. Dengan berkurangnya jumlah pengangguran berarti sektor UMKM banyak merekrut tenaga kerja yang berarti akan membantu pemerintah dalam upaya mengurangi angka kemiskinan. UMKM memegang peranan yang cukup besar dalam pembangunan perekonomian. UMKM selain berperan sebagai penggerak pertumbuhan serta pembangunan ekonomi juga mampu menyelesaikan permasalahan pengangguran karena dapat menyediakan lapangan pekerjaan yang luas sehingga mampu menyerap tenaga kerja dalam jumlah yang banyak. Oleh sebab itu, semakin baik dan banyak UMKM, maka semakin baik juga pertumbuhan ekonomi dan berkurangnya pengangguran. Hal tersebut dapat membantu dalam proses percepatan pemulihan perekonomian negara. Tenaga kerja merupakan unsur penting dalam sistem keterlaksanaan bisnis UMKM. Kebutuhan tenaga kerja didasarkan pada pemikiran bahwa tenaga kerja dalam masyarakat merupakan salah satu faktor yang potensial untuk pembangunan ekonomi secara keseluruhan, dengan demikian jumlah penduduk Indonesia yang cukup besar dapat menentukan percepatan laju pertumbuhan ekonomi. Kesempatan kerja yang tersedia dan kualitas tenaga kerja yang digunakan akan menentukan proses pembangunan ekonomi untuk menjalankan proses produksi dan juga sebagai pasar barang dan jasa. Penyerapan tenaga kerja merupakan jumlah tertentu dari tenaga kerja yang digunakan dalam suatu unit usaha tertentu atau dengan kata lain, penyerapan tenaga kerja adalah jumlah tenaga kerja yang bekerja dalam suatu unit usaha. Menurut Todaro (dalam Prasetyo dan Huda, 2019), penyerapan tenaga kerja merupakan permintaan tenaga kerja guna melaksanakan pekerjaan atau kondisi yang menggambarkan pencari kerja yang siap mengisi lapangan pekerjaan. Pandangan ini sejalan dengan Kawet et al., 2019) yang menyatakan bahwa penyerapan tenaga kerja adalah jumlah tenaga kerja yang dapat diserap oleh berbagai sektor pada suatu waktu. PENDAHULUAN Pada dasarnya, Indonesia merupakan negara dengan beranekaragam kekayaan alam yang ada. Hal tersebut mengharuskan masyarakat untuk dapat mengelola dan mengembangkan potensi atau kemampuannya sehingga mampu memanfaatkan potensi yang dimiliki untuk menghasilkan peluang usaha. Oleh karena itu, untuk mengelola potensi dan peluang tersebut dapat melalui pembentukan usaha mikro, kecil, dan menengah. Keberadaan UMKM sangat penting dalam pertumbuhan ekonomi negara serta dapat bermanfaat dan mendistribusikan pendapatan masyarakat (Aliyah, 2022). Sebutan UMKM merujuk pada kegiatan usaha yang dibentuk oleh warga negara, baik berupa usaha pribadi ataupun badan usaha. Menurut Tulus T.H. Tambunan (dalam Halim, 2020), usaha mikro, kecil, dan menengah (UMKM) adalah unit usaha yang berdiri sendiri yang dilakukan oleh perorangan atau kelompok badan usaha pada semua sektor ekonomi. Pada umumnya, perbedaan diantara usaha mikro (UMI), usaha kecil (UK), dan usaha menengah (UM) dasarnya dibedakan oleh dua hal aspek yaitu aspek pada nilai aset awal (tidak termasuk tanah dan bangunan) dan omset rata-rata per tahun atau jumlah pekerja tetap. Pengalaman ketika terjadi krisis yang terjadi dalam beberapa waktu lalu, berdampak pada banyak usaha besar mengalami perlambatan pertumbuhan bahkan berhenti aktifitasnya. Namun, sektor UMKM terbukti tetap survive dan tetap hidup dalam menghadapi krisis ekonomi dengan trend pertumbuhannya yang masih menunjukan positif. UMKM sebagai sektor yang menunjukkan mampu terus berkontribusi meski jika dilihat dari skala hasil yang disumbangkan terhadap PDB tidak 458 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 sebesar perusahaan besar namun kontinuitas dan konsistensi dari sumbangsih UMKM terhadap pendapatan negara mengindikasikan kemampuannya menopang kondisi perekonomian untuk dapat terus berjalan berkelanjutan secara konsisten dan menjadi lapangan pekerjaan bagi tenaga kerja produktif dengan pendidikan mereka yang tidak tinggi sebagai sumber pendapatan, pengurangan pengangguran, bentuk pemerataan distribusi pendapatan (Novitasari, 2022). B d k l l k i i l h j di I d i b \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 sebesar perusahaan besar namun kontinuitas dan konsistensi dari sumbangsih UMKM terhadap pendapatan negara mengindikasikan kemampuannya menopang kondisi \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jou http://e-journallppmunsa.ac.id/index.php/jeb sebesar perusahaan besar namun kontinuitas dan konsistensi dari sumbangsih UMKM terhadap pendapatan negara mengindikasikan kemampuannya menopang kondisi perekonomian untuk dapat terus berjalan berkelanjutan secara konsisten dan menjadi lapangan pekerjaan bagi tenaga kerja produktif dengan pendidikan mereka yang tidak tinggi sebagai sumber pendapatan, pengurangan pengangguran, bentuk pemerataan distribusi pendapatan (Novitasari, 2022). PENDAHULUAN Dalam kaitannya dengan penyerapan tenaga kerja, ada berbagai macam faktor yang mempengaruhi penyerapan tenaga kerja pada sektor UMKM, salah satunya adalah tingkat penjualan. Tingkat penjualan atau volume penjualan adalah hasil akhir yang dicapai perusahaan dari hasil penjualan produk yang dihasilkan oleh perusahaan tersebut. Volume penjualan tidak memisahkan secara tunai maupun kredit tetapi dihitung secara keseluruhan dari total yang dicapai. Menurut Kotler dan Amstrong (2019), tingkat penjualan sangat berpengaruh penting bagi penyerapan tenaga kerja, 459 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jou http://e-journallppmunsa.ac.id/index.php/jeb karena semakin tinggi peningkatan penjualan suatu produk, maka akan semakin banyak pula tenaga kerja yang dibutuhkan. karena semakin tinggi peningkatan penjualan suatu produk, maka akan semakin banyak pula tenaga kerja yang dibutuhkan. Hal ini sejalan dengan Menurut Swastha (2020) yang menyatakan bahwa tingkat penjualan adalah akumulasi dari kegiatan penjualan suatu produk barang-barang dan jasa yang dihitung secara keseluruhan selama kurun waktu tertentu secara terus menerus atau dalam satu proses akuntansi. Volume penjualan dapat diukur dengan dua cara, yaitu pertama, mencapai target penjualan, yang dapat diukur dengan unit produk yang terjual. Kedua, kenaikan jumlah penjualan, keuntungan yang meningkat di peroleh dari total nilai penjualan nyata perusahaan dalam suatu periode tertentu. Tingkat penjualan suatu usaha dapat mempengaruhi kebutuhan tenaga kerja, karena semakin besar tingkat penjualan, maka penerimaan perusahaan akan semakin banyak sehingga dapat berakibat pada penambahan tenaga kerja dengan tujuan menambah jumlah produksinya. Faktor lain yang dapat mempengaruhi penyerapan tenaga kerja pada sektor UMKM adalah tingkat upah. Upah yaitu pembayaran yang diperoleh karena berbagai bentuk jasa yang disediakan dan diberikan oleh tenaga kerja kepada para pengusaha. Menurut Sukirno (2017), upah adalah hak pekerja/buruh yang diterimah dan dinyatakan dalam bentuk uang sebagai imbalan dari pengusaha atau pemberi kerja kepada pekerja/buruh yang ditetapkan dan dibayar menurut suatu perjanjian kerja, kesepakatan, atau peraturan perundangan-undangan, termasuk tunjangan bagi pekerja/buruh dan keluarganya atas suatu pekerjaan dan jasa yang telah dilakuakan. Tujuan penetapan upah minimum adalah untuk meningkatkan taraf hidup pekerja sesuai dengan kebutuhan hidupnya. Upah merupakan hal yang paling utama dalam ketenagakerjaan. Pemberian upah yang adil dan setimpal akan memicu motivasi kerja yang tinggi sehingga kinerja para buruh/pekerja menjadi lebih baik dan tentunya pengaruh terhadap pendapatan perusahaan. Upah merupakan upaya yang dilakukan untuk memberi jaminan terpenuhinya kebutuhan hidup pekerja. Jika nilai upah yang ditawarkan oleh suatu perusahaan dinilai tidak mencukupi untuk memenuhi kebutuhan hidup pekerja, maka pekerja tersebut akan menolak pekerjaan yang ditawarkan (Junrillah, et al., 2021). PENDAHULUAN Kenaikan upah memang dapat meningkatkan kesempatan kerja melalui peningkatan konsumsi, namun dalam beberapa kasus kenaikan upah justru berdampak negatif terhadap penyerapan tenaga kerja itu sendiri. Tingginya tingkat upah yang ditetapkan kemungkinan besar akan menyebabkan pengusaha mengurangi penggunaan tenaga kerja sehingga akan berdampak pada berkurangnya penyerapan tenaga kerja. Hal ini sesuai dengan Mankiw (dalam Surianto, et al., 2021) yang menyatakan bahwa Upah tenaga kerja, bagi perusahaan merupakan biaya produksi sehingga dengan meningkatnya upah tenaga kerja akan mengurangi keuntungan perusahaan. Pada umumnya, perusahaan akan mengambil kebijakan pengurangan tenaga kerja guna mengurangi biaya produksi sehingga akan berdampak pada berkurangnya tenaga kerja yang dibutuhkan. Desa Penyaring yang menajdi lokasi dilaksanakannya penelitian ini adalah salah satu daerah di Kabupaten Sumbawa yang memiliki potensi dan kekayaan alam yang cukup melimpah, salah satunya adalah susu sapi dan kerbau. Susu sapi dan kerbau tersebut banyak dimanfaatkan oleh masyarakat sebagai bahan dasar untuk membuat produk olahan permen susu. Seiring pemasaran permen susu yang semakin luas, maka permintaan atas permen susu juga semakin meningkat. Dampaknya, para pengusaha permen susu dituntut agar dapat meningkatkan volume produksinya untuk memenuhi permintaan pasar sehingga kebutuhan terhadap faktor produksi semakin meningkat, salah satunya adalah tenaga kerja. 460 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jou Penyerapan tenaga kerja pada UMKM permen susu di Desa Penyaring memiliki dua faktor yang paling berpengaruh, yaitu tingkat penjualan dan tingkat upah. Tingkat penjualan permen susu di Desa Penyaring mengalami peningkatan sejak beberapa waktu yang lalu, hal ini dapat dilihat dari permintaan permen susu yang sudah menjangkau hingga keluar kota Sumbawa. Tingginya permintaan permen susu ini menyebabkan para pedagang menambah jumlah produksinya, sehingga secara otomatis kebutuhan akan tenaga kerja produksi juga bertambah. Begitu juga dengan tingkat upah tenaga kerja yang meningkat akan membawa dampak yang signifikan pada penyerapan tenaga kerja. Dengan adanya tingkat upah yang bertambah akan memicu motivasi kerja yang tinggi sehingga dapat meningkatkan partisipasi kerja. Peran usaha mikro, kecil dan menengah (UMKM) memiliki arti yang begitu penting bagi suatu daerah terutama sebagai salah satu penggerak pertumbuhan ekonomi daerah dan penyedia lapangan kerjas sehingga keberadaan UMKM dipandang sangat penting guna meningkatkan kesempatan kerja sebagai upaya untuk mengurangi angka pengangguran dan kemiskinan di daerah Kabupaten Sumbawa. PENDAHULUAN Oleh karena itu, peneliti tertarik untuk melakukan analisis lebih mendalam menggunakan faktor tingkat penjualan dan tingkat upah sehingga nantinya dapat diketahui apakah variabel tersebut berpengaruh atau tidak terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. METODE PENELITIAN Desain Penelitian Penelitian ini adalah penelitian asosiatif. Penelitian asosiatif menurut Darmanah (2019) merupakan penelitian yang bertujuan untuk mengetahui hubungan antara dua variabel atau lebih. Pada penelitian ini, jenis penelitian asosiatif dilakukan untuk menganalisis penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. Adapun faktor-faktor yang diduga mempengaruhi penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa adalah tingkat penjualan dan tingkat upah. Berdasarkan tujuan tersebut, maka kerangka pemikiran penelitian ini dapat digambarkan sebagai berikut. Gambar 1. Alur Penelitian. Tingkat Penjualan (X1) Tingkat Upah (X2) Penyerapan Tenaga Kerja (Y) Gambar 1. Alur Penelitian. http://e-journallppmunsa.ac.id/index.php/jeb Teknik Analisis Data Penelitian ini dilakukan dengan tujuan untuk menganalisis faktor-faktor yang mempengaruhi penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa yang terdiri atas tingkat penjualan dan tingkat upah. Seluruh data yang telah dikumpulkan akan diolah dengan menggunakan bantuan program SPSS untuk dikaji menggunakan teknik analisis regresi linier berganda, uji hipotesis parsial (uji t), uji hipotesis simultan (uji F), dan uji koefisien determinan (R2) (Ghozali, 2021). Populasi dan Sampel p p Populasi merupakan keseluruhan anggota, kejadian, atau objek yang memiliki karakteristik tertentu yang telah ditetapkan sebagai subjek penelitian. Menurut Arikunto (2019), populasi adalah keseluruhan subjek penelitian. Berdasarkan definisi tersebut, maka populasi yang menjadi subjek pada penelitian ini adalah seluruh UMKM permen susu yang berada di Desa Penyaring Kabupaten Sumbawa yang berjumlah 153 unit. Sedangkan sampel adalah bagian kecil dari anggota populasi yang diambil menurut prosedur tertentu sehingga dapat mewakili populasinya. Arikunto (2019) menyatakan bahwa sampel adalah sebagian atau wakil dari populasi yang diteliti. Apabila peneliti ingin meneliti semua elemennya yang ada dalam wilayah penelitian maka penelitiannya merupakan penelitian populasi. Sesuai dengan definisi tersebut, maka seluruh anggota populasi akan dijadikan sebagai sampel sehingga penelitiannya merupakan penelitian populasi. Penentuan sampel pada penelitian ini menggunakan teknik sensus sampling (sampel jenuh), yaitu teknik penentuan sampel bila semua anggota populasi digunakan sebagai sampel. Dengan demikian, maka sampel pada penelitian ini adalah seluruh UMKM permen susu yang berada di Desa Penyaring Kabupaten Sumbawa yang berjumlah 153 unit. Teknik Pengumpulan Data Dalam penelitian, peneliti menggunakan teknik dokumentasi untuk pengumpulan data sekunder. Menurut Nawawi (2019), teknik dokumentasi adalah cara mengumpulkan data yang dilakukan dengan kategorisasi dan klasifikasi bahan-bahan tertulis yang berhubungan dengan masalah penelitian, baik sumber dari dokumen maupun buku-buku, koran, majalah dan lain-lain. Berdasarkan pengertian tersebut, maka dapat dikatakan bahwa teknik dokumentasi merupakan teknik pengumpulan data yang membutuhkan dokumen-dokumen yang berkaitan dengan objek penelitian untuk dianalisis. Adapun dokumen-dokumen yang dikumpulkan pada penelitian ini merupakan data penjualan, upah dan jumlah tenaga kerja yang diperoleh dari dokumen atau arsip pencatatan laporan keuangan UMKM di Kabupaten Sumbawa. HASIL DAN PEMBAHASAN 1. Analisis Regresi Linear Berganda Jenis dan Sumber Data Jenis data yang digunakan dalam penelitian ini adalah data kuantitatif. Menurut Siregar (2017), data kuantitatif adalah adalah data yang berupa angka. Sesuai dengan bentuknya, data kuantitatif dapat diolah atau dianalisa dengan menggunakan teknik perhitungan stastik. Adapun data kuantitatif yang digunakan pada penelitian ini adalah data penjualan, upah dan jumlah tenaga kerja. Data yang digunakan dalam penelitian ini diperoleh dari sumber sekunder, yaitu sumber yang tidak langsung memberikan data kepada pengumpulan data. Data sekunder didapatkan dari sumber yang sudah ada, antara lain dari dokumentasi dan literatur (Sugiyono, 2021). Dalam penelitian ini, data sekunder yang digunakan adalah data penjualan, upah dan jumlah tenaga kerja yang diperoleh dari dokumen atau arsip pencatatan laporan keuangan UMKM di Kabupaten Sumbawa. 461 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jour 1. Analisis Regresi Linear Berganda Menurut (Sujarweni, 2020) menyebutkan bahwa analisis regresi linear berganda adalah regresi yang memiliki satu variabel dependen dan lebih dari satu variabel independent. Analisis regresi linear berganda digunakan untuk melihat pengaruh signifikan variabel independen terhadap variabel dependen dengan cara membuat persamaan garis regresi linear berganda. Pada penelitian ini, analisis regresi linear berganda digunakan untuk mengkaji pengaruh variabel independent, yaitu tingkat penjualan (X1) dan tingkat upah (X2) terhadap variabel dependen adalah penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y). 462 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jou Berdasarkan hasil pengujian yang telah dilakukan dengan bantuan aplikasi SPSS, diperoleh nilai koefisien regresi yang disajikan pada tabel berikut. Tabel 1. Hasil Pengujian Regresi Linear Berganda Coefficientsa Tabel 1. Hasil Pengujian Regresi Linear Berganda Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 1.112 .245 4.546 .000 Tingkat Penjualan .092 .022 .900 4.182 .000 Tingkat Upah .050 .010 .596 2.770 .006 a. Dependent Variable: Penyerapan Tenaga Kerja Sumber: Output SPSS (data sekunder diolah), 2023. Berdasarkan tabel tersebut, maka dapat dirumuskan persamaan regresi linear berganda sebagai berikut: Y = a + β1X1 + β2X2 + e Y = 1.112 + 0.092 (X1) + 0.050 (X2) + e Y = a + β1X1 + β2X2 + e Y = 1.112 + 0.092 (X1) + 0.050 (X2) + e Y = a + β1X1 + β2X2 + e Y = 1.112 + 0.092 (X1) + 0.050 (X2) + e Keterangan: Y = Penyerapan Tenaga Kerja (Variabel Dependent) β = Koefisien Regresi Variabel Independent β = Koefisien Regresi Variabel Independent X1 = Tingkat Penjualan (Variabel Independent 1) X1 = Tingkat Penjualan (Variabel Independent 1) = Tingkat Upah (Variabel Independent 2) e = Error term (faktor pengganggu) atau residu (5%). Persamaan regresi linear berganda tersebut dapat diinterpretasikan sebagai berikut: a. Nilai constanta (a) sebesar 1.112, hal ini menunjukan bahwa apabila nilai variabel-variabel independent, yaitu tingkat penjualan (X1) dan tingkat upah (X2) adalah konstan (0), maka nilai konsisten variabel dependent penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y) adalah sebesar 1.112. a. Nilai constanta (a) sebesar 1.112, hal ini menunjukan bahwa apabila nilai variabel-variabel independent, yaitu tingkat penjualan (X1) dan tingkat upah (X2) adalah konstan (0), maka nilai konsisten variabel dependent penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y) adalah sebesar 1.112. j p p ( ) b. 1. Analisis Regresi Linear Berganda β1 nilai koefisien regresi variabel tingkat penjualan (X1) adalah sebesar 0.092 dan bernilai positif. Nilai positif menunjukkan hubungan yang searah atau selaras yang berarti bahwa apabila nilai variabel tingkat penjualan (X1) mengalami kenaikan sebesar satu satuan, maka nilai variabel penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y) juga akan mengalami peningkatan sebesar 0.092. Demikian pula sebaliknya, dengan asumsi variabel independent lainnya, yaitu tingkat upah (X2) bernilai konstan (0). c. β2 nilai koefisien regresi variabel tingkat upah (X2) adalah sebesar 0.050 dan bernilai positif. Nilai positif menunjukkan hubungan yang searah atau selaras yang berarti bahwa apabila nilai variabel tingkat upah (X2) mengalami kenaikan sebesar satu satuan, maka nilai variabel penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y) juga akan mengalami peningkatan sebesar 0.050. Demikian pula sebaliknya, dengan asumsi variabel independent lainnya, yaitu tingkat penjualan (X1) bernilai konstan (0). 463 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jou http://e-journallppmunsa.ac.id/index.php/jeb 2. Uji Hipotesis Parsial (Uji-t) Menurut Sujarweni (2020), uji parsial atau uji-t adalah pengujian yang dilakukan untuk mengetahui hubungan variabel bebas terhadap variabel terikat secara parsial. Uji-t dapat dilakukan dengan membandingkan nilai t hasil perhitungan atau thitung dengan nilai ttabel pada taraf signifikan 5%. Variabel bebas dikatakan memiliki pengaruh terhadap variabel terikat secara parsial, jika nilai thitung lebih besar dari pada nilai ttabel dan nilai probabilitas yang dihasilkan lebih kecil dari 0.05. Uji-t pada penelitian ini digunakan untuk melihat signifikan pengaruh masing- masing variabel independent, yaitu tingkat penjualan (X1) dan tingkat upah (X2) terhadap variabel dependen penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y). Berdasarkan pengujian hipotesis yang telah dilakukan, diperoleh hasil yang disajikan pada tabel berikut. Tabel 2. Hasil Pengujian Hipotesis Parsial (Uji-t) Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 1.112 .245 4.546 .000 Tingkat Penjualan .092 .022 .900 4.182 .000 Tingkat Upah .050 .010 .596 2.770 .006 a. Dependent Variable: Penyerapan Tenaga Kerja Sumber: Output SPSS (data sekunder diolah), 2023. Tabel 2. Hasil Pengujian Hipotesis Parsial (Uji-t) Coefficientsa Berdasarkan hasil pengujian hipotesis parsial (uji-t) yang disajikan pada tabel di atas, maka dapat ditarik kesimpulan sebagai berikut: a. Pengaruh Tingkat Penjualan Terhadap Penyerapan Tenaga Kerja Berdasarkan hasil pengujian pengaruh tingkat penjualan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa, diperoleh nilai thitung sebesar 4.182 dan nilai ttabel pada derajat kebebasan (df=n-k=153- 3=150) dan α = 5% (0,05) adalah sebesar 1.976, sehingga nilai thitung lebih besar dari pada nilai ttabel (4.182>1.976) dan nilai signifikansi yang dihasilkan adalah sebesar 0.000 lebih kecil dari 0.05 (0.000<0.05). Hal ini berarti bahwa tingkat penjualan secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. b. Pengaruh Tingkat Upah Terhadap Penyerapan Tenaga Kerja Berdasarkan hasil pengujian pengaruh tingkat upah terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa, diperoleh nilai thitung sebesar 2.770 dan nilai ttabel pada derajat kebebasan (df=n-k=153-3=150) dan α = 5% (0.05) adalah sebesar 1.976, sehingga nilai thitung lebih besar dari pada nilai ttabel (2.770>1.976) dan nilai signifikansi yang dihasilkan adalah sebesar 0.006 lebih kecil dari 0.05 (0.006<0.05). Hal ini berarti bahwa tingkat upah secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 3. Uji Hipotesis Simultan (Uji F) 3. Uji Hipotesis Simultan (Uji F) Uji simultan atau uji F dalam analisis regresi linear berganda adalah pengujian signifikansi persamaan yang digunakan untuk mengetahui seberapa besar pengaruh variabel-variabel bebas secara bersama-sama terhadap variabel terikat. Uji F dapat 3. Uji Hipotesis Simultan (Uji F) Uji simultan atau uji F dalam analisis regresi linear berganda adalah pengujian signifikansi persamaan yang digunakan untuk mengetahui seberapa besar pengaruh variabel-variabel bebas secara bersama-sama terhadap variabel terikat. Uji F dapat 464 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 dilakukan dengan membandingkan nilai F hasil perhitungan atau Fhitung dengan nilai Ftabel pada taraf signifikan 5%. Variabel-variabel bebas dikatakan memiliki pengaruh secara bersama-sama terhadap variabel terikat, jika nilai Fhitung lebih besar dari pada nilai Ftabel dan nilai probabilitas yang dihasilkan lebih kecil dari 0.05 (Sujarweni, 2020). http://e-jou dilakukan dengan membandingkan nilai F hasil perhitungan atau Fhitung dengan nilai Ftabel pada taraf signifikan 5%. Variabel-variabel bebas dikatakan memiliki pengaruh secara bersama-sama terhadap variabel terikat, jika nilai Fhitung lebih besar dari pada nilai Ftabel dan nilai probabilitas yang dihasilkan lebih kecil dari 0.05 (Sujarweni, 2020). ) Uji-F pada penelitian ini bertujuan untuk menguji signifikansi pengaruh secara simultan variabel-variabel independent, yaitu tingkat penjualan (X1) dan tingkat upah (X2) terhadap variabel dependen penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y). Berdasarkan pengujian yang telah dilakukan, diperoleh hasil yang disajikan pada tabel berikut. y g j p Tabel 3. Hasil Pengujian Hipotesis Simultan (Uji-F) y g j p Tabel 3. Hasil Pengujian Hipotesis Simultan (Uji-F) Tabel 3. Hasil Pengujian Hipotesis Simultan (Uji-F) ANOVAb Model Sum of Squares Df Mean Square F Sig. 1 Regression 1.627 2 .814 14.223 .000a Residual 8.582 150 .057 Total 10.209 152 a. Predictors: (Constant), Tingkat Upah, Tingkat Penjualan b. Dependent Variable: Penyerapan Tenaga Kerja Sumber: Output SPSS (data sekunder diolah), 2023. Berdasarkan hasil pengujian hipotesis simultan (uji F) yang disajikan pada tabel di atas, diperoleh nilai Fhitung sebesar 14.223 dan nilai Ftabel pada derajat kebebasan (df1=k-1=3-1=2) dan (df2=n-k=153-3=150) sebesar 3.06, sehingga nilai Fhitung lebih besar dari pada nilai Ftabel (14.223˃3.06) dan nilai signifikansi yang dihasilkan 0.000 lebih kecil dari 0.05 (0.000˂0.05). Dengan demikian, maka dapat dinyatakan bahwa tingkat penjualan dan tingkat upah secara simultan berpengaruh signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 4. Uji Koefisien Determinasi (R2) 2 j Koefisien determinasi (R2) digunakan untuk mengetahui persentase perubahan variabel tidak bebas atau terikat (Y) yang disebabkan oleh variabel bebas (X). Nilai koefisien determinasi (R2) ditentukan dengan nilai Adjusted R Square. Jika nilai R2 semakin besar, maka persentase perubahan variabel tidak bebas (Y) yang disebabkan oleh variabel bebas (X) semakin tinggi. Tetapi jika nilai R2 semakin kecil, maka persentase perubahan variabel tidak bebas (Y) yang disebabkan oleh variabel bebas (X) semakin rendah (Sujarweni, 2020). 2 j Uji koefisien determinasi (R2) dilakukan dilakukan pada penelitian ini untuk melihat seberapa besar kemampuan variabel-variabel independent, yaitu tingkat penjualan (X1) dan tingkat upah (X2) dalam mempengaruhi perubahan variabel dependen penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y). Berdasarkan pengujian yang telah dilakukan, diperoleh hasil yang disajikan pada tabel berikut. 465 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 Tabel 4. Hasil Pengujian Koefisien Determinasi (R2) Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .854a .744 .741 .12401 a. Predictors: (Constant), Tingkat Upah, Tingkat Penjualan b. Dependent Variable: Penyerapan Tenaga Kerja Sumber: Output SPSS 16.0 (data sekunder diolah), 2023. \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 Tabel 4. Hasil Pengujian Koefisien Determinasi (R2) Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .854a .744 .741 .12401 a. Predictors: (Constant), Tingkat Upah, Tingkat Penjualan b. Dependent Variable: Penyerapan Tenaga Kerja Sumber: Output SPSS 16.0 (data sekunder diolah), 2023. \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jour Tabel 4. Hasil Pengujian Koefisien Determinasi (R2) Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .854a .744 .741 .12401 a. Predictors: (Constant), Tingkat Upah, Tingkat Penjualan b. Dependent Variable: Penyerapan Tenaga Kerja Sumber: Output SPSS 16.0 (data sekunder diolah), 2023. Tabel 4. Hasil Pengujian Koefisien Determinasi (R2) Model Summaryb Berdasarkan hasil pengujian koefisien determinasi (R2) yang disajikan pada tabel di atas, diketahui nilai Adjusted R Square pengaruh tingkat penjualan (X1) dan tingkat upah (X2) terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa (Y) adalah sebesar 0.741. Hal ini berarti bahwa kemampuan variabel tingkat penjualan dan tingkat upah dalam mempengaruhi perubahan variabel penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa adalah sebesar 74,1%, sedangkan sisanya sebesar 25,1% dipengaruhi oleh variabel lain yang tidak diteliti, seperti modal, tingkat produktifitas dan tingkat pendidikan tenaga kerja (Putri, et al., 2022). Pembahasan Dalam penelitian ini, diduga determinan tingkat pengangguran terdidik di Kabupaten Sumbawa Barat Tahun 2012-2021 adalah jumlah penduduk dan Upah Minimum. Berikut diuraikan hasil penelitian yang telah dilakukan. 1. Pengaruh Tingkat Penjualan Terhadap Penyerapan Tenaga Kerja Pengujian ini dilakukan dengan tujuan untuk mengetahui pengaruh tingkat penjualan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. Berdasarkan hasil analisis data yang telah dilakukan, dapat diketahui bahwa tingkat penjualan secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. Hasil ini mengandung arti bahwa semakin tinggi tingkat penjualan yang diperoleh UMKM, maka penyerapan tenaga kerja akan semakin meningkat. Demikian pula sebaliknya, semakin rendah tingkat penjualan yang diperoleh UMKM, maka penyerapan tenaga kerja akan semakin menurun. Tingkat penjualan berpengaruh terhadap penyerapan tenaga kerja, karena jika tingkat penjualan meningkat, maka perusahaan membutuhkan tenaga kerja lebih banyak lagi agar dapat menghasilkan produk untuk memenuhi permintaan pasar konsumen. Hal ini sesuai yang dijelaskan oleh Sumarsono (dalam Nugrahaeni dan Handayani, 2020), apabila permintaan akan barang hasil produksi perusahaan meningkat, maka produsen cenderung untuk menambah kapasitas produksinya. Untuk maksud tersebut, produsen akan menambah penggunaan tenaga kerjanya. Hasil penelitian ini sesuai dengan penelitian sebelumnya yang dilakukan oleh Pakpahan dan Lubis (2020). Hasil temuan penelitian ini menunjukkan bahwa tingkat penjualan berpengaruh positif terhadap penyerapan tenaga kerja pada usaha restoran di Kota Medan. Jumlah penjualan menjadi foktor penting dalam penyerapan tenaga kerja. Hal itu dikarenakan untuk meningkatkan jumlah penjualan atau penerimaan, maka upaya yang harus dilakukan perusahaan restoran di Kota Medan adalah dengan meningkatkan produksinya sehingga perusahaan restoran membutuhkan tenaga kerja yang lebih besar. 466 \|\|Volume\|\|11\|\|Nomor\|\|3\|\|Desember\|\|2023\|\| e-ISSN: 2580-7285 p-ISSN: 2089-1210 http://e-journallppmunsa.ac.id/index.php/jeb Pp. 458 - 469 http://e-jou http://e-journallppmunsa.ac.id/index.php/jeb 2. Pengaruh Tingkat Upah Terhadap Penyerapan Tenaga Kerja Pengujian ini dilakukan dengan tujuan untuk mengetahui pengaruh tingkat upah terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. Berdasarkan hasil analisis data yang telah dilakukan, dapat diketahui bahwa tingkat upah secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. Hasil ini mengandung arti bahwa semakin tinggi tingkat upah yang diberikan oleh UMKM, maka penyerapan tenaga kerja akan semakin meningkat. Demikian pula sebaliknya, semakin rendah tingkat upah yang diperoleh UMKM, maka penyerapan tenaga kerja akan semakin menurun. p y g p , p y p g j Tingkat upah berpengaruh positif terhadap penyerapan tenaga kerja, karena pemberian upah tinggi akan memicu motivasi kerja yang tinggi. Pembahasan Upah yang tinggi dapat memberikan jaminan terpenuhinya kebutuhan hidup pekerja. Jika nilai upah yang ditawarkan oleh suatu perusahaan dinilai tidak mencukupi untuk memenuhi kebutuhan hidup pekerja, maka pekerja tersebut akan menolak pekerjaan yang ditawarkan. Hal ini sesuai dengan pandangan Sukwiaty (dalam Jiuhardi dan Rahayu, 2023) yang menyatakan bahwa upah merupakan faktor yang sangat berpengaruh dalam masalah ketenagakerjaan. Menurut ekonomi klasik, upah adalah harga untuk faktor produksi tenaga kerja. Harga tersebut haruslah bisa memenuhi kebutuhan hidup dan menjamin kehidupan yang layak. Hal ini karena keinginan orang bekerja adalah untuk mendapatkan upah yang layak guna memenuhi kebutuhan hidup. Bila tingkat upah yang ditawarkan oleh pengusaha dinilai tidak mencukupi oleh pekerja, maka pekerja tersebut tidak akan menerima pekerjaan yang ditawarkan. Hasil penelitian ini sesuai dengan penelitian sebelumnya yang dilakukan oleh Lastiko (2019). Hasil penelitian menunjukkan bahwa tingkat upah memiliki pengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada industri kecil di Kabupaten Sidoarjo. Upah berpengaruh positif dapat dijelaskan bahwa apabila upah tinggi, menggambarkan bahwa perusahaan dalam kondisi laba yang tinggi dimana ketika perusahaan berada dalam kondisi laba yang tinggi maka akan diikuti dengan peningkatan kapasitas produksi-nya yaitu dengan meningkatan proporsi jumlah tenaga kerjanya demi mencukupi permintaan barang yang di produksi, sehingga mampu membayar upah dengan jumlah akumulasi yang tinggi. Laba yang tinggi menggambarkan bahwa perusahaan dalam kondisi keuangan yang baik sehingga memiliki kemampuan untuk menyerap tenaga lebih banyak. KESIMPULAN Berdasarkan hasil penelitian dan pembahasan yang telah dikemukakan sebelumnya, maka dapat dikesimpulkan sebagai berikut: 1. Tingkat penjualan secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 1. Tingkat penjualan secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 2. Tingkat upah secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 2. Tingkat upah secara parsial berpengaruh positif dan signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 3. Tingkat penjualan dan tingkat upah secara simultan berpengaruh signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 3. Tingkat penjualan dan tingkat upah secara simultan berpengaruh signifikan terhadap penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa. 4. Kemampuan variabel tingkat penjualan dan tingkat upah dalam mempengaruhi perubahan variabel penyerapan tenaga kerja pada sektor UMKM di Kabupaten Sumbawa adalah sebesar 74,1%, sedangkan sisanya sebesar 25,1% dipengaruhi oleh variabel lain yang tidak diteliti, seperti modal, tingkat produktifitas dan tingkat pendidikan tenaga kerja. 467 http://e-jou http://e-jou http://e-journallppmunsa.ac.id/index.php/jeb 1. Bagi Pemerintah 1. Bagi Pemerintah Perlu adanya perhatian dari pemerintah seperti sosialisasi, pemantauan dan pelatihan terhadap para pelaku UMKM misalnya dengan mengadakan sosialisai dan pelatihan peningkatan penjualan,pemasaran produk, agar pelaku UMKM ini paham dengan adanya perubahan teknik pemasaran konvensional menjadi pemasaran online, sehingga materi yang diberikan untuk meningkatkan penjualan dan pemasaran secara digital dapat memudahkan pelaku dalam meningkatkan penjualannya. g Perlu adanya perhatian dari pemerintah seperti sosialisasi, pemantauan dan pelatihan terhadap para pelaku UMKM misalnya dengan mengadakan sosialisai dan pelatihan peningkatan penjualan,pemasaran produk, agar pelaku UMKM ini paham dengan adanya perubahan teknik pemasaran konvensional menjadi pemasaran online, sehingga materi yang diberikan untuk meningkatkan penjualan dan pemasaran secara digital dapat memudahkan pelaku dalam meningkatkan penjualannya. g p p g p j y 2. Bagi UMKM Dalam upaya meningkatkan penjualan, hendaknya pemilik UMKM permen susu lebih mendalami lagi strategi untuk meningkatkan produksi penjualannya, hal ini supaya tingkat penjualan permen susu terus meningkat dan kebutuhan akan tenaga kerja akan terus meningkat. Selain ituuntuk meningkatkan potensi UMKM harus mempunyai strategi yang baik, seperti strategi peningkatan penjualan, pemasaran, maupun promosi yang lebih luas g p p g p j y 2. Bagi UMKM Dalam upaya meningkatkan penjualan, hendaknya pemilik UMKM permen susu lebih mendalami lagi strategi untuk meningkatkan produksi penjualannya, hal ini supaya tingkat penjualan permen susu terus meningkat dan kebutuhan akan tenaga kerja akan terus meningkat. Selain ituuntuk meningkatkan potensi UMKM harus mempunyai strategi yang baik, seperti strategi peningkatan penjualan, pemasaran, maupun promosi yang lebih luas SARAN Bertitik tolak dari uraian yang telah dikemukakan sebelumnya dari hasil penelitian serta kesimpulan yang diperoleh dari hasil analisis, maka penulis memberikan beberapa saran sebagai berikut. Swastha, B. (2020). Manajemen Penjualan, Ed. Ketiga. Yogyakarta: BPFE. DAFTAR PUSTAKA Aliyah, A.H. (2022). Peran Usaha Mikro Kecil dan Menengah (UMKM) Untuk Meningkatkan Kesejahteraan Masyarakat. Welfare: Jurnal Ilmu Ekonomi, Vol. 3(1): 64-72. Al-Farisi, S., Fasa, M.I., & Suharto. (2022). Peran UMKM (Usaha Mikro Kecil Menengah) Dalam Meningkatkan Kesejahteraan Masyarakat. Jurnal Dinamika Ekonomi Syariah, Vol. 9(1): 73-84. Arikunto, S. (2019). Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta: Rineka Cipta. Darmanah, G. (2019). Metodologi Penelitian. Lampung: CV. Hira Tech. Ghozali, I. (2021). Aplikasi Analisis Multivariate Dengan Program IBM SPSS 26 (Edisi 10). Semarang: Badan Penerbit Universitas Diponegoro. Halim, A. (2020). Pengaruh Pertumbuhan Usaha Mikro, Kecil Dan Menengah Terhadap Pertumbuhan Ekonomi Kabupaten Mamuju. GROWTH: Jurnal Ilmiah Ekonomi Pembangunan, Vol. 1(2): 157-172. Jiuhardi, & Rahayu, I.S. (2023). Pengaruh Investasi Dan Upah Minimum Terhadap Kesempatan Kerja. KINERJA: Jurnal Ekonomi dan Manajemen, Vol. 20(1): 107-114. Junrillah, T., Junaidi, & Nurhayani. (2021). Pengaruh produk domestik regional bruto sektor pertanian, upah minimum provinsi dan investasi terhadap penyerapan tenaga kerja pada sektor pertanian Kabupaten Batanghari. e-Jurnal Perspektif Ekonomi dan Pembangunan Daerah, Vol. 10(3): 169-176. Kawet, J.A., Masinambow, V.A.J., & Kawung, G.M.V. (2019). Pengaruh Jumlah Penduduk, Pendidikan Dan Tingkat Upah Terhadap Penyerapan Tenaga Kerja Di Kota Manado. Jurnal Pembangunan Ekonomi Dan Keuangan Daerah, Vol. 20(2): 62-79. 468 http://e-jou http://e-jou http://e-journallppmunsa.ac.id/index.php/jeb Kotler, P., & Armstrong, G. (2019). Prinsip-Prinsip Pemasaran. Edisi 12. Jakarta: Erlangga. Lastiko, R.Y. (2019). Pengaruh Tingkat Upah, Volume Penjualan, Lama Usaha, Pendidikan, Dan Modal Terhadap Penyerapan Tenaga Kerja Industri Kecil (Studi Kasus Sentra Industri Kulit Tanggulangin Kabupaten Sidoarjo). Jurnal Ilmiah Mahasiswa FEB Universitas Brawijaya, Vol. 7(2): 1-12. Nawawi, H. (2019). Metode Penelitian Bidang Sosial (Cetakan Kelima Belas). Yogyakata: Gajah Mada University Press. Novitasari, A.T. (2022). Kontribusi UMKM Terhadap Pertumbuhan Ekonomi Era Digitalisasi Melalui Peran Pemerintah. Journal of Applied Business and Economic (JABE), Vol. 9(2): 184-204. Nugrahaeni, D.W., & Handayani, H.R. (2020). Analisis Pengaruh Upah, Modal, dan Nilai Produksi Terhadap Penyerapan Tenaga Kerja di Industri Tahu Serasi Kecamatan Bandungan. Diponegoro Journal of Economics, Vol. 9(2): 56-65. Pakpahan, E., & Lubis, T.H. (2020). Pengaruh Upah Dan Hasil Penjualan Terhadap Penyerapan Tenaga Kerja Pada Usaha Restoran Di Kota Medan. Journal Economic and Strategy (JES), Vol. 1(1): 11-21. Prasetyo, A., & Huda, M. (2019). Analisis Peranan Usaha Kecil Dan Menengah Terhadap Penyerapan Tenaga Kerja Di Kabupaten Kebumen. Fokus Bisnis : Media Pengkajian Manajemen Dan Akuntansi, Vol. 18(1): 26-35. Putri, M.A., Nefri, J., & Setiawan, R. (2022). Faktor-Faktor Yang Mempengaruhi Penyerapan Tenaga Kerja Pada UMKM Industri Roti Di Kota Payakumbuh. Jurnal Cahaya Mandalika, Vol. 3(1): 16-26. Siregar, S. (2017). Metode Penelitian Kuantitatif Dilengkapi dengan Perbandingan Perhitungan Manual & SPSS. Jakarta: Kencana Prenadamedia Group. Sugiyono. (2021). Metode Penelitian Kuantitatif Kualitatif dan R & D. Bandung: Alfabeta. Sujarweni, V.W. (2020). Metodologi Penelitian Bisnis & Ekonomi. Yogjakarta: Pustaka Baru. Sukirno, S. (2017). Pengantar Bisnis. Jakarta: Prenada Media. Surianto, F., Razak, A.R., & Djam’an, F. (2021). Analisis Pengaruh Upah Minimum, Investasi, dan Nilai Produksi Terhadap Penyerapan Tenaga Kerja Industri Kecil dan Menengah di Kota Parepare. Jurnal Ekonomika dan Dinamika Sosial, Vol. 2(1): 56-76. Swastha, B. (2020). Manajemen Penjualan, Ed. Ketiga. Yogyakarta: BPFE. 469
https://openalex.org/W2897527177	https://www.intechopen.com/citation-pdf-url/61635	English	null	Cyber Security Body of Knowledge and Curricula Development	InTech eBooks	2,018	cc-by	11,061	Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 4 Abstract The cyber world is an ever-changing world and cyber security is most important and touches the lives of everyone on the cyber world including researchers, students, busi- nesses, academia, and novice user. The chapter suggests a body of knowledge that incor- porates the view of academia as well as practitioners. This research attempts to put basic step and a framework for cyber security body of knowledge and to allow practitioners and academicians to face the problem of lack of standardization. Furthermore, the chap- ter attempts to bridge the gap between the different audiences. The gap is so broad that the term of cyber security is not agreed upon even in spelling. The suggested body of knowledge may not be perfect, yet it is a step forward. Keywords: body of knowledge, cyber security, ciphering, compression, database, operating systems, computer network Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.77975 Evon M Abu-Taieh, Auhood Abd. Al Faries, Shaha T. Alotaibi and Ghadah Aldehim Evon M Abu-Taieh, Auhood Abd. Al Faries, Shaha T. Alotaibi and Ghadah Aldehim Additional information is available at the end of the chapter Additional information is available at the end of the chapter © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons ttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, stribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, di t ib ti d d ti i di id d th i i l k i l it d Cyber Security Body of Knowledge and Curricula Development Evon M Abu-Taieh, Auhood Abd. Al Faries, Shaha T. Alotaibi and Ghadah Aldehim Evon M Abu-Taieh, Auhood Abd. Al Faries, Shaha T. Alotaibi and Ghadah Aldehim 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons ttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, stribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction Cyber security is a newly developed concept, proving to be of significance in the cyber world. The concept of cyber security admitted by many is not clear hence not standardized. This chapter aims to suggest a body of knowledge (BOK) based on two aspects: practitioners and academia. The chapter studied previous work of ACM, NICE, NICCS, and major academia institutes that offer master programs in cyber security. Furthermore, the chapter attempts to bridge the gap between the different audiences. The gap is so broad that the term cyber security is not agreed upon even in spelling. Then, the chapter presents the suggested body of knowledge. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 32 Reimagining New Approaches in Teacher Professional Development The research first discusses the notation of body of knowledge: discussing the definition, incen- tive, and mechanism. The accreditation process is the application of the body of knowledge; hence accreditation and accreditation incentive are discussed and the ACM & IEEE develop- ment of body of knowledge to different disciplines in the Information Technology Arena. The research first discusses the notation of body of knowledge: discussing the definition, incen- tive, and mechanism. The accreditation process is the application of the body of knowledge; hence accreditation and accreditation incentive are discussed and the ACM & IEEE develop- ment of body of knowledge to different disciplines in the Information Technology Arena. The research then addresses duality of spelling of the term cyber security versus cybersecu- rity, both are used interchangeably. This discrepancy in spelling the term serves as the base gap among the cybersecurity community. While such a gap must be bridged commencing with reaching an agreement on one spelling, however, in the scope section, a framework of elements is suggested to at least limit the intrusions of some unrelated terms. Then, the research gives a presentation about cyber security in the academic arena. 1. Introduction The show- case of academic perspective of cyber security included 61 master programs and 17 different countries namely Australia, Cyprus, Czech Republic, Estonia, Finland, France, Germany, India, Italy, Lithuania, Malaysia, Malta, Netherlands, New Zealand, Spain, UK, USA. The showcase presented the many irregularities and anomalies of cyber security master programs. Next, the chapter sheds the light on cyber security from Practitioner Perspective, citing the work NICE to standardize the body of knowledge of cyber security. The research concludes with a suggested body of knowledge for cyber security. Based on a comprehensive defini- tion of cyber security, the chapter also suggested two matrices that represent the interaction among the different elements of computer system with physical and non-physical threats, and a matrix that shows the interaction of physical/ non-physical threats with conductor of the threat internal and external. Furthermore, the final section presents and explains the core elements of the cyber security. This research is an expansion from a paper titled Cyber Security discussed in SC2 IEEE conference [1]. Then, the research gives a presentation about cyber security in the academic arena. The show- case of academic perspective of cyber security included 61 master programs and 17 different countries namely Australia, Cyprus, Czech Republic, Estonia, Finland, France, Germany, India, Italy, Lithuania, Malaysia, Malta, Netherlands, New Zealand, Spain, UK, USA. The showcase presented the many irregularities and anomalies of cyber security master programs. 2. Body of knowledge This section covers four topics to be discussed: first, the definition of body of knowledge and what incites the development of body of knowledge, further, giving examples about the body of knowledge in the different disciplines. The next two sections discuss the accreditation bodies and the incentive of accreditations. The last section discusses ACM & IEEE efforts in developing body of knowledge to different disciplines in the Information Technology arena. 2.1. Body of knowledge: definitions, incentives, and examples Body of knowledge (BOK) is best stated by [2] “(1) structured knowledge that is used by members of a discipline to guide their practice or work.” (2) “The prescribed aggregation of knowledge in a particular area an individual is expected to have mastered to be considered or certified as a practitioner.” Another definition of BOK is by [3] “A BOK is a term used to rep- resent the complete set of concepts, terms, and activities that make up a professional Domain. It encompasses the core teachings, skills, and research in a field or industry.” Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 33 There are many incentives to build a BOK in different disciplines cited by many researchers: [4] said that “BOK describes relevant knowledge for a discipline and will need show the con- sensus in the Knowledge Areas (KA), and related disciplines”; in addition, the same source states that BOK is useful for curricula design for innovation while industry context present. [5] said that BOK is a practice to support education, research, professional development, and practice. Furthermore, [6] listed that BOK will allow to meet the challenge of rapidly chang- ing landscape and the challenge of accommodating the diversity of emerging technologies. Again, the same source recites that BOK is used in curricula development, and BOK is the basis of evaluating the knowledge and skills of the discipline graduates, hence providing a roadmap to follow. In different disciplines, there are many BOK, for example: Systems engineering (G2SEBoK), Information systems engineering (ISEBOK), Software engineering (SWEBOK) [5–7], Infor- mation Technology (ITBOK), Project management (PMBOK-1, PMBOK-2), Body of Quality Knowledge (BOQK), New Product Development Body of Knowledge (NPDBOK), Software Requirements Traceability Body of Knowledge [8], Canadian IT Body of Knowledge, Civil Engineering Body of Knowledge, Geographic Information Science and Technology Body of Knowledge, Project Management Body of Knowledge, Business Analysis Body of Knowledge, The requirements engineering body of knowledge (rebok) [3]. 2.2. Accreditation bodies The ultimate goal of accreditation bodies of higher education is to first standardize education and maintain the quality of education in the different educational institutes. The second is to enhance the credit transferability among different educational institutes and furthermore different countries. Next, we present some international and national quality assurance and accreditation organizations from Germany, Spain, Hong Kong, Pakistan, Canada, Swiss, Austria, and USA. In addition, there are two important information technology-based organi- zations: ABET which is IEEE-based organization and ACM. Internationally, there are two organizations: The International Network for Quality Assurance Agencies in Higher Education (INQAAHE) which has 280 members [9]. The second is US-based organization; Council for Higher Education Accreditation has 467 quality assur- ance bodies, accreditation bodies, and ministries of Education from 175 countries, and has 3000 member institutions [10]. CHEA is a member in INQAAHE. CHEA replaced Council on Postsecondary Accreditation (COPA) and Federation of Regional Accrediting Commissions of Higher Education (FRACHE). In Europe, there is European Association for Quality Assurance in Higher Education (ENQA) which has 51 organizations and 28 countries. ENQA [11] established The European Quality Assurance Register for Higher Education (EQAR), the European Students’ Union (ESI), the European University Association (EUA), and the European Association of Institutions in Higher Education (EURASHE), and ENIC-NARIC (National Academic Recognition Information Centre) comprises all countries of Europe as well as Australia, Canada, Israel, the United States of America, and New Zealand. Reimagining New Approaches in Teacher Professional Development 34 In Germany, Kultusministerkonferenz (KMK) [12] was founded in 1948, and then in 1957, German Council of Science and Humanities (Wissenschaftsrat) was founded. KMK established Accreditation Council (Akkreditierungsrat). Associated with the Accreditation Council, 10 agencies are as follows: Swiss Agency for Accreditation and Quality Assurance(AAQ), Accreditation, Certification and Quality Assurance Institute (ACQUIN), Accreditation Agency for Study Programmes in Health and Social Sciences (AHPGS), Agency for Quality Assurance and Accreditation of Canonical Study Programmes (AKAST), Agency for Quality Assurance and Accreditation Austria(AQ Austria), Agency for Quality Assurance by Accreditation of Study Programmes (AQAS), Accreditation Agency for Degree Programmes in Engineering, Informatics/Computer Science, the Natural Sciences and Mathematics (ASIIN), evaluation agency Baden-Württemberg(evalag), Foundation for International Business Administration Accreditation (FIBAA), and Central Evaluation and Accreditation Agency Hannover (ZEvA). In Spain, the Agencia Nacional de la Evaluación de la Calidad y Acreditación (National Agency for Quality Assessment and Accreditation), which is dubbed (ANECA), was founded in 2002 [13]. 2.2. Accreditation bodies There are six national accreditors (nation- wide not international): Accrediting Bureau of Health Education Schools (ABHES) (recognized by USDE), Accrediting Commission of Career Schools and Colleges (ACCSC) (recognized by USDE), Accrediting Council for Continuing Education and Training (ACCET) (recognized by USDE), Council on Occupational Education (COE) (recognized by USDE), Distance Education Accrediting Commission (DEAC) (recognized by USDE and CHEA), and Accrediting Council for Independent Colleges and Schools (ACICS) [21]. The specialized or programmatic accredi- tors are generally under CHEA or Department of Education US (USDE), and there are 76 agencies. In the USA, the Higher education accreditation in the United States is categorized: regional, national, programmatic, and faith-based accreditors. There are six regional accreditors namely Middle States Commission on Higher Education, New England Association of Schools and Colleges, Northwest Commission on Colleges and Universities (NWCCU), Higher Learning Commission (HLC) (formerly, North Central Association of Colleges and Schools (NCA)), Southern Association of Colleges and Schools (SACS) Commission on Colleges, and Western Association of Schools and Colleges (WASC-ACCJC). There are six national accreditors (nation- wide not international): Accrediting Bureau of Health Education Schools (ABHES) (recognized by USDE), Accrediting Commission of Career Schools and Colleges (ACCSC) (recognized by USDE), Accrediting Council for Continuing Education and Training (ACCET) (recognized by USDE), Council on Occupational Education (COE) (recognized by USDE), Distance Education Accrediting Commission (DEAC) (recognized by USDE and CHEA), and Accrediting Council for Independent Colleges and Schools (ACICS) [21]. The specialized or programmatic accredi- tors are generally under CHEA or Department of Education US (USDE), and there are 76 agencies. ABET is programmatic Accreditation established in 1932 and has 3369 programs [10]. ABET accredited 3852 programs at 776 colleges and universities in 31 countries according to [22]. ABET covers disciplines of applied and natural science, computing, engineering, and engi- neering technology at the associate, bachelor, and master degree levels. ABET has four accred- itation commissions: Applied and Natural Science Accreditation Commission (ANSAC), Computing Accreditation Commission (CAC), Engineering Accreditation Commission (EAC), and Engineering Technology Accreditation Commission (ETAC). ABET is a federation of 35 societies and organizations [22]; furthermore, ABET stemmed from seven engineering societies. The first computer engineering program accredited by ABET was in 1971 at Case Western Reserve University [25]. Another report was published for the body of knowledge for Information Technology; the report is 161 pages long. The Association for Computing Machinery (ACM) established in 1947 has 100,000 members. 2.2. Accreditation bodies ACM is an organization for academic and scholarly interest in computer science. ACM has 171 local chapters, 37 special interest groups, and more than 50 scholarly peer-reviewed journals [23]. 2.2. Accreditation bodies ANECA is a full member of European Association for Quality Assurance in Higher Education (ENQA), International Network for Quality Assurance Agencies in Higher Education (INQAAHE), and European Quality Assurance Register for Higher Education (EQAR). In the United Kingdom, there is Quality Assurance Agency (QAA) which is a member of INQAAHE and ENQA [14]. In Hong Kong, the Hong Kong Council for Accreditation of Academic and Vocational Qualifications (HKCAAVQ) replaced the Hong Kong Council for Academic Accreditation [15]. In India, there are 12 professional councils: All India Council for Technical Education (AICTE), Distance Education Council (DEC), Indian Council for Agriculture Research (ICAR), Bar Council of India (BCI), National Council for Teacher Education (NCTE), Rehabilitation Council of India (RCI), Medical Council of India (MCI), Pharmacy Council of India (PCI), Indian Nursing Council (INC), Dentist Council of India (DCI), Central Council of Homeopathy (CCH), and Central Council of Indian Medicine (CCIM) [16]. In Pakistan, under Quality Assurance Agency of Higher Education Commission of Pakistan, there are National Accreditation Council for Teachers Education (NACTE), National Agricultural Education Accreditation Council (NAEAC), National Business Education Accreditation Council (NBEAC), and National Computing Education Accreditation Council (NCEAC) [17–20]. In Canada, the Canada’s Association of I.T. Professional (CIPS) is a Full Member of the Association of Accrediting Agencies of Canada (AAAC). CIPS has established the Computer Science Accreditation Council (CSAC), the Information Systems and Technology Accreditation Council (ISTAC) and the Business Technology Management Accreditation Council (BTMAC) as autonomous bodies. CIPS was established with this name in 1968. CIPS accredits the University, college/applied degree programs. The programs include Computer Science Degree Programs, Software Engineering Degree Programs, Interdisciplinary Programs, Management Information Systems Degree Programs, Business Management Technology Programs, Computer Systems Technology type Diploma Programs, and Applied Information Technology Degree Programs. Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 3 35 In the USA, the Higher education accreditation in the United States is categorized: regional, national, programmatic, and faith-based accreditors. There are six regional accreditors namely Middle States Commission on Higher Education, New England Association of Schools and Colleges, Northwest Commission on Colleges and Universities (NWCCU), Higher Learning Commission (HLC) (formerly, North Central Association of Colleges and Schools (NCA)), Southern Association of Colleges and Schools (SACS) Commission on Colleges, and Western Association of Schools and Colleges (WASC-ACCJC). 2.4. ACM and IEEE efforts in body of knowledge ACM and IEEE developed a body of knowledge, explained in a report published in 15/ Dec/2016 as shown in [25]. The report was developed by two delegations, both represented ACM and IEEE computer society delegates came from USA, China, and Scotland from 10 universities and IBM. The delegates came from 10 universities namely Hofstra University (USA), Milwaukee School of Engineering (USA), Clarkson University (USA), University of Florida (USA), Georgia Institute of Technology (USA), Mississippi State University (USA), Tsinghua University (China), Peking University (China), University of Strathclyde (Scotland), and Auburn University (USA). The delegates were four from IEEE and seven from ACM. The report describes in five specialties pertaining to computers and leaves the sixth for future model. The report lists 14 underlying principles that guide the committee through the descrip- tion of the body of knowledge as seen in pages 14 and 15 of the report [25]. In Chapter 3 of the document, the body of knowledge of Computing engineering was described in detail. In 2013, a report was published for the computer science curricula [26], and the body of knowledge of computer science was discussed in Chapter 4 of the 518-page document. In 2016, C.C. Walrad said “the IEEE Computer Society’s (CS’s) Educational Activities Board merged with the Professional Activities Board to form the Professional and Educational Activities Board (PEAB) in 2015. This merger facilitated the development of the Guide to the Software Engineering Body of Knowledge (SWEBOK) and training in the SWEBOK knowledge areas, as well as coordination of IT curriculum development activities and the creation of a Guide to the Enterprise IT Body of Knowledge (EITBOK), which will be available in wiki form later this year. Moreover, the merger serves to strengthen the CS’s joint work with ACM” [27]. In 2017, a report was published for Information Technology curricula [28]. The body of knowl- edge was discussed in Chapter 6. Chapter 6 discussed four topics: structure of IT curricula framework, distilling the IT curricular framework, IT domain clusters, and contemporary illustration of IT. 2.3. Why accredit A body of knowledge “can be very useful to provide a comprehensive and integrative view of the discipline, for assessment of professionals and organizations, for self-assessment as well as for curriculum development for academic or professional development courses and degree programs” [2]. Accreditation in accordance to body of knowledge affects students, institutions, public, and professionals. In fact, accreditation is the process and implementa- tion phase of the body of knowledge. The ultimate goal of both the idea (body of knowledge) and the process (accreditation) is to create a better-educated computer professional. Such computer professional shall practice with an ethical manner to the well-being of the ordinary user, organization, establishment, and the public. Such professional is required in industry and government, and hence, all parties need to cooperate to create the proper environment for such professional to spring to life. Industry standards must be met by the Institutes in the supply–demand manners. Furthermore, properly accredited institute can provide computer Reimagining New Approaches in Teacher Professional Development 36 students with proper education that will prepare them to further advance their higher edu- cation. Also, institutes can self-evaluate, analyze, and bridge the gap between industry and education. The accreditation process must be fair, unswerving, confidential with clear process, trans- parent, and objective. The accreditation agency must be independent and autonomous from educational institute. The accreditation process must be carried out by qualified reviewers. Resources must be available to carry process effectively. Accreditation process, goals, steps, and time must be clear and set, and such guidelines are set by [24]. 3. Cyber security scope the scope section, a framework of elements is suggested to at least limit the intrusions of some unrelated terms; as such, in this section, the chapter presents the different discussions about the definition of the term “cyber security.” Moreover, the section illustrates the elements of the cyber security framework, as shown in Figure 1. Most importantly is to define cyber security, which entails cyber security as “The ability to protect or defend the use of cyberspace from cyber-attacks” [29], using the term as one word. However, in 2014, [30] conducted a whole research to clarify the ambiguity of the term. In this context, the authors of the chapter found nine different definitions and came up with the 10th definition, denoted as “Craigen’s definition” as follows: “Cyber security is the organiza- tion and collection of resources, processes, and structures used to protect cyberspace and cyberspace-enabled systems from occurrences that misalign de jure from de facto property rights.” Cyber security entails protecting what from who and means of protection; [31, 32]. The three parts of the definition drive each other; the what part of the definition includes hardware, software, network, and data, the means of protection are practices, technology, and process, while the from who part of the definitions includes internal and external/hostile or naïve threats and attacks. In this token, the different attacks rely on the technology and its development, and so the means of protection relies on the technology and how attacks are dealt with, thereby creating a vicious cycle. 4. Cyber security in academia This section discusses the flounder of the academic arena regarding cyber security. The study reviewed a total of 61 master programs in 61 institute, 19 of them were studied with details of courses offered. Geographically, the study covered 17 different countries that are con- sidered well developed, with respect to Information Technology namely Australia, Cyprus, Czech Republic, Estonia, Finland, France, Germany, India, Italy, Lithuania, Malaysia, Malta, Netherlands, New Zealand, Spain, UK, and USA. 3. Cyber security scope The term cyber security has a duality: some write the term as one word, while others write the term as two words; accordingly, this is the key gap within all players in the cyber security community, which could be overcome by reaching a unanimous decision on one spelling. In Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 37 the scope section, a framework of elements is suggested to at least limit the intrusions of some unrelated terms; as such, in this section, the chapter presents the different discussions about the definition of the term “cyber security.” Moreover, the section illustrates the elements of the cyber security framework, as shown in Figure 1. Most importantly is to define cyber security, which entails cyber security as “The ability to protect or defend the use of cyberspace from cyber-attacks” [29], using the term as one word. However, in 2014, [30] conducted a whole research to clarify the ambiguity of the term. In this context, the authors of the chapter found nine different definitions and came up with the 10th definition, denoted as “Craigen’s definition” as follows: “Cyber security is the organiza- tion and collection of resources, processes, and structures used to protect cyberspace and cyberspace-enabled systems from occurrences that misalign de jure from de facto property i ht ” C b it t il t ti h t f h d f t ti [31 32] Figure 1. Cyber security elements. Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 37 the scope section, a framework of elements is suggested to at least limit the intrusions of some unrelated terms; as such, in this section, the chapter presents the different discussions about the definition of the term “cyber security.” Moreover, the section illustrates the elements of the cyber security framework, as shown in Figure 1. Figure 1. Cyber security elements. Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 37 Figure 1. Cyber security elements. Figure 1. Cyber security elements. the scope section, a framework of elements is suggested to at least limit the intrusions of some unrelated terms; as such, in this section, the chapter presents the different discussions about the definition of the term “cyber security.” Moreover, the section illustrates the elements of the cyber security framework, as shown in Figure 1. 4. Cyber security in academia This section discusses the flounder of the academic arena regarding cyber security. The study reviewed a total of 61 master programs in 61 institute, 19 of them were studied with details of courses offered. Geographically, the study covered 17 different countries that are con- sidered well developed, with respect to Information Technology namely Australia, Cyprus, Czech Republic, Estonia, Finland, France, Germany, India, Italy, Lithuania, Malaysia, Malta, Netherlands, New Zealand, Spain, UK, and USA. Table 1 illustrates the number of master programs distributed geographically, which high- lights that UK and USA have the most master programs pertaining to cyber security, where USA has 25 master programs, followed by UK with 16 master programs, whereas Spain, Estonia, Finland, France, and Netherlands follow with two programs, and the remaining 10 countries with only one program each. Reimagining New Approaches in Teacher Professional Development 38 38 Country Number of programs Australia 1 Cyprus 1 Czech Republic 1 Estonia 2 Finland 2 France 2 Germany 1 India 1 Italy 1 Lithuania 1 Malaysia 1 Malta 1 Netherlands 2 New Zealand 1 Spain 2 UK 16 USA 25 Total 61 Table 1. Master programs in different countries. Number of programs Table 1. Master programs in different countries. In light of the abovementioned, it is worth noting that a report, by professor Andrew McGettrick, the Chair of Education Board in ACM, titled “Toward Curricular Guidelines for Cyber security“ within “Report of a Workshop on Cyber security Education and Training “clearly stated that “Cyber security is currently an immature and ill-defined subject and not a true discipline since it lacks some of the criteria normally applied to disciplines” [33]. 4.1. Cyber security trending in academia Academia is supposed to lead the world in standardization and setting the rules. Yet, in this case, academia is at loss for the following reasons: first, there is a discrepancy in the wording of the term cyber security; some use “Cyber Security,” others use “Cybersecurity,” and others use the term “security.” Second, the offering of cyber security master program is under the umbrella of Master of Science, Master of Arts, engineering, criminal justice. Third, the faculty conducting the program is computer science, engineering, business, management, Arts and Social Sciences. Fourth, the master program is offered online, traditional, and distance learn- ing. Fifth, in research, the only paper tackled the subject was [34], which discussed the need from JOB POSTING perspective. In the next section, each of reasons will be presented along Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 39 with 61 different master programs offered worldwide with each program the university, fac- ulty, and country, shown in Appendix A. In respect to the term cyber security, being used as two separate words “Cyber Security,” 17 universities used it, namely KL University, North Umbria University London Campus, The University of Waikato, University of Westminster, University of Greenwich, Tallinn University of Technology, The University of Warwick, Harbour Space, Saint Peter’s University, Estonian Information Technology College, University of Southern California, The University of San Diego, Wright State University, University of York, University of south Australia, Temple University, George Mason University. On the other hand, 16 institutes used the term “Cybersecurity,” as one word, namely The George Washington University, St. Mary’s University, University of Central Missouri, Webster University Leiden, University of Maryland, University of Dallas College of Business, Sacred Heart University, Johns Hopkins, University of Maryland, NYU Tandon School of Engineering, University Of South Florida, Fordham University, University of Dallas, Villanova University, Embry-Riddle Aeronautical University, John Jay College of Criminal Justice. However, other terms referring to the term “Cybersecurity” like “Information Security” and “Digital Security” were used in the naming of the master program namely University of Turku, University of Kent, Eurecom, Asia Pacific University of Technology & Innovation (Apu), Cardiff University, Ferris State University, Vilnius Gediminas Technical University. 4.1. Cyber security trending in academia Moreover, the term “security” was used in nine program names to show that the program is related to the issue, namely: The University of Findlay, Eit Digital Master School, Cranfield University, Edinburgh Napier University, University of Amsterdam, Brunel University, Leeds Beckett University, Aalto University, and Esiea Graduate Engineering School. The programs were offered as Master of Science (MSC) in the faculty of computer science like University of Maryland, University of York or engineering like Villanova University, University of Southern California; still, others offered their program as a Master of Arts (MA) or under the faculty of Business like Brunel University and Temple University or faculty of management like University of San Francisco or faculty of criminal justice like John Jay College of Criminal Justice. On a different note, while Brunel University program is a distance learning type, some institu- tions offer “Cyber security” programs online such as University of Liverpool, University of The Cumberlands, Norwich University, NYU Tandon, whereas others require a traditional way of teaching. According to numerous academic programs, “Cyber security” is referred to as digital secu- rity. In fact, there are 19 different names that pertain to cyber security according to different academic programs, inter alia: Digital Forensics, Network Security, Applied Security and Analytics, Security and Privacy, Information Security and Cryptography, Forensics, Electronic Warfare, Counter Terrorism and Organized Crime, Information Security and Biometrics, Security and Management, Information Technologies Security, Intelligence and Security Studies, Information Security & Privacy, Information Assurance, in Network Security and Pen Testing, Cyber Security Engineering, Network & Information Security, Operations and Leadership, IT Auditing and Cyber Security. Reimagining New Approaches in Teacher Professional Development 40 The chapter reviewed the 61 master programs listed in Appendix A, of which 19 disclosed their detailed classes offered within their respective programs. 4.1. Cyber security trending in academia In turn, the researcher studied 110 different classes offered within the 19 programs and found the following: (1) six programs listed a course pertaining to programming and algorithms; (2) five programs listed a course pertaining to assurance using titles like Computer Systems Assurance Units, Development of high assurance systems, Information Assurance, Information Assurance and Security; (3) one master program offered a course about auditing entitled “Software Security Assessment”; (4) seven programs offered a course about cryptography under different titles like Applied Cryptography, Mathematics for Cryptography; (5) one program covered database courses; (6) seven programs offered Forensics under different titles like Cyber-crime and Computer Forensics, Digital Forensics, e-Crime, e-Discovery and Forensic Readiness, Forensic Computing, Network and Internet Forensics; (7) four programs offered Identity with titles like Identity and Access Management, and Identity Management for Federal IT; (8) four programs offered courses per- taining to Law and Ethics, such as Information Security and Ethics, Cyber Security Operational Policy, Human Aspects of Cyber security: Law, Ethics and Privacy as well as Introduction to Ethical Hacking; (9) 13 programs offered curses on Networks, ranking the topic as the highest to be offered under different titles: Advanced Network and Data Communication, Computer networking, Cryptography & Network Security, Cyber Network Security, Network and Internet Forensics, Net- work Essentials Intensive, Network Security, Network Visualization and Vulnerability Detection, Networks and Protocols, and Security applications in networking and distributed systems; (10) five programs covered the topic Operating systems (OS) under related titles: Operating Systems Security, Practical Unix, and Secure Operating Systems; (11) eight programs offered Windows Administration; (12) eight programs offered Software engineering under differ- ent titles: Information System Infrastructure Lifecycle Management, Complex Systems Engineering Management, Secure Software Design and Development, Software Engineering & Design, Software engineering, Software Security Lifecycle, Trusted System Design, Analysis and Development Units; (13) four programs covered Digital logic and microprocessors design under different descrip- tions: Computer Architecture, Secure Systems Architecture, Securing Digital Infrastructure, and Computer systems; (14) more significantly is that 14 master programs used the term security vaguely in a number of courses, utilizing it as follows: Information Security, Application Security, Best Practices Managing Security and Privacy for Cloud Computing, Computer Security and Privacy, Computer Security Fundamentals, Computer Security, Critical Infrastructure and Control System Security, Cyber Network Security, Cyber security Essentials, Foundations of Cyber Security, Hardware Security, Host Computer Security, Information, Security and Privacy, as well as Management and Cyber Security. 4.1. Cyber security trending in academia In all the 19 master programs, there are 18 courses that are of special interest to cyber security: In all the 19 master programs, there are 18 courses that are of special interest to cyber s 1. Intrusion Detection 2. Advanced Penetration Testing 3. Cyber Fraud and Theft 4. Cyber Incident Handling and Response Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 41 5. Cyber Incident Response and Computer Network Forensics 6. Cyber Intelligence 7. Cyber Intelligence & Counterintelligence 8. Cyber Security: Emerging Threats and Countermeasures 9. Cyber Terrorism 10. Cyber Security: Threats and Defense 11. Electronic Evidence Analysis and Presentation 12. Imaging for Security Applications Watermarking & Biometrics 13. Incident Detections & Responses 14. Intrusion Analysis and Response 15. Malware and Intrusion Detection 16. Security Attacks and Defenses 17. Security Tools for Information Security 18. System Exploitation and Penetration Testing 18. System Exploitation and Penetration Testing 5. Cyber security from practitioner perspective In this section, the chapter discusses the body of knowledge represented from a practitioner approach. Accordingly, the chapter researched the perspective of National Initiative for Cyber Security Education (NICE) and National Initiative for Cyber Security Careers and Studies (NICCS). Managed by the Cyber Security Education and Awareness Branch (CE&A) within the Department of Homeland Security’s (DHS) Office of Cyber security and Communications (CS&C), NICCS is an online resource for cyber security training that connects government employees, students, educators, and industry with cyber security training providers through- out the nation to ensure that the government workforce has the appropriate training and education in the cyber security field. Likewise, NICE is an initiative led by National Institute of Standards and Technology in the USA department of Commerce with partnership between government and academia focusing on cyber security training and education and workforce development; the NICE Program Office operates under the Applied Cyber security Division, positioning the program to support the ability of USA to address current and future cyber security challenges through standards and best practices [35], with the strategic intent to entice a nationwide dialogue, thereby leading an action on how to address the critical short- age of a skilled cyber security workforce. Reimagining New Approaches in Teacher Professional Development 42 Moreover, the NICE Cyber security Workforce Framework (NIST Special Publication 800–181) serves as a fundamental reference resource to improve the communication needed to identify, recruit, and develop cyber security talent. To reverse engineer and attempt to define the core body of knowledge, NICE limited The Cyber Security Work Categories to the following seven categories of common cyber security functions: 1. Operate and maintain 2. Securely provision 3. Protect and defend 4. Oversee and govern 1. Operate and maintain 2. Securely provision 4. Oversee and govern 5. Analyze 6. Investigate 7. Collect and operate. In addition, NICE had in mind the following audience for the framework: employer, employee, training, education, and technology providers. Within this context, NICCS developed 17 focus areas of education used as guidelines to education institutions [36]: 1. Cyber Investigations: focuses on analyses of computer incidents and intrusions to determine attacker/source, infiltration path, mechanism, system modifications and effects, damages, exfiltration path, data exfiltrated, and residual effects. 2. Data Management Systems Security: concentrates on secure configuration, operation, and maintenance of databases and database management systems housing sensitive data. 3. 5. Cyber security from practitioner perspective 16. Systems Security Administration: focuses on secure configuration, operation, and mainte- nance of a computer system (host or workstation). 16. Systems Security Administration: focuses on secure configuration, operation, and mainte- nance of a computer system (host or workstation). 17. Systems Security Engineering: involves using system development life cycle while embed- ding and taking into account security issue. 17. Systems Security Engineering: involves using system development life cycle while embed- ding and taking into account security issue. 5. Cyber security from practitioner perspective Data Security Analysis: the analysis of data (e.g., system logs, network traffic) aims to iden- tify suspected malicious activities. 4. Digital Forensics: the analysis of computer systems (hosts, servers, network components) aims to determine the effects that malware has had on the system. 5. Health-Care Security: focuses on design, development, operation, and maintenance of com- puter systems used in health-care applications. 6. Industrial Control Systems—SCADA Security: concentrates on design, development, opera- tion, and maintenance of industrial control systems used in real-time infrastructures. 6. Industrial Control Systems—SCADA Security: concentrates on design, development, opera- tion, and maintenance of industrial control systems used in real-time infrastructures. 7. Network Security Administration: focuses on secure configuration, operation, and operation of an enterprise computer network. 7. Network Security Administration: focuses on secure configuration, operation, and operation of an enterprise computer network. 8. Network Security Engineering: concentrates on the design of secure network infrastructures and security analysis of network traffic. 8. Network Security Engineering: concentrates on the design of secure network infrastructures and security analysis of network traffic. 9. Secure Cloud Computing: targets design, development, operation, and maintenance of secure cloud architectures. 9. Secure Cloud Computing: targets design, development, operation, and maintenance of secure cloud architectures. Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 43 43 10. Secure Embedded Systems: concentrates on design, development, utilization, and manage- ment of secured embedded systems technologies. 11. Secure Mobile Technology: focuses on design, development, utilization, and management of secure mobile technologies, devices, and services. 11. Secure Mobile Technology: focuses on design, development, utilization, and management of secure mobile technologies, devices, and services. 12. Secure Software Development: revolves around the development of secure software 13. Secure Telecommunications: involves design, development, and secure use of secure tel- ecommunications systems whether the system is digital and analog. 13. Secure Telecommunications: involves design, development, and secure use of secure tel- ecommunications systems whether the system is digital and analog. 14. Security Incident Analysis and Response: examines system vulnerability analysis and devel- oping the right future response. 14. Security Incident Analysis and Response: examines system vulnerability analysis and devel- oping the right future response. 15. Security Policy Development and Compliance: revolves around the IT policy of an organiza- tion and the monitoring and evaluation tools related to such policy. 15. Security Policy Development and Compliance: revolves around the IT policy of an organiza- tion and the monitoring and evaluation tools related to such policy. 6. Suggested cyber security body of knowledge Cyber security encompasses physical and non-physical security of data, software, and hard- ware, from harm by both authorized and non-authorized access, whether access is internal or external. Cyber security is conducted via technology through predetermined processes. Since technology is advancing expeditiously, it is not only challenging but it is also imperative to predetermine the process of security; as such, several best practices and lessons learned are traded among practitioners. The physical security of data software and hardware from authorized and non-authorized access includes but not limited to, protecting the server room, its location, the switches, the cable, data and data storage devices from fire, and excessive heat intruders. As such, server rooms are typically equipped with fire distinguisher, air conditioned, insulated from fire, and its floor is raised to docket the cables. In addition, the switches are usually installed in hidden high places to limit the reachability, and cables are docketed in walls or under a raised floor. The location of the server room is another issue that is part of physical security. The major issues that pertain to this subject are summarized in the subsequent matrix. For authorized personnel, another layer of protection to access the IT systems should be in place, varying from setting up password-protected access or magnetic card to retina scan to lock and key, as illustrated in Figure 2. However, oftentimes, physical security and non- physical security are not confined to external threats and attackers and could be consid- ered internally, in which case, if the assessed damage is considered either intentional or non-intentional. For intentional damage, a rigorous policy should be established to ensure employee compliance and discipline like cameras and employee follow-up; yet, non-inten- tional damage is alleviated by proper training and teaching. Reimagining New Approaches in Teacher Professional Development 44 44 Figure 3. Matrix that shows the interaction of physical/non-physical threats with conductor of the threat internal and external. Figure 2. Matrix of interaction computer system with physical and non-physical threats. Figure 2. Matrix of interaction computer system with physical and non-physical threats. Figure 3. Matrix that shows the interaction of physical/non-physical threats with conductor of the threat internal and external. Figure 3. Matrix that shows the interaction of physical/non-physical threats with conductor of the threat internal and external. 6. Suggested cyber security body of knowledge The non-physical damage is not only more sensitive but it is immensely difficult to follow, in view that the more significant threat comes from unlawful use and access to the system. Viruses are a major threat in this case, hence, limiting the access to the system by using strict policy and good antivirus may halt the effect. The external threat of damage like hacking and viruses poses as the most challenging, albeit, software tools like firewall and antivirus may protect the system, as illustrated in Figure 3; however, external physical threat, such as attacking and looting ATM machines or physical attacks on server room, switches, cables, and data, can be overcome by setting up tangible measures. These methods can include, but not limited to, setting up servers in protected rooms, ensuring that switches and cables are not visible to external entities, and storing data in secure locations with copy and the use of enormous storage. 1. Cyber Security Assurance 2. Cyber Security Assessment 6.1. Core element in cyber security The core elements in cyber security are the following 12 elements; these are the pillars or the base to any cyber security program: 1. Cyber Security Assurance 2. Cyber Security Assessment Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 45 Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 45 Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 45 3. Ciphering 4. Algorithms 5. Networks 6. Digital Logic and Microprocessors Design 7. Operating Systems 8. Database 9. Cyber Law & Ethics 10. Viruses & Hacking 11. Software Tools & Techniques 12. Software Auditing & Software Engineering. 12. Software Auditing & Software Engineering. The following will further explain the 12 pillars (see Figure 4) and core elements in the cyber security body of knowledge. 6.1.1. Cyber security assurance Cyber security assurance is best defined by Lipner [37] and he also explained how to achieve security assurance. To define assurance, Lipner stated that “assurance: making systems that can resist attack.” And he added “Assurance is achieved by integrating security into the process of designing, building, and testing systems” [37]. In a research conducted by [38] to develop Software Assurance Curriculum for master level and again in [39] to develop for Software Assurance Curriculum for master’s level, the authors proposed a comprehensive curriculum specialized for software assurance. Cyber security special must learn method like Mission Risk Diagnostic (MRD) described by [40] used to assess risk in systems across the life cycle and supply chain. In addition, specialist must learn SQUARE. Security Quality Requirements Engineering (SQUARE) is a nine-step process that helps organizations build security, including privacy, into the early stages of the production lifecycle [41–43]. 6.1.2. Cyber security assessment Cyber security assessment is a process to assess an organization’s level of risk and prepared- ness. The process is repeatable and measurable. The process has two parts: Inherent Risk Profile & Cyber security Maturity [44]. The assessments are conducted in domains accord- ing to five levels of maturity according to FFIEC [44] suggested model. According to [45], an assessment framework was developed and was adopted by 30% of US organizations. The framework provides a risk-based approach for cyber security through five core func- tions: identify, protect, detect, and respond and recovery. Furthermore, the cyber security Reimagining New Approaches in Teacher Professional Development 46 professional should be able to create and conduct a cyber security assessment by understand- ing the various methodologies across all industries on how to conduct and manage a cyber security assessment, risk analysis, and how to mitigate various cyber security threats by conducting the following: first, understand and write reports on cyber threat attack analysis Second, understand and write cyber security policy based on assessments. Third, detect and analyze incidents of action of attacks and threats. Fourth, establish cyber security controls based on established models and frameworks. Fifth, manage attack countermeasures. Sixth mitigate risks of threats and attacks. Seventh, cycling of reports and evidence procedures for prosecution after such assessments have been produced. Figure 4. Pillars of cyber security. Figure 4. Pillars of cyber security. Figure 4. Pillars of cyber security. Figure 4. Pillars of cyber security. professional should be able to create and conduct a cyber security assessment by understand- ing the various methodologies across all industries on how to conduct and manage a cyber security assessment, risk analysis, and how to mitigate various cyber security threats by conducting the following: first, understand and write reports on cyber threat attack analysis. Second, understand and write cyber security policy based on assessments. Third, detect and analyze incidents of action of attacks and threats. Fourth, establish cyber security controls based on established models and frameworks. Fifth, manage attack countermeasures. Sixth, mitigate risks of threats and attacks. Seventh, cycling of reports and evidence procedures for prosecution after such assessments have been produced. Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 47 6.1.3. Ciphering Ciphering is an essential part of security that allows the data to be transferred from point to point safely and without allowing anyone to look at the data being transferred. 6.1.2. Cyber security assessment Ciphering is an old technique yet still needed for security. There are many types of ciphering that entails hardware and software, tools and techniques in addition to ciphering algorithms. Ciphering algorithms can be classified according to key as symmetrical and a symmetrical, according to type of operations conducted: substitution, transposition, bit manipulation, and to the cipher process block or stream cipher. Hence, public key ciphering RSA, and block cipher algorithms like IDEA, RC2, RC5, CAST, ElGamel, DSA, and Skipjack are important for the cyber secu- rity specialist. Topics like cryptanalysis, hash functions, digital signatures, and web security should be covered in detail. 6.1.4. Algorithms Algorithms are the backbone of software. To develop any software, one must understand the logic behind the building blocks of the software. Algorithms deal with data taking into account speed, space storage, and time complexity. Search, sort, compression, and data structure are all based on algorithms. Algorithm is the language that a programmer, analyst, designer speaks with the computer to materialize their idea into working software. Hence, developing the log- ical sense to security specialist is a must trait. Typical course must include the following in the syllabus: sort & search algorithms, graph algorithms (Graph traversal (DFS, BFS) and applica- tions, Connectivity, strong connectivity, bi-connectivity, Minimum spanning tree, Shortest path, Matchings, Network flow), and hard problems (Traveling salesman problem, Longest path, Hamilton cycle, Boolean circuit satisfiability, Clique, Vertex cover). Algorithm design: Divide-and-conquer, Graph traversal, Greedy, Dynamic Programming, Reductions, Use of advanced data structures. Algorithm correctness: Proofs and proof techniques (assumptions, basic logic inference and induction), Tree and graph properties. Algorithm analysis: Time and space complexity, Asymptotic analysis: Big Oh, Little oh, Theta, Worst case and average case analysis, Lower bounds. Tractable and intractable problems: Polynomial time algorithms, NP algorithms, NP hardness and NP completeness, NP Reductions. 6.1.7. Operating systems Operating system (OS) is the layer of software that lay between hardware and applications. Through OS, a person can speak to the computer hardware using a programming language. The essentials under this topic are processes and threads, mutual exclusion, CPU scheduling, deadlock, memory management, and file systems, distributed systems. 6.1.5. Networks Networks are a backbone of the data transfer; it is the roads to cars. The types and standards of networks are essential to anyone working in the cyber security. Networks are not only hardware they are standards, and routing algorithm, in addition to hubs, switches, cables, and jacks. Furthermore, the security models of computer networks along with ISO standard of these models, that is, Open Systems Interconnection (OSI), and transmission control pro- tocol and Internet protocol (TCP/IP). Typically, a network course covers the following topics: Fundamentals, Link Layer, Media Access, Internetworking, Routing, Transport Layer, and Application Layer. The Fundamentals & Link Layer which includes Building a network, Layering and protocols—Internet Architecture, Network software, Performance; Link layer Services: Framing, Error Detection, Flow control. The Media Access & Internetworking which Reimagining New Approaches in Teacher Professional Development 48 includes Media access control—Ethernet (802.3), Wireless LANs 802.11, Bluetooth, Switching and Bridging, Basic Internetworking (IP, CIDR, ARP, DHCP, and ICMP). The routing topic which includes Routing (RIP, OSPF, metrics), Switch basics—Global Internet (Areas, BGP, IPv6), Multicast addresses, multicast routing (DVMRP, PIM). The Transport Layer topic which includes Overview of Transport layer, UDP, Reliable byte stream (TCP), Connection management, Flow control, Retransmission, TCP Congestion control, Congestion avoid- ance (DECbit, RED), QoS, Application requirements. The Application Layer which includes Traditional applications: Electronic Mail (SMTP, POP3, IMAP, MIME), HTTP, Web Services, DNS, and SNMP. 6.1.9. Cyber law and ethics 6.1.9. Cyber law and ethics Knowing the difference between cyber laws and regulations to cyber security is like knowing the laws and regulations to policeman. It is essential to know cyber laws and regulation since borders do not exist in the cyber world. There also many ethical issues that pertain to the topic not to mention the power that comes with such territory. 6.1.6. Digital logic and microprocessors design Digital logic and microprocessors design are basic and fundamental for cyber security. Under this topic, things like the principles of programmable logic devices, combinational and sequential circuits, and the principles of hardware design, the structure and electronic design of modern processors. In addition, logical gates, flip-flop, and binary world are included. 6.1.7. Operating systems 6.1.8. Database Database is where data are stored in a computer system, topics under database are (but not lim- ited to) data models like Entity Relations (ER), relational; query languages including relational algebra, Structure Query Language (SQL); implementation techniques of database manage- ment systems including index structures, concurrency control, recovery, and query process- ing; management of semi-structured and complex data; distributed and NoSQL databases. 6.1.10. Viruses, warms, and hacking Cyber security specialist must be aware of the methods and tools and techniques used to counter affect viruses, warms, hacking the malware software in general. For a policeman to be good at his job, he must be aware of the criminal acts and how they are conducted. Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 49 Cyber security must know the different types of viruses, warms, and hacking methods in order to deal with such problems. Malware ranges from annoying malicious software to cyber-weapon that attacks and destroys. Furthermore, detection, analysis, control, and eradication of such software are essential part of cyber security education. Tools like Dependency Walker, Fakenet, FileAlyzer 2.0, HxD, IDA Free, ImpREC, LordPE, Malcode Analyst Pack, OllyDbg, PEiD, PEview, Regshot, Resource Hacker, Sysinternals Suite, UPX, Visual Studio, Windbg, Wireshark, System Monitor, Process Explorer, CaptureBAT, Regshot, VMware, BinText, LordPE, QuickUnpack, Firebug, PELister, PEiD, IDA Pro, OllyDbg and plug-ins such as OllyDump, HideOD, Rhino, Malzilla, SpiderMonkey, Jsunpack-n. Internet Explorer Developer Toolbar, cscript Honeyd, NetCat, Wireshark, curl, wget, xorsearch OfficeMalScanner, OffVis, Radare, FileInsight, malfind2, apihooks, SWFTools, Flare, and shellcode2exe are essential for cyber security expert. 6.1.11. Software tools and techniques There are tremendous amount of software tools and techniques that is especially developed for cyber security. The simplest is antivirus software, database management software, operat- ing systems management software, network management software, and so on. Hence, cyber security specialist must be familiar with these tools and techniques. 6.1.12. Software auditing and software engineering 6.1.12. Software auditing and software engineering Software auditing and software engineering is the 12th pillar that is of core importance to Cyber security. Most attacks on cyber systems are viruses or hacks coming from internal or external source, thereby misusing a vulnerability of software. For example, a programmer forgot to take into account certain case or a port. Software engineering should take the right measure so that such case does not occur. Regular auditing to software and data and build- ing self-tests within the software will catch such problem beforehand. Hence, cyber security specialist must be aware of the tools, techniques, and methods of software engineering and auditing. Furthermore, security specialist must have the knowledge of Software Assurance Framework (SAF) explained in [46]. Software Assurance Framework (SAF) is a collection of cyber security practices that programs can apply across the acquisition lifecycle and sup- ply chain. Hence, cyber security specialist must be aware of software security framework such as IMAF. IMAF is a framework suggested by [47] that aligns drivers with software security codes of practices. There are many codes of practices listed by [47]: Building Security in Maturity Model (BSIMM), Open Web Application Security Project (OWASP), Software Assurance Maturity Model (SAMM), Department of Homeland Security Measurement work and Assurance for CMMI Process Reference Model, and CERT Resilience Management Model. None-core competencies and elements are important but since technology keeps changing, further courses can be developed through course training or self-training or both. Such ele- ments include but not limited to Intrusion Detection (Analysis and Response), penetration testing, Intelligence & counterintelligence, and Electronic Evidence Analysis. 50 Reimagining New Approaches in Teacher Professional Development 7. Conclusion MSc in Applied Security and Analytics The University of Findlay USA Master in Security and Privacy (S&P) Eit Digital Master School Ger Master’s Degree Programme in Information Security and Cryptography University of Turku Fin Computer Engineering International University Alliance USA MSc Cyber Security and Forensics University of Westminster UK Science and Technology Electronic Warfare, Information and Cyber Degrees Cranfield University UK International Security Degrees Cranfield University UK Resilience, Counter Terrorism and Organized Crime Degrees Cranfield University UK MSc Computer Forensics & Cyber Security University of Greenwich UK MSc Advanced Security and Digital Forensics Edinburgh Napier University UK MSc in Cyber Security Tallinn University of Technology Estonia MSc Information Security and Biometrics University of Kent UK Master in Cyber Security and Management (CSM) The University of Warwick UK Warwick Manufacturing Group Wmg Master in Information Security Harbour.Space Spain Master of Information and Information Technologies Security Vilnius Gediminas Technical University LT 1. MSc System and Network Engineering: Security University of Amsterdam NL MSc in Digital Security and Forensics Asia Pacific University of Technology & Innovation (APU) Mal MA Intelligence and Security Studies (Distance Learning) Brunel University UK College of Business, Arts and Social Sciences Master in Cybersecurity St. Mary’s University USA Master in Cybersecurity University of Central Missouri USA MS Cybersecurity Webster University Leiden NL MSc Cyber Security Engineering (CSE) The University of Warwick UK Warwick Manufacturing Group Wmg MSc in Information Security & Privacy Cardiff University UK Master in Embedded Systems Masaryk University Czech R. MSc Digital Forensics and Security Leeds Beckett University U K Name Institute Country Faculty Master of Cyber Security The University of Waikato New Z. 7. Conclusion This research first discussed the body of knowledge notion and scope of cyber security defini- tion and then gave a presentation about cyber security in the academic arena. The showcase of academic perspective of cyber security included the 61 master programs from 17 differ- ent countries namely Australia, Cyprus, Czech Republic, Estonia, Finland, France, Germany, India, Italy, Lithuania, Malaysia, Malta, Netherlands, New Zealand, Spain, UK, and USA. The showcase presented the many irregularities and anomalies of cyber security master programs. Next, the chapter presented cyber security from practitioner perspective citing the work NICE to standardize the body of knowledge of cyber security from practitioner perspective. The last part of the chapter presents a suggested body of knowledge for cyber security. Based on a comprehensive definition of cyber security, the chapter also suggested two matrices that represent the interaction among the different elements of computer system with physical and non-physical threats, and a matrix that shows the interaction of physical/non-physical threats with conductor of the threat internal and external. Furthermore, the section presents and explains the core elements of the cyber security. A. Master programs, their affiliation, country, and faculty Name Institute Country Faculty MSc in Computer Science University of Nicosia Cyp MSc in cyber security in CS The George Washington University USA Engineering & Applied Science Master in Digital Security Eurecom Fr Master of Engineering in Cybersecurity University of Maryland USA School of Engineering MSc in Computer, Communication and Information Sciences - Security and Mobile Computing Aalto University Fin Mastère Spécialisé SIS: Sécurité de l’Information et des Systèmes Esiea Graduate Engineering School F Master of Information Systems University Of San Francisco USA School of Management Masters of Science in Engineering in Artificial Intelligence and Robotics Sapienza University of Rome It Digital Forensics and Cybersecurity programs John Jay College of Criminal Justice USA Master of Technology in Cyber Security and Digital Forensics K L University Ind Network Security and Pen Testing MSc Middlesex University London UK MSc Cyber Security Northumbria University London Campus UK A. Master programs, their affiliation, country, and faculty Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 51 Name Institute Country Faculty Master of Cyber Security The University of Waikato New Z. 7. Conclusion MSc in Applied Security and Analytics The University of Findlay USA Master in Security and Privacy (S&P) Eit Digital Master School Ger Master’s Degree Programme in Information Security and Cryptography University of Turku Fin Computer Engineering International University Alliance USA MSc Cyber Security and Forensics University of Westminster UK Science and Technology Electronic Warfare, Information and Cyber Degrees Cranfield University UK International Security Degrees Cranfield University UK Resilience, Counter Terrorism and Organized Crime Degrees Cranfield University UK MSc Computer Forensics & Cyber Security University of Greenwich UK MSc Advanced Security and Digital Forensics Edinburgh Napier University UK MSc in Cyber Security Tallinn University of Technology Estonia MSc Information Security and Biometrics University of Kent UK Master in Cyber Security and Management (CSM) The University of Warwick UK Warwick Manufacturing Group Wmg Master in Information Security Harbour.Space Spain Master of Information and Information Technologies Security Vilnius Gediminas Technical University LT 1. MSc System and Network Engineering: Security University of Amsterdam NL MSc in Digital Security and Forensics Asia Pacific University of Technology & Innovation (APU) Mal MA Intelligence and Security Studies (Distance Learning) Brunel University UK College of Business, Arts and Social Sciences Master in Cybersecurity St. Mary’s University USA Master in Cybersecurity University of Central Missouri USA MS Cybersecurity Webster University Leiden NL MSc Cyber Security Engineering (CSE) The University of Warwick UK Warwick Manufacturing Group Wmg MSc in Information Security & Privacy Cardiff University UK Master in Embedded Systems Masaryk University Czech R. 7. Conclusion MSc Digital Forensics and Security Leeds Beckett University U K Reimagining New Approaches in Teacher Professional Development 52 52 Name Institute Country Faculty Master of Science in Cyber Security Saint Peter’s University USA MSc in Cybersecurity (Information Assurance) University of Dallas USA College of Business MSc in Network Security and Pen Testing Middlesex University Malta Malta MSc in Information Security and Intelligence Ferris State University USA Masters in Cyber Security Engineering Estonia Information Technology College MSc Network & Information Security Kingston University London UK Máster en Ciberseguridad UCAV- DELOITTE * Universidad Católica De Ávila Spain Master of Science: Cybersecurity Sacred Heart University USA MSc in Cybersecurity Johns Hopkins USA Johns Hopkins Engineering Master of Engineering in Cybersecurity University of Maryland USA Computer Science Master of Science Cyber Security Engineering University of Southern California USA Viterbi School of Engineering Cybersecurity Online NYU Tandon School of Engineering USA Cybersecurity Master’s Degree University of South Florida USA MSc in Cybersecurity Fordham University USA Master of Science in Cyber Security Operations and Leadership The University of San Diego USA MSc in Cybersecurity University of Dallas USA College of Business MSc in Cyber Security Wright State University USA The College of Engr & C S MSc in Cybersecurity Villanova University USA College of Engineering MSc in Cyber Security University of York UK Department of Comp. Sci. MSc (Cyber Security and Forensic Computing) University of South Australia Aus IT Auditing and Cyber Security Temple University USA Fox School of Business Applied Information Technology, Cyber Security Concentration (MS) George Mason University USA School of Engineering MSc in Cybersecurity Engineering Embry-Riddle Aeronautical University USA College of Engineering Author details Evon M Abu-Taieh1, Auhood Abd. Al Faries2,3, Shaha T. Alotaibi2 and Ghadah Aldehim2 Address all correspondence to: abutaieh@gmail.com 1 The University of Jordan, Aqaba, Jordan 2 Princess Nourah Bin Abdulrahman University, Saudi Arabia 3 King Saud University, Saudi Arabia Evon M Abu-Taieh1, Auhood Abd. Al Faries2,3, Shaha T. Alotaibi2 and Ghadah Aldehim2 Address all correspondence to: abutaieh@gmail.com 1 The University of Jordan, Aqaba, Jordan 2 Princess Nourah Bin Abdulrahman University, Saudi Arabia 3 King Saud University, Saudi Arabia Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 53 Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 53 Cyber Security Body of Knowledge and Curricula Development http://dx.doi.org/10.5772/intechopen.77975 53 References [1] Abu-Taieh E. Cyber Security Body of Knowledge. In: SC2 IEEE Conference; Japan. 2017 [2] Oren TI. Toward the body of knowledge of modeling and simulation. In: Interservice/ Industry Training, Simulation, and Education Conference (I/ITSEC) 2005; Orlando, FL. 2005 [3] Penzenstadler B, Fernandez M, Richardson D, Callele D, Wnuk K. The requirements engineering body of knowledge (rebok). 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https://openalex.org/W2150640296	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_Dual_Blockade_of_PD-1_and_CTLA-4_Combined_with_Tumor_Vaccine_Effectively_Restores_T-Cell_Rejection_Function_in_Tumors/22395077/1/files/39840698.pdf	French	null	Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors	Cancer research	2,013	cc-by	48	Supplemental Figure 2 Supplemental Figure 2 PD-L1 PD-L2 B7.1 CT26 EpCAM+ Tumor cells CD45+ Tumor leucocytes PD-L1 PD-L2 B7.1 ID8-VEGF EpCAM+ Tumor cells ID8-VEGF CD45+ Tumor leucocytes A B A CT26 EpCAM+ Tumor cells CD45+ Tumor leucocytes B B ID8-VEGF EpCAM+ Tumor cells ID8-VEGF CD45+ Tumor leucocytes B7.1
https://openalex.org/W2006566646	https://bmccomplementmedtherapies.biomedcentral.com/counter/pdf/10.1186/s12906-015-0604-9	English	null	Intervention development for integration of conventional tobacco cessation interventions into routine CAM practice	BMC complementary and alternative medicine	2,015	cc-by	12,802	* Correspondence: myram@email.arizona.edu 1Department of Family and Community Medicine, University of Arizona College of Medicine, P.O. Box 245052, Tucson, AZ 85724, USA Full list of author information is available at the end of the article © 2015 Muramoto 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. Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 DOI 10.1186/s12906-015-0604-9 Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 DOI 10.1186/s12906-015-0604-9 Open Access Intervention development for integration of conventional tobacco cessation interventions into routine CAM practice Myra L Muramoto1*, Eva Matthews1, Cheryl K Ritenbaugh1 and Mark A Nichter2 Abstract Background: Practitioners of complementary and alternative medicine (CAM) therapies are an important and growing presence in health care systems worldwide. A central question is whether evidence-based behavior change interventions routinely employed in conventional health care could also be integrated into CAM practice to address public health priorities. Essential for successful integration are intervention approaches deemed acceptable and consistent with practice patterns and treatment approaches of different types of CAM practitioners – that is, they have context validity. Intervention development to ensure context validity was integral to Project CAM Reach (CAMR), a project examining the public health potential of tobacco cessation training for chiropractors, acupuncturists and massage therapists (CAM practitioners). This paper describes formative research conducted to achieve this goal. Methods: Intervention development, undertaken in three CAM disciplines (chiropractic, acupuncture, massage therapy), consisted of six iterative steps: 1) exploratory key informant interviews; 2) local CAM practitioner community survey; 3) existing tobacco cessation curriculum demonstration with CAM practitioners; 4) adapting/tailoring of existing curriculum; 5) external review of adaptations; 6) delivery of tailored curriculum to CAM practitioners with follow-up curriculum evaluation. Results: CAM practitioners identified barriers and facilitators to addressing tobacco use with patients/clients and saw the relevance and acceptability of the intervention content. The intervention development process was attentive to their real world intervention concerns. Extensive intervention tailoring to the context of each CAM discipline was found unnecessary. Participants and advisors from all CAM disciplines embraced training content, deeming it to have broad relevance and application across the three CAM disciplines. All findings informed the final intervention. Conclusions: The participatory and iterative formative research process yielded an intervention with context validity in real-world CAM practices as it: 1) is patient/client-centered, emphasizing the practitioner’s role in a healing relationship; 2) is responsive to the different contexts of CAM practitioners’ work and patient/client relationships; 3) integrates relevant best practices from US Public Health Service Clinical Practice Guidelines on treating tobacco dependence; and 4) is suited to the range of healing philosophies, scopes of practice and practice patterns found in participating CAM practitioners. The full CAMR study to evaluate the impact of the CAMR intervention on CAM practitioners’ clinical behavior is underway. Keywords: Context validity, Intervention protocol, Curriculum development, Training, Interprofessional education, Tobacco cessation, Chiropractic, Acupuncture, Massage therapist, Community based participatory research Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 2 of 14 Background integration to be effective, interventions would at once need to be tailored into real world CAM practices; yet maintain their conceptual integrity and be subject to established evaluation criteria. Complementary and alternative medicine (CAM) ther- apy use is an increasingly important factor in the health care landscape [1]. A number of national surveys indi- cate substantial [2-6] and in some cases growing use [7] of CAM therapies. In the United States (U.S.), between the 2002 and 2007 National Health Interview Survey (NHIS), the proportion of adults reporting use of at least one form of CAM increased from 25.7% to 29.4% - a rela- tive increase of 14.2%. Provider-based CAM (pbCAM, in- cluding all those services which require the presence of a practitioner as contrasted with CAM treatments that can be self-administered, such as herbal medicines) use saw a relative increase of 29.6%. The most significant increases in pbCAM use were seen in chiropractic care, acupuncture, massage therapy, and folk medicine [7]. Harris and colleagues [6] note in their international re- view of CAM use studies that more population-based assessment (i.e. through government sponsored health surveys) of CAM use is necessary to provide a more accurate picture of trends in prevalence of CAM use over time. Project CAM Reach Context validity of the research intervention is a key as- pect of Project CAM Reach (CAMR), a National Cancer Institute (NCI) sponsored study examining the public health potential of tobacco cessation training for chiro- practors, acupuncturists and massage therapists (CAM practitioners). The CAMR study has two main aims. First, develop an intervention protocol, a tobacco cessa- tion brief intervention training and practice-system intervention that includes appropriate tobacco cessation best practices from the U.S. Public Health Service Guideline on Treatment of Tobacco Dependence (PHS Guideline) [19] and is tailored for the needs of CAM practitioners. Second, in the real world of CAM prac- tices, evaluate the impact of the CAMR intervention on CAM practitioners’ knowledge, attitudes and practice behaviors with respect to integration of tobacco cessa- tion practices recommended by the PHS guideline [19]. Context validity of interventions in CAM practices The inspiration for CAMR is three-fold. First, the growing burden of chronic disease is at the heart of the US health care crisis. Chronic disease accounts for more than 75% of health care costs in the US and the steady escalation of the nation’s health care bill is driven in large part by the increasing costs of caring for chronic disease [20-22]. Globally, chronic diseases are the largest cause of death. The leading chronic diseases share com- mon life-style related major risk factors of tobacco use, unhealthy diet, physical inactivity, and alcohol use [23,24]. Second, CAM practitioners have characteristics and practice patterns that make them well suited to ad- dressing lifestyle-related chronic disease risk factors. Third, local CAM practitioners participating in a tobacco-cessation training project for lay community members (described below) requested that tobacco ces- sation training be made more available to their disci- plines [25]. Context validity of interventions in CAM practices Growth in CAM use has inspired innovative attempts to offer patients holistic care through integrating CAM into conventional medical practice [8-10]. By contrast, there has been much less exploration of how interventions widely used in conventional medicine and behavioral health might be effectively incorporated into pbCAM practice as a means of advancing the preventive and pro- motive health goals of both CAM and conventional medicine [11]. CAM practitioners and practices differ markedly from conventional medicine practitioners and practices with respect to professional training, practice patterns, business models, treatment and healing para- digms, philosophical orientation to the patient/client – practitioner relationship and perceptions of treatment effectiveness [11-18]. A central question that arises is whether evidence-based health care and/or behavior change interventions widely employed in conventional medical and behavioral health care could be integrated into the CAM practice context in ways that ensure val- idity within the specific context of a CAM practice while maintaining the conceptual integrity of the evidence- based intervention. In this paper we refer to this concept as “context validity”. Intervention development to ensure context validity requires addressing such questions as: Will these interventions be acceptable to practitioners in different CAM disciplines? Do the interventions “fit” or make sense within the training and healing traditions, scope of practice, and practice patterns relevant to prac- titioners of the specific CAM disciplines who would be asked to implement the intervention? In order for Tobacco cessation and CAM practitioners These CAM practitioners rec- ognized the value of such training for their own practices, and at the end of the study approached the research team with the recommendation and request that cessation training, tailored to the needs of CAM practitioners, be offered to their disciplines. The purpose of this paper is to describe the iterative CBPR process we used to develop the CAMR intervention protocol and the resulting inter- vention that included both a patient/client centered tobacco cessation BI training and a practice system intervention tailored for CAM practitioners. We note that the CAM disciplines participating in the CAMR study customarily use different terms to refer to per- sons seeking their care. Chiropractors and acupunc- turists usually refer to “patients”, whereas massage therapists usually say “clients”. For simplicity, we will use “patients” throughout this paper. CAM practitioners have characteristics and practice patterns that may make them better suited to health and wellness promotion than conventional practitioners. Compared to conventional biomedical practitioners, visits with CAM practitioners are often longer and more frequent [13,34,35], providing more time to ad- dress complex lifestyle issues. They often see patients for regular health maintenance/wellness care, allowing for repeated follow-ups and reassessment of behavioral changes [13]. g Analysis of 2002 and 2007 data from the National Health Interview Survey in the U.S. found that CAM practitioners provide care for significant numbers of smokers [36]. A population-based survey of CAM use in an eastern region of Germany also found that a signifi- cant proportion of CAM users were current smokers (28.6%) [37] Published English-language reports of population-based surveys of CAM use in non-U.S. popu- lation are sparse. Most published reports focus on spe- cific clinical populations, e.g. outpatients to a health center, cancer survivors. Some clinical population stud- ies have reported significant rates of tobacco use among CAM users. A Swedish health centre-based survey of 1442 patients found that among users of manual ma- nipulative CAM therapies, 14.7% were current smokers, and 18.8% were current snuff takers. Of those using ma- nipulative CAM therapies and herbs, 25.6% were current smokers, and 37.5% were current snuff takers [38]. In the U.S., as in some other countries, some populations with higher rates of CAM use are also at higher risk for tobacco use. Tobacco cessation and CAM practitioners Tobacco cessation and CAM practitioners Even after decades of public health tobacco control ef- forts, tobacco remains the single largest preventable cause of death globally [26]. In the U.S., where the current work was conducted, tobacco cessation brief in- terventions (BIs) based on the 5A’s framework (Ask, Advise, Assess, Assist, Arrange) [27], and that also in- clude intra-treatment social support, continue to form the backbone of practice-based conventional healthcare intervention. More recently, BIs are being evaluated in developing nations [28,29]. That said, despite clear evi- dence from the U.S. that BIs by health care providers Page 3 of 14 Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 3 of 14 result in increased tobacco cessation rates [19], and that such BIs are the most cost-effective preventive health services [30], implementation of BIs by biomedical phy- sicians fall far short of the ideal [31]. For nearly 3 de- cades, cessation training in the US has focused on conventional biomedical health practitioners, primarily physicians. Only more recently has cessation training in- cluded non-physicians, e.g. nurses, respiratory therapists, dentists, and dental hygienists [27,32]. But with rare ex- ceptions [33], the focus remains on training biomedical health professionals. from conventional biomedical practitioners, existing tobacco cessation training programs designed for con- ventional practitioners may not be well-suited for CAM practitioners. To ensure that the CAMR intervention had context validity for the three CAM disciplines engaged in this study, we used an iterative and community based partici- patory research (CBPR) approach to develop an interven- tion protocol integrating conventional tobacco cessation interventions recommended by the PHS Guideline into real world CAM practice. The CAMR intervention builds on an existing program of research [25,50-53] that devel- oped the Helpers Program (“Helpers”), the foundational curriculum for the CAMR intervention. Helpers is a community-based brief intervention (BI) training program that teaches lay community members how to offer a struc- tured, four-step “helping conversation” to tobacco users. The helping conversation uses active listening skills and motivational communication strategies to encourage quit- ting tobacco (i.e. permanently stopping/giving up tobacco) without confrontation, or “nagging” [53]. One of the inspi- rations for CAMR came from local CAM practitioners (chiropractor, acupuncturist, and massage therapist) who had participated in a prior NCI-sponsored study of community-based tobacco cessation BI training for the lay public (Project Reach) [25]. Tobacco cessation and CAM practitioners These populations include: the uninsured/ underinsured [4,7,39]; some low-income and rural popu- lations [40-42]; some ethnic/racial minority and new immigrant groups and persons living with specific condi- tions such as HIV/AIDS [43,44], mental illness [45,46] and cancer [47-49]. Step 1 – Exploratory interviews with key informants Methods The purpose of the key informant interviews was two- fold: to gain a better understanding of potential barriers and facilitators that practitioners may have to conduct- ing helping conversations so that these factors could be directly addressed within the intervention, and to inform development of the community survey of local CAM practitioners for Step 2. Initial key informants (chiro- practic, acupuncture and massage therapy) were identi- fied through project investigators’ existing relationships with CAM practitioners in the local Tucson community. These informants referred investigators to additional practitioners for invitation to participate in individual, semi-structured interviews. A total of eleven practi- tioners participated (3 chiropractors, 3 acupuncturists, and 5 massage therapists). The three chiropractors were in private solo practice. One chiropractor was primarily providing locum tenens coverage for other chiropractors in the community. One chiropractor was also a licensed acupuncturist. He used acupuncture and nutrition ad- vice within his practice, but self-identified as a chiro- practor. One chiropractor had originally trained and practiced as a massage therapist before going to chiro- practic school, but self-identified as a chiropractor. He predominately used chiropractic manipulation treat- ments and rarely used massage therapy in his practice. All three acupuncturists were trained in acupuncture and Traditional Chinese Medicine (TCM). Two were in private solo practice; one was also part-time faculty at a local acupuncture/TCM school. The third acupuncturist was president and faculty of an acupuncture/TCM School. He had an active practice within the school and was also faculty for the University of Arizona College of Medicine’s Program in Integrative Medicine. The five massage therapists represented a range of practice con- texts and used a range of massage/bodywork treatments (Swedish, deep tissue, shiatsu, ergonomic evaluation). Three massage therapists were working in relatively large group practices (>5 practitioners); one was a prac- tice owner/practitioner, one as an independent contrac- tor and one as an employee (who also had a mobile private solo practice, delivering treatments in patients’ homes). The other two massage therapists were in pri- vate solo practice; one also had a mobile practice. Three Investigators conducted open-ended, semi-structured interviews with key informants, usually at the practi- tioner’s office. The interview guide addressed the follow- ing domains: practitioners’ attitudes, knowledge, prior training, experience and practice behaviors regarding tobacco dependence and cessation. Methods The CAMR intervention development was designed as an iterative process in which the outcomes from each step were used to inform and shape the next step. The six steps for each type of CAM practitioner were: 1) ex- ploratory interviews with key informants; 2) survey of local CAM practitioner community members; 3) test demonstration of existing curriculum with CAM practi- tioners; 4) adapting and tailoring existing curriculum; 5) external review of adaptations; 6) delivery of tailored curriculum to CAM practitioners with follow-up Despite their increasingly important role in healthcare, and potential to promote tobacco cessation, CAM practitioners have largely been overlooked in the pub- lic health tobacco control agenda. Further, because of the different professional background and training, clinical practice models, scope of practice and practice patterns that clearly distinguish CAM practitioners Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 4 of 14 massage therapists were current or former faculty at local massage therapy schools. curriculum evaluation by trainees. Methods and process outcomes for each developmental step are presented below. The resulting curriculum and intervention then moved into the main research study protocol, described elsewhere. The University of Arizona Human Subjects Protection Program approved the study (Protocol No. 0900000349R002). Informed consent was obtained from each participant involved in the study. These practitioners were chosen because they were: 1) experienced practitioners in their CAM discipline; 2) in active clinical practice at least half-time; 3) known and respected by their professional peers in the local com- munity; and 4) had experience with either CAM pro- fessional education, clinical research, and/or elected leadership in one of their discipline’s professional orga- nizations. Only one practitioner (chiropractor) had prior special interest in tobacco control and had sought out conventional tobacco cessation training. Three practitioners identified members of their office staff as having key roles in practice flow or patient education; these staff members were also interviewed (n = 4). Office staff roles were receptionist (n = 2) and treatment assistant/patient educator (n = 2). Several of the key informant practitioners continued their en- gagement with the project by becoming members of CAMR’s Local Advisory Panel. This panel provided on-going local practitioner input and pilot testing of the subsequent project intervention and evaluation. The panel consisted of 2–4 practitioners from each of the three CAM disciplines involved in the study. Step 2 – Survey of local CAM practitioner community Methods empathy toward tobacco users struggling to quit; com- munication skills (e.g. active listening) that specifically guide Helpers away from confrontation or nagging; as- sessment of readiness to quit (to reduce the inclination to push tobacco users with low readiness to quit); and referral skills to connect tobacco users with established cessation services (e.g. telephone-based tobacco cessa- tion counseling services, or “quit lines”) along with basic information regarding cessation aids recom- mended in the PHS Guideline [19]. The survey of local CAM practitioners was designed to query respondents on domains that might impact ac- ceptability of the CAM Reach intervention and that were found to be salient in the key informant interviews in Step 1. Findings of this step were intended to inform questions that would be asked of participants in the demonstration training in Step 3 so that curriculum con- tent could be tailored for each CAM discipline. The sur- vey was pilot tested with key informants from Step 1, augmented by approximately eight additional local prac- titioners recruited through Step 1 key informants and research team’ personal contacts. A letter introducing the practitioner survey was mailed to all practitioners with an address in the Tucson, Arizona metro area. Address lists were obtained from the Arizona state licensing boards for chiropractors, acupuncturists and massage therapists (N = 1560; chiropractors (N = 187), acupuncturists (N = 126), massage therapists (N = 1247). An introductory email was also sent out through an alumni email list for two local massage therapy schools. The one-page practitioner survey was mailed out two weeks after the introductory communications. The sur- vey queried: years in practice, which CAM disciplines are practiced, prior tobacco cessation training, screening for tobacco use, cessation advice, interest in receiving cessation training, and interest in research participation. No incentives were offered for survey completion. Sur- vey non-responders received one follow-up telephone call, asking practitioners to complete the survey. Helpers incorporates key components of the PHS Guideline recommended 5As [19], but transforms the traditional application of the 5As into a less proscriptive approach that is more tobacco-user centered, and fo- cuses on encouraging tobacco user behavior change that is aligned with the tobacco user’s current willingness/ readiness to take action toward giving up tobacco. This is because the 5As’ proscriptive approach to tobacco cessation was originally developed to guide allopathic physicians in helping patients quit tobacco. Step 2 – Survey of local CAM practitioner community Methods The 5As presumes a provider-patient relationship context and places much emphasis on advising a tobacco user to quit, regardless of their readiness to do so. In contrast, the helping conversation is a 4-step approach that does not presume a particular health care context, is motiv- ational and more tobacco-user centered, and focuses on encouraging behavior change that is in the direction of quitting, yet aligned with the tobacco user’s current willingness/readiness to take action toward quitting. To garner feedback about Helpers training for CAM practitioners, the research team held a one-day Helpers training demonstration and debrief/critique session with seven local CAM practitioners (two chiropractors, three acupuncturists, and two massage therapists) followed by a focus group-type feedback/debriefing session. Practi- tioners were recruited from key informants, key inform- ant referral and our research team’s personal contacts with local CAM practitioners. The number of practi- tioners recruited was based on practical considerations of having representation of each of the CAM disciplines in the study, and a range of practice styles to likely achieve saturation of feedback themes. Practice styles of participants were very similar to those of key informants Step 1. Participants were asked to participate as practitioners-trainees, but also to take notes on feedback forms for the afternoon debrief/critique session. Forms were structured to help elicit specific and detailed feed- back on each section of the training. One CAM practice staff member (receptionist) also participated. After the training demonstration, participants were also shown materials from a 5A’s tobacco cessation training devel- oped for chiropractors (patient handouts, practitioner guides, and display posters) [33]. Participants gave feed- back about the utility of these materials in their practices We described the survey data using means and stand- ard deviations, tabulating for sub-populations using Stata 11 [54]. Step 1 – Exploratory interviews with key informants Methods Also queried were: attitudes, practices and experience with counseling pa- tients/clients on other lifestyle-related issues; patient and practice characteristics; and practice flow and lo- gistical issues important to the intervention model. We probed particular specific areas of possible practi- tioner concern: e.g. how they felt about talking to pa- tients about tobacco when they come in with a specific presenting problem; whether in their experience patients see questions about tobacco as odd or intrusive, or as a routine part of a holistic practitioner’s intake evaluation; whether practitioners were concerned about losing pa- tients if practitioners became more proactive in tobacco cessation counseling, and if practitioners saw their dis- cipline as having a role in public health in general and tobacco control specifically. Interviews were audiotaped and transcribed for later thematic analysis. All transcribed interviews were coded manually for a basic set of themes identified from an ini- tial reading of all interviews. Themes reflected both key investigator interest areas as well as issues that emerged from unprompted discourse. Attention was paid to the multi-vocality of informants who expressed divergent opinions at different points of the same interview indi- cating shifts in context. Page 5 of 14 Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Step 3 – Demonstration of existing tobacco cessation curriculum to CAM practitioners Methods In order to evaluate the extent to which an existing training might be useful as a foundational curriculum for CAM Reach and to identify key areas for tailoring, we demonstrated the Helpers Program (Helpers) an existing tobacco cessation training developed in our prior research [53], for a group of local CAM practi- tioners in Step 3, including some of the key informants from Step 1. The aims of Step 3 were to assess overall acceptability of tobacco cessation training content based on the PHS Guideline, and gather practitioner feedback on the type and extent of revisions needed to tailor the training for each CAM discipline. Helpers is a multi- media, interactive training that emphasizes a tobacco- user centered, non-confrontational and motivational approach to tobacco cessation [53]. More specifically, this curriculum addresses: tobacco addiction (to build Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 6 of 14 and agreed to pilot test the CAM Reach system inter- vention components in their practices (display materials, chart reminders, practitioner guides, patient handouts). Investigators analyzed their notes from the demonstra- tion session (direct observations and practitioner verbal comments) and practitioners’ written feedback for over- arching and convergent themes of (e.g. acceptability of training content) as well as specific critiques and sugges- tions for revision. Approximately 1–2 weeks after the demonstration training, project staff went to each practi- tioner’s office to conduct observations of the practice flow and practice environment. At this visit staff also pilot tested the feasibility and acceptability of employing an in-office “practice patient” (standardized patient) as a final learning activity/readiness assessment to conclude the CAMR training. Feasibility was assessed by conduct- ing the practice patient role-play in each practitioner’s practice location with research staff noting the ease (or difficulty) of completing the practice patient role-play. Immediately after concluding the role-play activity, staff solicited practitioner acceptability feedback about the practice patient experience, e.g. practitioner comfort/ discomfort with activity, perceived value as a learning experience, recommendations for inclusion/exclusion as a learning activity in the final CAMR intervention, and suggestions for improvement. Approximately 2 months later, practitioners participated in a follow-up focus group discussion and feedback about experiences using the new tobacco intervention skills and practice support /system intervention materials. Findings from this step were used in the adaptation and revision of the existing curriculum in Step 4. Step 3 – Demonstration of existing tobacco cessation curriculum to CAM practitioners Methods panel members were selected for their nationally/ internationally recognized subject matter expertise in: education and/or research in one of the three CAM disciplines included in the study (two chiropractors, two acupuncture/Oriental medicine practitioners, and two massage therapists); two integrative medicine (two practitioners); and tobacco cessation research and policy (two experts). Advisors provided structured feedback on: 1) adequacy and appropriateness of desired learner com- petencies, 2) overall instructional approach; 3) learning goals, objectives and instructional activities; and 4) com- pleteness of content. All advisors were sent a curriculum review package containing: 1) reviewer instructions, 2) draft of core competencies for learners’ training goals, 3) detailed out- line of the adapted training curriculum and descriptions of learning activities, 4) reviewer feedback forms which queried: the CAMR intervention’s validity/suitability for the CAM practice context; training content appropriate- ness and completeness; instructional design, length and format acceptability; and opinions regarding dissemin- ation potential. The curriculum development team compiled and analyzed national advisor feedback for over-arching and recurrent themes as well as conver- gence of specific critiques and recommendations. National advisors then participated in a follow-up con- ference call to discuss, clarify and expound upon the panel’s collective feedback. The findings of this stage were integrated into the curriculum in preparation for final pilot testing in Step 6. Step 6 – Pilot test of revised curriculum Methods The new CAM Reach (CAMR) training (1-day training workshop and the follow-up in-office standardized pa- tient exercise) was tested in a pilot of the full training with a second (new) group of CAM practitioners (N = 8), along with investigators (N = 2), and project staff (N = 4) to con- firm integration of results from prior development steps and fine-tune presentation timing and use of multimedia. Practitioners were recruited through key informants, key informant referrals, and the research teams’ personal con- tacts with local practitioners. The number of practitioners recruited was based on practical considerations of having representation of each of the CAM disciplines in the study, and a range of practice styles thought to likely achieve saturation of feedback themes. Practice styles of participants were similar to those of key informants Step 1. The workshop also distributed a number of practice support printed materials described in more detail below (e.g. display posters, informational bro- chures, stickers flag charts of tobacco users). CAM practitioner attendees at this pilot training had not par- ticipated in the demonstration training described above Step 4 – Adaptation and revision of existing curriculum Methods The aim of Step 4 was to adapt the existing Helpers cur- riculum for context validity in each of the three types of CAM practices included in this study, in preparation for external subject matter expert review in Step 5. Practi- tioner feedback from Step 4 was analyzed for consistent themes and convergent recommendations for change. The Helpers training curriculum was deconstructed into major and minor topical areas to facilitate adaptations of existing content areas, addition of new content, and re- arranging the order of topic presentation. The basic organization of the training modules corresponded to each of the four steps of the helping conversation (Awareness, Understanding Helping, Relating) was retained. Step 5 – External subject matter expert review Methods A ten member national advisory panel reviewed the adapted curriculum with the purpose of providing feed- back and advice for further necessary revisions. Advisory Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 7 of 14 Page 7 of 14 Page 7 of 14 chiropractors to be seen as doing more of a public service. [RS, chiropractor] chiropractors to be seen as doing more of a public service. [RS, chiropractor] nor had prior exposure to the training content. The data collection process was the same as in Step 3. Practitioners were asked to participate as trainees/reviewers, while tak- ing written notes on forms specific to each section of the training to provide feedback in the debriefing/critique ses- sion held immediately after completion of the 1-day work- shop. The debriefing session was audio-recorded to provide back up for investigator and project staff notes taken during the session. Research team notes and practi- tioners’ written feedback were analyzed for consistent themes, convergent critiques and recommendations, which informed the revisions leading to the final CAMR training workshop component of the CAMR intervention protocol. Two frequently cited barriers to addressing tobacco use with patients/clients were similar to those encoun- tered among conventional practitioners, i.e. time con- straints [55] and lack of training. “Some [chiropractors] are high volume and won’t take much time, but others will.” [KS, chiropractor, talking about barriers to talking to patients about tobacco cessation] “I hadn’t really thought about why is it I’m not seeing smoking cessation and like I said, I never felt that successful at it, initially, and then, so people have called me and I’ve started to deflect; ‘Why don’t you see someone else who specializes in this?’” [LM, acupuncturist, talking about why she does not routinely address tobacco use in her practice] Step 1 results– Exploratory interviews with key informants Step 1 aimed to better understand CAM practitioners’ potential barriers and facilitators to conducting helping conversations. Major themes from the interviews are summarized in Table 1. “I bet there’s a lot of new information that I’m not aware of, the whole neuro-transmitter thing, I bet there’s a lot of great stuff that I should know. And it would probably prompt me to think about how I use acupuncture and how I might go, OK, if I can understand it in this neurological way, this modern way, how would I bring my acupuncture ideas to bear on that, that would interest me a lot. ‘Cause I think that that piece about any addictive substance is so interesting.” [LM, acupuncturist, talking about her interest in receiving cessation training] Practitioners uniformly felt that tobacco use was detri- mental to patient’s health and that cessation training was relevant to their practices. Three practitioners also viewed engaging in tobacco cessation as a public service or public health role for their CAM discipline. “And I think there’s a lot of chiropractors there, and they see a lot of patients, and this [tobacco cessation] would be one way—chiropractic is supposed to be about creating a healthier body, and therefore, I think chiropractors are perfect for this [promoting tobacco cessation]. Step 1 results– Exploratory interviews with key informants And I think the profession as a whole, if some chiropractors got involved, the American Chiropractic Association, would throw their full support to chiropractors doing something like this, because I would think it would only help “ I probably do not bring it up, um, and I let the client bring it up first, then maybe would go into, the physical effects of that and how its affecting the condition that maybe they’re complaining about, but I think I would Table 1 Major themes from key informant interviews Interest in CAMR Study Thought tobacco cessation was relevant and important to practice; CAMR and participation in tobacco cessation viewed as a public health service Experience w/ Tobacco use (TU) Conversations TU conversations not typically initiated by practitioners unless requested by client/patient; TU not uniformly assessed among new clients/patients; practitioners felt most comfortable with initiating conversations about TU with established clients/patients Barriers to TU Conversations Patient might perceive TU conversations as intrusive - potential client alienation or confrontation; being perceived as giving a “sales pitch”; time constraints; cost effectiveness of TU conversations; scope of practice concerns (massage therapists); potential for patient to be dissatisfied and leave the practice Training Content Desired Tobacco use effects on health and the healing process, link between tobacco use and common presenting problems; TU conversation starters; biomedical and psychological perspectives of tobacco addiction; TU cessation referral resources Tone Desired Encouraging, supportive, focused on listening and referral Environmental/System Change Intake appointments typically long, allow for lifestyle conversations; return client flow allows for follow up conversations; intake forms could be modified to include TU questions; posters and handouts welcomed in practice Research/Training logistics No-cost training and CEUs extremely desirable; practice patient protocol acceptable and positively regarded Table 1 Major themes from key informant interviews Barriers that differed from those commonly cited by conventional practitioners were: perceived intrusiveness or potential patient/client social discomfort or alienation– i.e. social risk [34] and concerns over whether addressing tobacco use fit within their scope of practice. “… one woman I’ve seen off and on for many years, I tried to help her quit smoking with acupuncture and it didn’t work. Now she’s finally quitting. She’s tried and tried and tried. Finally, she’s quitting with that drug Chantix.” [LM, Acupuncturist, talking about difficulty of sustained behavior change] “… when [they are] on the massage table, because they’re naked, and there's a sheet over and they're laying down and I’m standing up and I'm clothed, I've tried to avoid anything that would increase that power differential or increase, maybe, a shame level.” [GA, Massage Therapist] “But what I felt like was, some of the people I worked with who were smoking cigarettes, they were really hoping it [acupuncture] would be magic, and that they wouldn’t have to do any of the emotional work of really looking at the addiction.” [LM, Acupuncturist] “But I do have cases where people are not ready. I think people believe that this [acupuncture] can make them quit. I said, nothing under the sun can make you quit, when you are ready to quit, then you can come to me, and I’ll help you quit. But don’t think that these cigarettes can erased your memory; that you’ve never been smoking before, that you never knew what smoking is all about.” [SL, Acupuncturist] “… so we really have to be extremely careful when we’re making suggestions. There is a away that you can make suggestions based on your own personal experience, or somebody else’s experience that sounds similar to theirs…just passing on that information, you are not prescribing or diagnosing and so we do that sometimes, but we really have to be careful with that.” [CR, massage therapist, talking about discussing tobacco use with client and scope of practice] Informants were also asked whether they thought that a learning/assessment activity that featured an in-office “practice patient” (standardized patient) as a way to evaluate and clinical skills and receive feedback would be useful and acceptable. Participants thought this an in- teresting idea, likely to be clinically useful and well ac- cepted. Table 1 Major themes from key informant interviews Thought tobacco cessation was relevant and important to practice; CAMR and participation in tobacco cessation viewed as a public health service TU conversations not typically initiated by practitioners unless requested by client/patient; TU not uniformly assessed among new clients/patients; practitioners felt most comfortable with initiating conversations about TU with established clients/patients Patient might perceive TU conversations as intrusive - potential client alienation or confrontation; being perceived as giving a “sales pitch”; time constraints; cost effectiveness of TU conversations; scope of practice concerns (massage therapists); potential for patient to be dissatisfied and leave the practice Tobacco use effects on health and the healing process, link between tobacco use and common presenting problems; TU conversation starters; biomedical and psychological perspectives of tobacco addiction; TU cessation referral resources Tobacco use effects on health and the healing process, link between tobacco use and common presenting problems; TU conversation starters; biomedical and psychological perspectives of tobacco addiction; TU cessation referral resources Encouraging, supportive, focused on listening and referral Intake appointments typically long, allow for lifestyle conversations; return client flow allows for follow up conversations; intake forms could be modified to include TU questions; posters and handouts welcomed in practice Intake appointments typically long, allow for lifestyle conversations; return client flow allows for follow up conversations; intake forms could be modified to include TU questions; posters and handouts welcomed in practice No-cost training and CEUs extremely desirable; practice patient protocol acceptable and positively regarded No-cost training and CEUs extremely desirable; practice patient protocol acceptable and positively regarded Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 8 of 14 also need more information about that too, I don’t think—My study at massage school, I actually did research on using massage with alcoholism, but not with tobacco.” [CR, Massage therapist, talking about barriers to talking about tobacco use with clients] also need more information about that too, I don’t think—My study at massage school, I actually did research on using massage with alcoholism, but not with tobacco.” [CR, Massage therapist, talking about barriers to talking about tobacco use with clients] Practitioners conveyed frustration with the difficulty of motivating patient behavior change related to lifestyle is- sues, the associated paucity of sustained behavior change, and patients’ frequent expectations of a “quick and easy” fix – echoing sentiments often expressed by conventional medical practitioners. Table 1 Major themes from key informant interviews Practitioners reported two factors that would encourage their participation in tobacco cessation train- ing: being free of charge and practitioners would receipt continuing education credits for training participation. Practitioners expressed more hesitancy to bring up tobacco use with new patients, preferring to defer ad- dressing tobacco use until later in the relationship. Two practitioners were concerned that raising the issue of to- bacco could potentially to be perceived by patient as a “sales pitch” for additional CAM services. “I think it’s easier done [bringing up smoking] when you have a patient relationship, which is built over the years, it’s much easier to deal with it. You know, if you bring it up to a new patient on a second visit, then it’s sort of you don’t have the trust bond that you do with your older patients.” [RS, chiropractor] Data from Step 1 led us to develop a sensitive and context-driven approach to how and when to approach different patients about tobacco use. It also led us to document that participating practitioners found their pa- tients to be receptive to tobacco conversations. “I could see myself doing it in the clinic maybe after a session, if the conversation had come up, if we were talking about—if they were asking me questions about it, then definitely. Or if I had to approach it with them, I would do it very carefully, in a roundabout way most likely, and then try to have them bring themselves into it. …. I’d want to make sure that they obviously are interested in quitting because it really needs to be them. That’s why I usually let them come to me.” [DD, Massage therapist, talking about speaking to clients about quitting tobacco] Step 3 results - Demonstration of existing tobacco cessa- tion curriculum This step aimed was to evaluate an existing training (Helpers) as a foundational curriculum for CAM Reach and to identify key areas for tailoring. Practitioners reacted positively to the Helpers overall training content and instructional approach, including the patient- centered, motivational focus of the structured helping conversation. They demonstrated keen interest in the pathophysiology of tobacco’s health effects as well as the conventional/PHS guideline-based therapies, particularly cessation medications, wanting to know more so that they would feel comfortable responding to patients’ questions. In the training debriefing, practitioners asked numerous questions and recommended expansion of these two content areas of the training. Despite prompt- ing by investigators, practitioners showed much less interest in hearing more about CAM therapies for cessa- tion. Practitioners wanted inclusion of new and different training tools and patient handouts (e.g. handouts ad- dressing the link between tobacco use and common pre- senting problems of patients; a detailed handout about medications that could be provided to interested pa- tients; and a quick reference of benefits of quitting) and recommended additional skill-building activities in the instructional design. They also made suggestions for types of video role-plays (e.g. depicting practitioner in- teractions with patients who were more resistant to talk- ing about their tobacco use, as well as receptive patients) Specific content was added to address second-hand smoke, and third-hand smoke exposure, and to provide minor expansion of CAM therapies content to address current research about CAM therapies for cessation. Content was added on screening for second-hand smoke exposure in non-tobacco users. A referral resource for patients who were interested in helping the sources of their second-hand smoke exposure – usually a friend or family member - to give up tobacco was also added. This resource is the Helpers Program on-line training, de- scribed above [53]. Finally, learning activities were ex- panded and arranged so that participants would have progressive practice with helping conversation skills over the course of the training, with a summative skills prac- tice role-play at the end of the training. A standardized “practice patient” experience was added as a summative learning activity/skills evaluation to be administered in the practitioner’s office approximately two weeks after the training workshop. The workshop content was reconfigured into an introduction and four modules (Table 3). The total training length was expanded to eight contact hours (7 hour workshop plus 1 hour in- office standardized patient). Step 2 results - Survey of local CAM practitioner community Step 2 aimed to gather information from the local CAM practitioner community on domains potentially effecting acceptability of the CAM Reach intervention (based on results of Step 1). Overall survey response rate was 23% (n = 356), with differences in response rate by discipline: chiropractors, 30% (n = 56); acupuncturists 50% (n = 63); massage therapists, 19% (n = 237). Overall, nearly two thirds (64.6%) of those responding reported no previous Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 9 of 14 Table 2 Prior tobacco cessation training, interest in training by practitioner type Overall Acs DCs MTs (n = 356) (n = 63) (n = 56) (n = 237) % % % % Prior Cessation Training None 64.6 8.5 66.1 78.9 In professional school 19.9 72.9 17.9 6.6 Cont. Education 11.1 35.6 16.1 3.5 Learned on own 17.3 37.3 12.5 13.2 Interest in Cessation Training Yes 66.4 62.3 66.7 67.3 No 10.3 13.2 4.4 10.8 Unsure 23.5 24.5 28.9 22.0 Acs = acupuncturists; DCs = doctors of chiropractic; MTs = massage therapists. Table 2 Prior tobacco cessation training, interest in training by practitioner type and practitioner interview clips for the multi-media as- pects of the training. Despite the differences in profes- sional backgrounds and scope of practice among the three CAM disciplines, there were no recommendations for discipline-specific tailoring other than inclusion of interview clips from the same CAM discipline as the practitioner audience. Practitioners also saw value in keeping the interview clips from different CAM disci- plines and did not recommend limiting clips to practi- tioners from the same discipline as the audience. Practitioners uniformly viewed the in-office “practice pa- tient” (standardized patient) learning activity as a posi- tive, informative experience and supported its inclusion in the final study intervention protocol. Step 4 results – Adaptation and revision of existing curriculum This step aimed to adapt the existing Helpers curri- culum for context validity for each of the three CAM disciplines included in this study. A key conceptual adaptation of the curriculum was to emphasize the role of the relationship between practitioners and patients/ clients. The CAM Reach training was framed as based on three fundamental principles: 1) tobacco cessation is a process, not an event; 2) practitioners can offer helping conversations to a tobacco user at any stage in the process of quitting; 3) helping conversations are part of a supportive, healing relationship. cessation training. Prior cessation training was most common among acupuncturists and least common among massage therapists. Practitioners reported infre- quently advising patients/clients to quit tobacco. Ap- proximately two-thirds of practitioners responding were interested in receiving cessation training. See Table 2. Step 3 results - Demonstration of existing tobacco cessa- tion curriculum Scope of the tobacco problem, tobacco’s effects on health and healing, importance of linking effects of tobacco use to patient’s health concerns, practice systems to identify tobacco use, harm from second hand and third hand smoke exposure, the CAM practitioner’s role in helping, context of helping, getting the helping conversation started, skills practice role play Tobacco products and their harmful constituents, aspects of tobacco addiction (biological, psychological, social), active listening and communication skills (open-ended questions, reframing, body language), motivators and barriers to quitting (i.e. giving up tobacco), assessing readiness to quit, skills practice role play PHS guideline, types of cessation behavioral support services, cessation medications, referral skills, CAM approaches for tobacco cessation, motivational strategies (i.e. motivating and clarifying questions, eliciting ‘change talk’, ‘rolling with resistance’, emphasizing benefits of quitting, negotiating action), importance of continuing to offer helping conversations – even with patients not ready to quit, components of a simple quit plan, skills practice role play Finishing the helping conversation on a positive note, setting the stage/leaving door open to have future helping conversations, tips and strategies for following up, two final skills practice role play Distribution of printed practice support materials, discussion of how to use/implement printed materials to engage patients and promote practitioner’s willingness to help tobacco users quit, explanation of practice patient (standardized patient) office visit activity that was felt to be overly technical and not help- ful to explain or reinforce content. In particular, CAM practitioners in attendance were very positive about the new content on pathophysiology of tobacco health ef- fects and cessation medications. Practitioners commen- ted that although they felt any recommendation to use medications was outside of their scope of practice, they noted that patients frequently ask them about medica- tions (both over-the-counter and prescription). Practi- tioners found the medication information interesting and useful in that they were now more comfortable with offering the CAMR patient handouts about medications and/or directing their patients to physicians, pharmacists or “quit lines” (free telephone-based stop smoking coun- seling services that are widely available in all U.S. states) for more information and assistance with cessation med- ications. Practitioners liked that the CAMR training resulted in new knowledge and skills that were immedi- ately applicable in their practices. Other feedback in- cluded recommendations for minor re-ordering of slides, video role-plays, and practitioner testimonials for better instructional flow. Step 3 results - Demonstration of existing tobacco cessa- tion curriculum The final workshop was accepted for eight hours of continuing education units by the Arizona licensing boards for chiropractic, acu- puncture and massage therapy. Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 10 of 14 Table 3 CAM reach training curriculum modules Training module Content Introduction Overall knowledge and skills goals for the training, three guiding principles of Reach training, four steps of a Helping conversation, video example of helping conversation between practitioner and patient. Module 1 - Awareness Scope of the tobacco problem, tobacco’s effects on health and healing, importance of linking effects of tobacco use to patient’s health concerns, practice systems to identify tobacco use, harm from second hand and third hand smoke exposure, the CAM practitioner’s role in helping, context of helping, getting the helping conversation started, skills practice role play Module 2 - Understanding Tobacco products and their harmful constituents, aspects of tobacco addiction (biological, psychological, social), active listening and communication skills (open-ended questions, reframing, body language), motivators and barriers to quitting (i.e. giving up tobacco), assessing readiness to quit, skills practice role play Module 3 - Helping PHS guideline, types of cessation behavioral support services, cessation medications, referral skills, CAM approaches for tobacco cessation, motivational strategies (i.e. motivating and clarifying questions, eliciting ‘change talk’, ‘rolling with resistance’, emphasizing benefits of quitting, negotiating action), importance of continuing to offer helping conversations – even with patients not ready to quit, components of a simple quit plan, skills practice role play Module 4 - Relating Finishing the helping conversation on a positive note, setting the stage/leaving door open to have future helping conversations, tips and strategies for following up, two final skills practice role play Closure Distribution of printed practice support materials, discussion of how to use/implement printed materials to engage patients and promote practitioner’s willingness to help tobacco users quit, explanation of practice patient (standardized patient) office visit Table 3 CAM reach training curriculum modules Training module Content roduction Overall knowledge and skills goals for the training, three guiding principles of Reach training, four steps of a Helping conversation, video example of helping conversation between practitioner and patient. Final CAMR intervention protocol Final CAMR intervention protocol The final CAMR intervention protocol and content is outlined in Table 4. Broadly, the protocol called for both practitioner education and system change components that create a welcoming and information rich environ- ment for patients. For example, there were seven differ- ent display posters, stickers with tobacco screening questions for intake forms, chart stickers (to signify to- bacco users). The display posters depicted a variety of people with text encouraging patients/clients to ask their Step 6 results – Pilot test of revised curriculum Step 3 results - Demonstration of existing tobacco cessa- tion curriculum As in Step 3, the in-office stan- dardized patient exercise was uniformly viewed as a positive and very helpful learning experience. Step 5 results - External subject matter expert review The purpose was to gather feedback and advice for fur- ther necessary revisions from nationally/internationally recognized experts in the three CAM disciplines, to- bacco cessation, and integrative medicine. Congruent with results from Step 3, national advisors also sup- ported the interprofessional education approach, recom- mending only a minor amount of tailoring for each practitioner type. There was also strong support for the conceptual shift toward a relationship-centered interven- tion approach with an instructional design and activities emphasizing progressive skills building. National advi- sors also provided substantive contextual input on spe- cific issues including: typical content/training received in typical CAM school curricula, professional scopes of practice, integration of conventional therapies, and po- tential practitioner role in discussing/providing informa- tion on cessation medications. National advisor feedback and contextual information informed additional tailoring of curriculum content, patient handouts, and instruc- tional design for the unique needs of chiropractors, acupuncturists and massage therapists. Advisors also commented on the dissemination potential of the pro- posed CAMR intervention and recommended explor- ation of online training possibilities as well as integration of CAMR tobacco cessation training into CAM primary professional education settings. Discussion Researchers conducting CAM research have consistently faced methodological critiques of interventions that lack context validity within real world CAM clinical practice. The CAMR intervention protocol development process addressed context validity from both the perspective of CAM practitioners as well as conventional biomedicine. Incorporation of the latest thinking in tobacco cessation from conventional research as well as formative research with CAM practitioners was essential to the formulation of the three guiding principles of the CAMR interven- tion: 1) tobacco cessation is a process, 2) practitioners can offer helping conversations to a tobacco user at any stage in the process; 3) helping conversations are part of a supportive, healing relationship. The iterative development process also yielded some interesting outcomes. First, the participating practi- tioners expressed much more interest in having more in- formation about biomedical models of the mechanisms of tobacco’s health effects and also cessation medica- tions, than additional information about CAM therapies specifically for tobacco cessation. Discussion with na- tional advisors, indicated that practitioners were likely to be already familiar with therapies from their own system of treatment. Second, the development process did not identify a need to extensively tailor the CAMR intervention for each CAM discipline. Rather, participating practitioners’ and advisors’ comments confirmed earlier formative re- search results about the course content (information and skills training) as having relevance and clinical appli- cation across different CAM disciplines (e.g. body- system specific health consequences of tobacco use, communication skills). Notably, practitioners spontan- eously identified other health behaviors that might be addressed using the same communication skill set. Prac- titioners also pointed to the potential for interprofes- sional education – the opportunity for practitioners from different CAM disciplines to learn from one an- other vis a vis such conduits as videos modeling how practitioners from another CAM discipline approached patients about tobacco their use. An interprofessional approach to training is particularly relevant for those who practice with CAM practitioners from other By attending to context validity, the CAMR interven- tion was able to bridge a gap between the proscriptive 5 A’s approach the PHS Guideline recommends for con- ventional biomedical practitioners (i.e. ask about tobacco use at every visit and advise the user to quit) and the relatively greater hesitancy of CAM practitioners to bring up tobacco use with new patients. CAMR training workshop 7 hour, in-person continuing education workshop (7 CEUs) Practice patient/system change visit 1 hour in-office visit to conduct practice patient assessment and help implement office system changes (1 CEU) Patient education materials 10 brochures: Tobacco and Your Body: Surprising things that you may not know; Secondhand and Thirdhand Smoke: Surprising things that you need to know; Thinking of Quitting Tobacco? We Can Help; Medications that Help with Quitting Tobacco; The Personal Quit Plan; Simple Quit Plan; Quit Line brochure; Helpers Brochure (for those wishing to help others quit tobacco); Roadmap for Quitting Tobacco; Benefits of Quitting Timeline 7 different display posters, stickers with tobacco screening questions for intake forms, chart stickers (to signify tobacco users). Display posters depicted a variety of people with text encouraging patients/clients to ask their practitioner about quitting tobacco or second hand smoke, e.g. “Ask your [practitioner type] about quitting tobacco”, and “Got pain? Did you know that quitting tobacco can help? Ask us how”. Understanding step helps the practitioner to attend to the relationship by asking about the patient’s reasons for wanting to quit tobacco and their readiness to quit to- bacco before offering Helping (e.g. advice, information, motivational strategies) that is in alignment with the pa- tient’s acceptance and readiness to take action. Finally, Re- lating emphasizes the practitioner’s role in attending to the relationship by seeking permission to follow-up and providing ongoing support for behavior change. practitioner about quitting tobacco or second hand smoke, e.g. “Ask your [practitioner type] about quitting tobacco”. One poster’s text addressed pain: “Did you know that smoking can increase your pain? Ask us for help to quit”. Step 6 results – Pilot test of revised curriculum Step 6 aimed to confirm integration of results from prior development steps and identify last revisions needed to produce the final CAMR training intervention. Partici- pant feedback confirmed that national and local advisor recommendations had been effectively incorporated and also recommended the elimination of one learning Page 11 of 14 Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Table 4 Final CAM reach intervention protocol Intervention component Description CAMR training workshop 7 hour, in-person continuing education workshop (7 CEUs) Practice patient/system change visit 1 hour in-office visit to conduct practice patient assessment and help implement office system changes (1 CEU) Patient education materials 10 brochures: Tobacco and Your Body: Surprising things that you may not know; Secondhand and Thirdhand Smoke: Surprising thing that you need to know; Thinking of Quitting Tobacco? We Can Help; Medications that Help with Quitting Tobacco; The Personal Quit Plan; Simple Quit Plan; Quit Line brochure; Helpers Brochure (for those wishing to help others quit tobacco); Roadmap for Quitting Tobacco; Benefits of Quitting Timeline Practice support materials Display posters, intake form stickers, chart stickers, brochure holders 7 different display posters, stickers with tobacco screening questions for intake forms, chart stickers (to signify tobacco users). Display posters depicted a variety of people with text encouraging patients/clients to ask their practitioner about quitting tobacco or second hand smoke, e.g. “Ask your [practitioner type] about quitting tobacco”, and “Got pain? Did you know that quitting tobacco can help? Ask us how”. Discussion The final CAMR inter- vention emphasizes a motivational, relationship-centered approach to the helping conversation, in which the four steps of a helping conversation (Awareness, Understand- ing, Helping, Relating) are sequenced to help the practi- tioner address tobacco use, while attending to the relationship. For example, the Awareness step prompts the practitioner to identify links between the patient’s to- bacco use and their reasons for seeking treatment and to offer the patient information, thus laying groundwork for addressing tobacco use now, or at a future visit. The Page 12 of 14 Muramoto et al. BMC Complementary and Alternative Medicine (2015) 15:96 Page 12 of 14 the focus of the practice-based CAMR study and must be answered be answered before wider adoption of the CAMR intervention protocol. A related research ques- tion follows: Could the same common focus on patient- centered care and the patient –practitioner relationship also help bring together both conventional and CAM Practitioners in collaborative efforts to help patients give up tobacco use? With the growing interest by conven- tional health practitioners and the public in integrative medicine, and CAM practitioners’ growing interest in ways to enhance their contributions to public health education and promotion, this question also deserves further research. disciplines – a common scenario [56]. A third interest- ing outcome were the similarities between the frustra- tions expressed by CAM practitioners and conventional practitioners over the challenges of motivating patients/ clients to make and sustain healthy behavior changes. A limitation of the study is that the participating CAM practitioners self-selected to be in a research study on tobacco cessation, and thus may not be fully representa- tive of the general population of CAM practitioners. There were a limited number of CAM practitioners par- ticipating in the development steps (other than the mail survey). These practitioners also self-selected to partici- pate in an intervention development process, so their re- sults may not be generalizable. Another limitation is the low response rate of chiropractors and massage thera- pists in Step 2. It is possible that the high proportion of respondents with no previous cessation training, and an interest in receiving cessation training is over estimated. Such practitioners may have been more likely to answer a survey about tobacco cessation training and may not reflect the actual need or demand for cessation training among the general population of CAM practitioners. Conclusions CAM practitioners are well suited to delivering tobacco cessation brief interventions to their patients and clients - they have access to tobacco users, motive to take action (desire to promote health, healing and wellness) and op- portunity to intervene (patient/client contact time). An in- clusive and iterative process to develop the CAMR training curriculum and practice intervention, with much formative research, resulted in an intervention protocol that has context validity for CAM practitioners in that it: 1) is patient-centered and emphasizes the practitioner’s role in a healing relationship; 2) is practitioner-friendly in that it is responsive to the different contexts of CAM practitioner practices and their patient relationships; 3) in- tegrates relevant best practices from U.S. PHS Clinical Practice guideline on treatment of Tobacco Dependence; and 4) is suited to the differing contexts of healing phil- osophy, scope of practice and practice patterns found among CAM practitioners. The CAMR practice-based mixed-methods research study currently underway in a larger sample of CAM Practitioners (N = 99) will evaluate the effectiveness of this intervention protocol in changing CAM practitioners’ clinical practice behavior. In conclusion, the CAMR intervention protocol, with its focus on patient-centered care and the role of the patient-practitioner relationship, has potential to serve as a common touchstone that has context validity yet could generalize across three vastly different CAM disci- plines and their varied practice contexts – and connect practitioners in a way conducive to interprofessional education and practice. More importantly, can the CAMR intervention change CAM Practitioner clinical behavior in real-world practice settings? This question is Discussion Shared frustrations over motivating patients to make and sustain healthy behavior change are common among practitioners of all types, providing a departure point for productive dialogue and exchange of experiences. A common desire for more effective ways to promote healthy behavior change provides an opportunity for col- laboration in what we have elsewhere described as a community of cessation practice [57]. This desire can serve as the basis for cessation training in a shared rep- ertoire of behavior change strategies and tools, e.g. helping conversations, active listening skills, and motiv- ational communication strategies that could help bring CAM and conventional practitioners together toward a common goal of reducing tobacco use [57]. Acupuncturists’ higher response rate to the commu- nity CAM practitioner survey may be a reflection of more acupuncturists reporting having had prior training in tobacco cessation, either in their primary professional training or as continuing education. This may indicate greater interest and/or familiarity with the topic of to- bacco cessation and a higher likelihood of responding to a survey about tobacco cessation training. Of the three CAM disciplines participating in our study, only acu- puncturists have specific treatments within their core practices that are for treatment of drug withdrawal. Our national advisors indicated that there is a well-known acupuncture protocol for treating drug withdrawal that can be applied to nicotine withdrawal, and that this protocol is typically taught in acupuncture school. The present study was conducted in the U.S. Primary profes- sional training, scope of practice, and government or industry regulation of CAM practitioners in other coun- tries may be different. Accordingly, care must be taken in any transferability and generalizability of study find- ings and the resulting CAMR intervention protocol to CAM practitioners in other countries. Abbreviations Acs: Acupuncturists; CAM: Complementary and alternative medicine; CAM: Practitioners (chiropractors, acupuncturists, massage therapists); Competing interests The authors declare that they have no competing interests 16. Hibbard JH, Greene J. What the evidence shows about patient activation: better health outcomes and care experiences; fewer data on costs. 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Enabling and sustaining the activities of lay health influencers: lessons from a community-based tobacco cessation intervention study. Health Promot Pract. 2010;11(4):483–92. 51. Castañneda H, Nichter M, Muramoto M. Enabling and sustaining the activities of lay health influencers: lessons from a community-based tobacco cessation intervention study. Health Promot Pract. 2010;11(4):483–92. 52. Yuan NP, Castaneda H, Nichter M, Wind S, Carruth L, Muramoto M. Lay health influencers: How they tailor brief tobacco cessation interventions. Health Educ Behav. 2012;39(5):544–54. First published on October 10, 2011. 52. Yuan NP, Castaneda H, Nichter M, Wind S, Carruth L, Muramoto M. Lay health influencers: How they tailor brief tobacco cessation interventions. Health Educ Behav. 2012;39(5):544–54. First published on October 10, 2011. 53. Muramoto ML, Wassum K, Connolly T, Matthews E, Floden L. Helpers program: a pilot test of brief tobacco intervention training in three corporations. 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References 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: 56. Floden L, Matthews E, Govidajaran L, Muramoto ML. Characterizing Complementary and Alternative Medicine Practices to Identify Opportunities for Health Promotion. Abstract 282549. In: 141st Meeting of the American Public Health Association Annual Meeting. Boston, MA; 2013. https://apha.confex.com/apha/141am/webprogram/Paper282549.html (accessed March 27, 2015). • Convenient online submission 57. Practitioners CAM. Public health partners for promoting healthy lifestyles. Boston, MA: 141st Meeting of the American Public Health Association; 2013. 57. Practitioners CAM. Public health partners for promoting healthy lifestyles. Boston, MA: 141st Meeting of the American Public Health Association; 2013.
https://openalex.org/W3204304457	https://discovery.ucl.ac.uk/id/eprint/10156347/1/Dodds_The%20role%20of%20new%20nuclear%20power%20in%20the%20UKs%20net-zero%20emissions%20energy%20system_VoR.pdf	English	null	The role of new nuclear power in the UK's net-zero emissions energy system	Energy	2,023	cc-by	11,044	The role of new nuclear power in the UK’s net-zero emissions energy system James Price a,, Ilkka Keppo b, Paul E. Dodds a James Price a,, Ilkka Keppo b, Paul E. Dodds a UCL Energy Institute, University College London, London, United Kingdom b Department of Mechanical Engineering, Aalto University, Espoo, Finland a UCL Energy Institute, University College London, London, United Kingdom b Department of Mechanical Engineering, Aalto University, Espoo, Finland A R T I C L E I N F O Keywords: Power system Net-zero energy system Nuclear power Variable renewable energy Swift and deep decarbonisation of electricity generation is central to enabling a timely transition to net-zero emission energy systems. While future power systems will likely be dominated by variable renewable energy (VRE) sources, studies have identified a need for low-carbon dispatchable power such as nuclear. We use a cost- optimising power system model to examine the technoeconomic case for investment in new nuclear capacity in the UK’s net-zero emissions energy system and consider four sensitivity dimensions: the capital cost of new nuclear, the availability of competing technologies, the expansion of interconnection and weather conditions. We conclude that new nuclear capacity is only cost-effective if ambitious cost and construction times are assumed, competing technologies are unavailable and interconnector expansion is not permitted. We find that bioenergy with carbon capture and storage (BECCS) and long-term storage could reduce electricity system costs by 5–21% and that synchronous condensers can provide cost-effective inertia in highly renewable systems with low amounts of synchronous generation. We show that a nearly 100% variable renewable system with very little fossil fuels, no new build nuclear and facilitated by long-term storage is the most cost-effective system design. This suggests that the current favourable UK Government policy towards nuclear is becoming increasingly difficult to justify. inertia is needed to stabilise system frequency and avoid instances of a large rate of change of frequency (RoCoF) which can lead to generator, load and interconnector disconnections. i 1. Introduction There is significant debate in the literature [5–10] regarding the future value of low-carbon dispatchable (LCD) power (nuclear, biomass, hydrogen and fossil fuels with carbon capture and storage) as the share of variable renewables increases. LCD plants can help to ensure that the supply of electricity is adequate (i.e. supply is able to meet demand during the normal operation of the system) and secure (i.e. to meet demand in light of unexpected contingency events such as a generator going offline). A number of studies have shown how LCD plants can reduce total system costs by limiting the need for the overcapacity of VREs and other flexibility options [11,12]. Many studies have highlighted the need for a concerted effort to rapidly transition power systems across the world from fossil fuels to wards low-carbon technologies and particularly renewables (e.g. Ref. [1]. This transition is considered a precursor to electrifying heat and transport and should be largely complete by 2035–2040 in a Paris-aligned world [2]. Wind and solar photovoltaic (PV) generation are already cost- competitive with fossil generation [3] and their global capacities are growing rapidly; for example, the combined wind and solar shares were 29% and 24%1 of total annual electricity generation in 2019 for Ger many and the United Kingdom (UK), respectively. However, power production from VRE is driven by the weather and so it can vary rapidly in time and space. This leads to significant intermittency of supply and a marked paradigm shift from the dispatchable power systems of the recent past. Heuberger and Mac Dowell [4] conclude that a 100% var iable renewable UK system would run into significant operational dif ficulty, with periods of unmet demand and a lack of system inertia. This * Corresponding author. E-mail address: james.price@ucl.ac.uk (J. Price). 1 https://yearbook.enerdata.net/renewables/wind-solar-share-electricity-production.html. Energy 262 (2023) 125450 Energy 262 (2023) 125450 Contents lists available at ScienceDirect Available online 15 September 2022 0360-5442/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. E-mail address: james.price@ucl.ac.uk (J. Price). 1 https://yearbook.enerdata.net/renewables/wind-solar-share-electricity-production.html. https://doi.org/10.1016/j.energy.2022.125450 Received 8 October 2021; Received in revised form 9 September 2022; Accepted 11 September 2022 Available online 15 September 2022 0360-5442/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.energy.2022.125450 Received 8 October 2021; Received in revised form 9 September 2022; Accepted 11 September 2022 1.1. The value of new nuclear power generation Of all LCD technologies, nuclear power generates perhaps the greatest controversy. Although nuclear plants have been operating in several countries for 60 years, the future of nuclear power across Europe is unclear, with France seeking to scale it back from around 70% of Energy 262 (2023) 125450 J. Price et al. Nomenclature Abbreviations BECCS Bioenergy with carbon capture and storage BEIS Department for Business, Energy and Industrial Strategy CCC Committee on Climate Change EDF Electricit´e de France EIA Energy Information Administration ENTSO-E European Network of Transmission System Operators for Electricity EPR European Pressurised Reactor FOAK First of a kind GAMS General Algebraic Modelling System H2-CCGT Hydrogen fired combined cycle gas turbine H2-OCGT Hydrogen fired open cycle gas turbine highRES High spatial and temporal resolution electricity system model HTGRs High temperature gas reactors Hydro-RoR Run-of-river hydropower IEA International Energy Agency IRENA International Renewable Energy Agency JRC Join Research Centre LCD Low carbon dispatchable power LCOE Levelised cost of electricity MAF Mid-term Adequacy Forecast MAF Mid-term Adequacy Forecast NGCCGT-CCS Natural gas fired combined cycle gas turbine with carbon capture and storage NGOCGT Natural gas fired open cycle gas turbine NOAK Nth of a kind O&M Operating and maintenance PyPSA Python for Power System Analysis model RAB Regulated Asset Base REMIX Renewable Energy Mix model RoCoF Rate of change of frequency SC Synchronous condenser Solar PV Solar photovoltaic TSO Transmission system operator UK United Kingdom VRE Variable renewable energy Notation h Hour of the year z Spatial zone, see Fig. 1 s Set of storage technologies Hlevel Amount of energy stored in each zones aggregated reservoir in GWh Hinflow Natural inflow into each zones reservoir in GWh Hspill Hourly power generation from reservoir hydro Hcap Installed power generating capacity in each zone Havail fac Availability factor of reservoir hydropower U Committed units in cluster N Total units in cluster UP Num. units in cluster starting up DOWN Num. 1.1. The value of new nuclear power generation units in cluster shutting down P Power generated (GW) OR Operating reserve provided (GW) FR Frequency response provided (GW) ORquick Quick start OR (GW) FRreq Frequency response required (GW) af Availability factor pmax Maximum power (GW) pmin Minimum power (GW) ormax Maximum OR in reserve time window (GW) frmax Maximum FR in response time window (GW) minup/down Minimum up/down time gen Generation store Storage trade Interconnection trans Within country transmission capex Capital cost fom Fixed operating and maintenance cost varoom Variable operating and maintenance cost crent Congestion rent trade_cost Net of import costs – export revenues annual electricity generation today to 50% by 2035, and Germany intending to phase out nuclear altogether. Studies on the economics of new nuclear investments have reached differing conclusions, with little economic rationale in Sweden [13] and Saudi Arabia [14], but with nuclear a part of the lowest-cost future energy system in Switzerland [15] and Finland [16]. Various studies have taken a Europe wide perspective and found that high renewable penetration and no nuclear power could be a cost-effective solution (see e.g. Refs. [17–19]. F¨alth et al. [20] find that nuclear in Europe could reduce total system costs by 0–19% depending on technology cost assumptions. While others have demonstrated that a highly renewable European system without nuclear and carbon capture could be 30% more expensive [12] with this figure growing further still at very high VRE penetrations [21]. It has also been shown that flexibly operated nuclear power in power systems with high renewable shares can lower system operating costs [22]. the timescale have escalated,2 and it is unclear whether other planned investments at Moorside (Cumbria), Wylfa (Anglesey) and Sizewell (Suffolk) will go ahead. The UK Government has agreed a guaranteed fixed price (“strike price”) for electricity produced from Hinkley Point C of £93/MWh,3 which is substantially higher than the £40/MWh strike price agreed for offshore wind farms opening in 2023–25.4 Nevertheless, in the Energy White Paper published in December 2020, the UK Gov ernment has made clear its continued support for new nuclear capacity [23], in part based on the assertion that nuclear is a “reliable” source of electricity that is needed to help manage VRE intermittency and can reduce system costs. 2 https://www.ft.com/content/fbc43de5-d3ae-49fd-9f5f-9e84f1db508d. 3 https://www.lowcarboncontracts.uk/cfds/hinkley-point-c. 4 https://www.gov.uk/government/publications/contracts-for-difference-c fd-allocation-round-3-results. 4 https://www.gov.uk/government/publications/contracts-for-difference-c fd-allocation-round-3-results. 1.1. The value of new nuclear power generation units in cluster shutting down P Power generated (GW) OR Operating reserve provided (GW) FR Frequency response provided (GW) ORquick Quick start OR (GW) FRreq Frequency response required (GW) af Availability factor pmax Maximum power (GW) pmin Minimum power (GW) ormax Maximum OR in reserve time window (GW) frmax Maximum FR in response time window (GW) minup/down Minimum up/down time gen Generation store Storage trade Interconnection trans Within country transmission capex Capital cost fom Fixed operating and maintenance cost varoom Variable operating and maintenance cost crent Congestion rent trade_cost Net of import costs – export revenues Nomenclature Abbreviations BECCS Bioenergy with carbon capture and storage BEIS Department for Business, Energy and Industrial Strategy CCC Committee on Climate Change EDF Electricit´e de France EIA Energy Information Administration ENTSO-E European Network of Transmission System Operators for Electricity EPR European Pressurised Reactor FOAK First of a kind GAMS General Algebraic Modelling System H2-CCGT Hydrogen fired combined cycle gas turbine H2-OCGT Hydrogen fired open cycle gas turbine highRES High spatial and temporal resolution electricity system model HTGRs High temperature gas reactors Hydro-RoR Run-of-river hydropower IEA International Energy Agency IRENA International Renewable Energy Agency JRC Join Research Centre LCD Low carbon dispatchable power LCOE Levelised cost of electricity MAF Mid-term Adequacy Forecast MAF Mid-term Adequacy Forecast NGCCGT-CCS Natural gas fired combined cycle gas turbine with carbon capture and storage NGOCGT Natural gas fired open cycle gas turbine NOAK Nth of a kind O&M Operating and maintenance PyPSA Python for Power System Analysis model RAB Regulated Asset Base REMIX Renewable Energy Mix model RoCoF Rate of change of frequency SC Synchronous condenser Solar PV Solar photovoltaic TSO Transmission system operator UK United Kingdom VRE Variable renewable energy Notation h Hour of the year z Spatial zone, see Fig. 1 s Set of storage technologies Hlevel Amount of energy stored in each zones aggregated reservoir in GWh Hinflow Natural inflow into each zones reservoir in GWh Hspill Hourly power generation from reservoir hydro Hcap Installed power generating capacity in each zone Havail fac Availability factor of reservoir hydropower U Committed units in cluster N Total units in cluster UP Num. units in cluster starting up DOWN Num. 1.2. Aims and structure of this study As part of our sensitivity analysis, we also make biomass with carbon capture and storage (BECCS) available to the model. All generation capacity online today is assumed to be retired by 2050 apart from Hinkley Point C and Sizewell B, resulting in a total of 4.5 GW of existing nuclear capacity. We note that while Sizewell B is currently due to retire in the mid-2030s, its owner EDF are targeting an extension of its operation lifetime until 2055. 1. Important security of supply options are not considered. The ability for options other than LCD plants to provide inertia and frequency response services are neglected, ignoring the development of syn chronous condensers and batteries. 2. Long-term storage with its potentially important role in providing a multitude of services is not considered. 3. The full role and value of interconnection with Europe is not considered. We represent two energy storage options: grid-scale Lithium-Ion batteries with an 8-h discharge duration, and in our sensitivity analysis we include long-term storage which uses hydrogen produced by elec trolysis as the energy storage medium in salt caverns and H2 burning CCGT and OCGTs for power output (H2-CCGT and H2-OCGT, respec tively). The ratio between the energy and power components (i.e. the discharge duration) for the latter is free for the model to optimise. The technical energy storage potential for hydrogen storage in salt caverns for each country where available is taken from Caglayan et al. [33]. 4. They lack the necessary spatiotemporal resolution to appropriately model highly renewable electricity systems. There is a paucity of work conducted within academia on this important policy question in a UK context, i.e. what is the role of new nuclear in the country’s net-zero energy system, and the aforementioned UK studies from the wider literature have one or more of the short comings outlined. Set against the existing literature which has looked at this question for other individual countries in Europe, focusing on the UK is of interest because it has a considerably higher electricity demand than those nations with its own particular context in terms of, for instance, wind energy and long-term energy storage potential. Furthermore, none of the previous studies looking at new nuclear in other countries, or across Europe as a whole, address all four of the key issues we identify. For instance, while Kan et al. 1.2. Aims and structure of this study [13] model Sweden at high spatiotemporal resolution and embedded within an interconnected European system, they do not consider long-term storage or inertia re quirements for secure system operation. For this work the model is free to expand transmission between zones within the UK while the capacity of interconnection between the UK and Ireland/Europe and between other European countries is a sensitivity dimension that we explore. Cost assumptions for all technologies are given in the Supplementary material. For run-of-river (Hydro-RoR), reservoir hydropower and pumped hydro storage we fix power capacities (i.e. no further investment is permitted) based on data from various sources including the ENTSO-E Transparency Platform5 and Power Statistics,6 and national trans mission system operators (TSO) in the first instance, with some gaps filled based on the JRC’s hydropower database.7 Energy storage ca pacities for reservoir and pumped storage are taken from Schlachtberger et al. [19] and Geth et al. [34] respectively. Inflow into run-of-river and reservoir plants is modelled following the methodology of H¨orsch et al. [35]: run-off data from the European Centre for Medium Range Weather Forecasting ERA5 reanalysis used to set the hourly shape of the inflow time series across the year for each country, with total annual generation fixed to that reported by the Energy Information Administration (EIA),8 in this case for 2012. Therefore, we take the novel step of using a modelling framework, the high spatial and temporal resolution electricity system model (highRES), which addresses the four points raised above and captures a range of sensitivities in terms of weather conditions, technological availability and costs to address our research question. highRES is a member of a class of models, which also includes such examples at REMIX [28], PyPSA [5,6], Calliope [29] and Balmorel [30], that is specifically designed to represent the challenges of VRE intermittency and the solutions to overcome it. It is therefore ideally suited to examine the case for new nuclear power capacity in a net-zero emissions UK energy system. . One important addition we make to the technologies represented in highRES for this study are synchronous condensers (SC). As discussed previously, real world electricity systems need inertia to resist system frequency changes and limit RoCoF, thereby contributing to system se curity. Today, inertia is provided to the system by the kinetic energy in large rotating machines within conventional synchronous generators (e. g. nuclear or gas plants) that are synchronised to the grid. 1.2. Aims and structure of this study In this paper, we explore whether new nuclear beyond Hinkley Point C is likely to be economically-viable or necessary for an adequate and secure UK electricity system embedded within a wider whole energy system that achieves the country’s net-zero emissions goal by 2050. That In the UK, which recently put in place a legally-binding commitment to achieve net-zero greenhouse gas emissions across the whole economy by 2050, the picture for nuclear power is just as unclear. The majority of the existing 9 GW of nuclear capacity is set to retire in the next few decades, with only Sizewell B possibly still generating by 2050. Work has begun on a new nuclear power plant, Hinkley Point C, but costs and 2 J. Price et al. Energy 262 (2023) 125450 as the principal focus of this study, is modelled as multiple zones, with neighbouring countries mostly represented as individual nodes and those further away as regional groups. highRES is a cost-minimising model of this entire system written in the General Algebraic Modelling System (GAMS) language. It simultaneously optimises spatially-explicit capacity investment, based on annualised costs, and hourly dispatch in each of the 18 zones as well as interconnection between them. In addition, highRES also schedules hourly frequency response and oper ating reserve, which can both be provided by thermal generators and storage but not VREs (for details see Ref. [31]. is, does nuclear represent a cost-effective LCD option to support inter mittent renewables? We focus on the third generation European Pres surised Reactor (EPR) pressurised water reactor design that is being built at Hinkley Point C and assume that any other third generation reactors would have similar costs and performance. We do not consider fourth generation modular reactors as these require substantial research and development and have not been constructed commercially, so have no credible cost or performance data. Studies focusing on the future of the UK’s electricity system in the context of its net-zero emissions objective either envisage a sizable build out [24–26] or at least the replacement of existing nuclear capacity [27]. However, these efforts all have some combination of the following methodological issues: We model capacity planning and dispatch for solar PV, on and offshore wind, nuclear, natural gas combined cycle turbines with CCS (NGCCGT-CCS) and open cycle turbines (NGOCGT). 5 https://transparency.entsoe.eu/. 6 https://www.entsoe.eu/data/power-stats/. 7 https://github.com/energy-modelling-toolkit/hydro-power-database. 8 https://www.eia.gov/international/data/world. 1.2. Aims and structure of this study As VRE re places such generators, new sources of inertia provision need to be identified to maintain system stability. SC are large rotating machines without a prime mover, i.e. a local source of initial motive power. Once synchronised to the system frequency, their large spinning mass pro vides inertia to the grid in a similar manner to a synchronous generator, This paper is structured as follows: in the next section we describe the model and methodology we use here, we follow that with a description and discussion of the results and finally we summarise the insights emerging from this study. 9 https://www.nationalgrideso.com/news/latest-boost-stability-pathfinder- construction-flywheel-begins. the formulation is as follows: the formulation is as follows: while drawing a small amount of power from it to overcome frictional losses. They can be based on retrofitting generators so as to utilise existing equipment or built from scratch (here we model the latter op tion). In this study we model an hourly minimum inertia for the UK system, which is driven by an assumed RoCoF limit of 1 Hz/s which leads to a minimum inertia of 41.25 GWs given a largest loss of 1.65 GW (one unit of Hinkley Point C), and an hourly frequency response requirement that is linked to total system inertia (for details see Ref. [31]. SC are well-established and used in Denmark, Italy and Australia because of the inertia they can provide to highly VRE powered systems with low amounts of synchronous generation. Indeed, the UK’s TSO National Grid is currently in the process of procuring SC capacity to support renewable integration in the country’s power system.9 while drawing a small amount of power from it to overcome frictional losses. They can be based on retrofitting generators so as to utilise existing equipment or built from scratch (here we model the latter op tion). In this study we model an hourly minimum inertia for the UK system, which is driven by an assumed RoCoF limit of 1 Hz/s which leads to a minimum inertia of 41.25 GWs given a largest loss of 1.65 GW (one unit of Hinkley Point C), and an hourly frequency response requirement that is linked to total system inertia (for details see Ref. [31]. SC are well-established and used in Denmark, Italy and Australia because of the inertia they can provide to highly VRE powered systems with low amounts of synchronous generation. 2.1. highRES electricity system model 2.1. highRES electricity system model The implementation of highRES we employ in this study is based on that used previously (see Refs. [31,32] but spatially reconfigured and extended to cover the whole of the UK, broken down into 9 zones, and a further 27 European countries aggregated into an additional 9 zones as shown in Fig. 1. The rationale behind this aggregation is a trade-off between spatial detail and computational burden. Therefore, the UK, 3 J. Price et al. Energy 262 (2023) 125450 Energy 262 (2023) 125450 Fig. 1. Map showing the spatial zones used in this work. The UK is composed of 9 interconnected zones with the remaining countries in Europe in various states of aggregation as described in the text. J. Price et al. Fig. 1. Map showing the spatial zones used in this work. The UK is composed of 9 interconnected zones with the remaining countries in Europe in various states of aggregation as described in the text. d in this work. The UK is composed of 9 interconnected zones with the remaining countries in Europe in various states of 2.3. Sensitivities Here we consider a number of important sensitivity dimensions which, while not exhaustive, do represent some of the key uncertainties relevant to our research question. First, given our focus on the prospects for nuclear power in the UK’s net-zero aligned electricity system, we consider two capital cost levels for nuclear capacity. Both are based on analysis conducted for the Department for Business, Energy and Indus trial Strategy (BEIS) by Leigh Fisher and Jacobs [39] and consider a first of a kind (FOAK) overnight capital cost of 3927 £2010/kW and a nth of a kind (NOAK) cost of 3520 £2010/kW, or an approximate reduction of 10% for subsequent plants. This is to capture the uncertainty over whether the build out of new nuclear units will be relatively small in number, and hence a FOAK capital cost more appropriate, or more substantial, and so a NOAK costing more representative. Furthermore, we also assume a 10 and 6 year construction time for the FOAK and NOAK cases, respectively, when calculating addition capital costs from interest during construction. Finally, we represent the planning and operation of thermal plants and long-term storage as clustered units following Palmintier [36] whose integer decision variables are relaxed to be linear, which offers a significant computational speed up with only a limited loss of accuracy. For similar reasons, and given our focus on the UK, we opt to only apply these linearised unit commitment constraints, including the operating reserve and frequency response equations, to the UK system. The Eu ropean system is then modelled using the standard linear approach as in Zeyringer et al. [32]; where technologies are represented as continuous lumps of capacity rather than units. Second, we model the four technology availability scenarios listed in Table 1. We begin with a BASE case of technologies that are either available at scale today or, have been well demonstrated as for NGCCGT- CCS. We then add two more options that are at a somewhat earlier stage in their development: BECCS and long-term storage. BECCS is currently being trialled in the UK by Drax at their site in North Yorkshire and has been identified as being important for a net-zero energy system due to the negative emissions it can provide. 2.3. Sensitivities Long-term storage is also being seen as potentially crucial, with BEIS recently announcing a competition with a total funding pot of £68 million to support the demonstration of long-term storage.11 An “ALL” scenario then considers the simultaneous availability of both BECCS and long-term storage. 10 Open Power System Data. 2020. Data Package Time series. Version 2020-10- 06. https://doi.org/10.25832/time_series/2020-10-06. the formulation is as follows: Indeed, the UK’s TSO National Grid is currently in the process of procuring SC capacity to support renewable integration in the country’s power system.9 Hlevel h,z = Hlevel h−1,z + Hinflow h,z −Hgen h,z −Hspill h,z (1) Hlevel h,z ≤Hmax level z (2) Hgen h,z ≤Hcap z × Havail fac (3) Hlevel h,z = Hlevel h−1,z + Hinflow h,z −Hgen h,z −Hspill h,z Hlevel h,z = Hlevel h−1,z + Hinflow h,z −Hgen h,z −Hspill h,z (1) Hlevel h,z ≤Hmax level z Hlevel h,z ≤Hmax level z (2) Hgen h,z ≤Hcap z × Havail fac (3) where Hlevel is the amount of energy stored in each zones aggregated reservoir in GWh, Hinflow is the natural inflow into each zones reservoir in GWh, Hgen is the hourly power generation, Hspill is the water spilled by the reservoir in each hour in GWh, Hcap is the installed power generating capacity in each zone and Havail fac the availability factor of reservoir hydropower. The subscripts h and z are indices for hour and spatial zone, respectively. where Hlevel is the amount of energy stored in each zones aggregated reservoir in GWh, Hinflow is the natural inflow into each zones reservoir in GWh, Hgen is the hourly power generation, Hspill is the water spilled by the reservoir in each hour in GWh, Hcap is the installed power generating capacity in each zone and Havail fac the availability factor of reservoir hydropower. The subscripts h and z are indices for hour and spatial zone, respectively. The formulation of highRES has previously been described in detail in Price et al. [31] and Zeyringer et al. [32]; so here we only describe additional equations developed for this study. These have been added to: i) model the operation of reservoir hydropower, and ii) model the unit operability of CCGTs and OCGTs coupled to long-term storage … For i), For ii), the formulation is: For ii), the formulation is: Uh,z,s ≤Nz,s (4) Uh,z,s = Uh−1,z,s + UPh,z,s −DOWNh,z,s (5) Uh,z,s ⋅pmax s ⋅≤afs ≥Ph,z,s + ORh,z,s + FRh,z,s (6) Uh,z,s ≤Nz,s (4) Uh,z,s = Uh−1,z,s + UPh,z,s −DOWNh,z,s (5) Uh,z,s ⋅pmax s ⋅≤afs ≥Ph,z,s + ORh,z,s + FRh,z,s (6) (4) Uh,z,s ≤Nz,s Uh,z,s ≤Nz,s Uh,z,s = Uh−1,z,s + UPh,z,s −DOWNh,z (5) (6) 4 J. Price et al. Energy 262 (2023) 125450 Ph,z,s ≥Uh,z,s . (12) (12) FRh,z,s ≤Uh,z,s⋅frmax s Here (4) limits the number of committed storage units to the number actually deployed, (5) is a commitment balance between hours, (6) limits maximum generation, (7) limits minimum generation, (8)–(9) ensure minimum up and down times, respectively, are enforced, (10) limits the provision of operating reserve while offline to H2-OCGT only due to its rapid start up capability and (11)–(12) limits the provision of operating reserve and frequency response by cluster to the maximum ramp achievable in the relevant time window (20 min for operating reserve, 10 s for frequency response). In these equations the subscript s are the set of storage technologies which are represented as units, i.e. H2- OCGT and H2-CCGT. 2.2. System boundaries To model the design of UK power systems embedded within a wider energy system that achieves the countries net-zero emissions target by 2050, we constrain annual grid CO2 intensity to be ~2 gCO2/kWh based on the Balanced Pathway from CCC [27]. We also extend this definition of a net-zero compatible power system to cover Europe to ensure that the UK is not importing high-carbon electricity. p g g y Hourly annual demand for 2050 for both the UK and the European zones is based on metered data from Open Power System Data10 for 2012. As noted in Section 1, a substantial proportion of heat in buildings and road transport is likely to be electrified in the future, which would change the shape of the demand profile. Furthermore, the electrification of the former energy service demand, combined with the likely deployment of large amounts of VREs, will act to increase the coupling between supply and demand under a common meteorology. To capture both the change in total demand and the hourly shape, we take our 2012 demand profiles and develop a regression model for each country, based on an hourly extension of the approach used by Scapin et al. [37] and informed by the methodology of Wang and Bielicki [38]; to separate out electricity demand into a temperature dependent and independent portions. We then augment this set of weather-independent profiles with an assessment of hourly electrified heat, ensuring that the weather driving both VRE supply and heat demand are the same, and electric vehicle demands. For further details, see the Supplementary material. Third, previous studies have highlighted the potentially critical role that the expansion of interconnection between countries can play in supporting the integration of high VRE shares. However, wider factors beyond simple least-cost solutions, such as Brexit, mean there are un certainties as to the expansion of the UK’s interconnection with Europe. We explore two situations in an attempt to capture this sensitivity. In the 11 https://www.gov.uk/government/publications/longer-duration-energy-sto rage-demonstration. the formulation is as follows: pmin s (7) Uh,z,s ≥ ∑ h h−minup s UPh,z,s (8) Nz,s −Uh,z,s ≥ ∑ h h−mindown h DOWNh,z,s (9) ( Ns −Uh,z,s ) ⋅pmax s ⋅afs ≥ORquick h,z,s s = H2 −OCGT (10) ORh,z,s ≤Uh,z,s⋅ormax s (11) FRh,z,s ≤Uh,z,s⋅frmax s (12) J. Price et al. Ph,z,s ≥Uh,z,s . pmin s (7) Uh,z,s ≥ ∑ h h−minup s UPh,z,s (8) Nz,s −Uh,z,s ≥ ∑ h h−mindown h DOWNh,z,s (9) For the UK, this methodology leads to annual heat in buildings de mand that ranges from 74 to 107 TWh depending on the weather year, EV demand of 84 TWh, and total demand from 628 to 661 TWh. The total demand figure includes an additional 155 TWh from the Climate Change Committee’s (CCC) Balanced Pathway which stems from manufacturing and construction, fuel supply, other demands and hydrogen production via electrolysis (not for use in the power sector). We assume these demands have a flat profile over the year. (9) li When BECCS is available to the model we limit the biomass potential to ~61 TWh, as used by the CCC’s Balanced Pathway in 2050, permit ting ~17 TWh per year of electricity generation given a technology ef ficiency of ~28%. As shown by Fig 2.8 of CCC [27]; this is similar to today’s use of biomass in the UK’s power sector and ensures a degree of sustainability around this controversial technology. ( Ns −Uh,z,s ) ⋅pmax s ⋅afs ≥ORquick h,z,s s = H2 −OCGT (11) ORh,z,s ≤Uh,z,s⋅ormax s Table 1 Definition of the technology availability scenarios used in this work. 11 https://www.gov.uk/government/publications/longer-duration-energy-sto rage-demonstration. 5 J. Price et al. Energy 262 (2023) 125450 more conservative case, we fix the capacity of interconnection between countries to the 2027 net transfer capacities proposed by ENTSO-E’s Mid-term Adequacy Forecast (MAF) in 2018. This means the UK has 16.5 GW of interconnection with Europe (including land-based trans mission between Northern Ireland and Ireland) in our modelling (we update the MAF data for links between the UK to Norway and Denmark down from 4 GW to 2.8 GW which represents Viking Link and NSL). In addition, we limit the UK’s maximum hourly net imports to 30% of the country’s total hourly demand, noting that to date during 2021 this peaked at ~23% and will very likely rise in future as new inter connectors come online. The second, more ambitious case, allows highRES to optimise interconnection capacity up to a limit of 50 GW per link between countries. Here we allow hourly net imports into the UK to peak at 50% of total hourly demand. Note that in both situations the model can expand the transmission system within the UK to its optimal level. i ~300 GW or more, and run-of-river and pumped hydro, whose capacity is fixed and does not change across the cases. The dashed line represents the total capacity of existing nuclear power expected to be online by 2050 (4.5 GW), thereby making any new investment clear. The results for the H2 scenario are not plotted as these are the same as the ALL technology set, as BECCS is not deployed in any cases where long-term energy storage is also available. In an average weather year, new nuclear is only cost-effective under the BASE technology scenario with NOAK nuclear capex and no inter connector expansion allowed, with 4 GW of new capacity installed. Permitting interconnector expansion under NOAK costs leads to no new nuclear capacity and the same is true under a switch to FOAK capital costs, regardless of the interconnector dimension. Furthermore, the remaining 8 cases plotted in the average weather year panel do not have any new build nuclear capacity. A similar pattern is observed for the worst weather year available in our sample of historic conditions but with a greater build out of nuclear than under average conditions, with the BASE-NOAK-NO case seeing an additional 13 GW of capacity. Table 1 The best year sees no new build nuclear across all cases, which highlights the impact that the variability of weather within different weather years can have on VREs and, as a result, the wider power system design. Never theless, these panels clearly show that new nuclear power only features when the sensitivity dimensions are most in its favour, i.e. NOAK capex, no interconnection expansion and BECCS and long-term storage being unavailable. If any of these change then new nuclear is not seen to be cost-effective. Our fourth and final sensitivity dimension is the choice of weather year used to drive the production of wind and solar power and the de mand for heat in our model. Past studies have demonstrated that different weather years lead to different optimal system designs [32] and it may be the case that a relatively “poor” weather year, in which low wind/solar output coincides with cold temperatures and hence higher heating demands, would result in a more prominent role for nuclear power. However, running all of the 25 weather years (1993–2017) that highRES currently includes in conjunction with the other sensitivity dimensions discussed above would be computationally expensive. While there are many options for identifying the “worst”, “average” and “best” years, here we take the route of running all the available weather years, one at a time, with the BASE technology options, FOAK nuclear costs and fixed interconnection capacity. From this we obtain a distribution of 25 total system levelised cost of electricity (system LCOE; that is total sys tem cost divided by demand for the combined UK and European system). We assume the “worst” weather year (2010) for the modelled system has the highest LCOE, as it requires the highest combined expenditure for infrastructure planning (i.e. generators, storage, transmission and interconnection) and operational costs (fuel costs). The “average” (1995) and “best” (2014) years are then the median and lowest system LCOE, respectively. i Fig. 2 also shows a number of other insights. First, when intercon nection expansion is permitted, the model consistently opts to sub stantially increase its capacity while reducing the role for batteries and, where available, long-term storage. This indicates the cost-effective nature of this form of flexibility for VRE integration. Table 1 That said, this does lead to an import dependence for the UK under the BASE and BECCS scenarios of up to ~6% and ~14% of annual demand, respec tively, in the case of average weather conditions. While cost-effective, it is a political decision as to whether this amount of annual net imports is acceptable from a domestic energy security standpoint. Second, across all the weather conditions studied here, the addition of BECCS to the system drives a pronounced reduction in the deploy ment of NGCCGT-CCS in favour of NGOCGT and an increase in gener ation from the latter (see Fig. 3). This shows that when the annual CO2 emissions budget can be extended by the negative emissions afforded by BECCS, open-cycle turbines represent a cheaper option to manage VRE intermittency and meet system adequacy/security requirements. Furthermore, our results indicate that at most 2.2 GW of BECCS is needed to drive this switch and to make new build nuclear cost ineffective. Exploring all permutations of the sensitivity dimensions defined above results in 48 model runs (4 technology scenarios x 2 nuclear capex levels x 2 interconnection states x 3 weather years) which are executed using GAMS 27.2 and CPLEX 12.9 on a cluster computing environment. 2.4. UK system cost As discussed by Kan et al. [13]; while the total cost of the combined system is a foundational output of optimisation models, here we wish to focus on the implications of our sensitivity dimensions for a specific country, the UK. To do that we draw from that work and make use of their definition of a total nodal system cost (equation (3) from that paper) and nodal LCOE (equation (4) from that paper). This approach captures all investments and operational costs incurred within the UK power system, considers the cost impact of the net electricity trade balance over the year and includes the share of congestion rent earned by the UK. Third, 1.5–6.3 GW of SC capacity is deployed in the BASE cases where new nuclear is not built and under every combination of the ALL technology scenario. This is because it provides inertia to support secure high VRE penetration and is seen to be more cost effective than nuclear power or BECCS to provide this service when long-term storage is available, despite consuming a small amount of electricity when online. This underscores the potentially important role this technology can have in future highly renewable systems which are inherently low on con ventional synchronous generation. Finally, when available, long-term storage displaces all or nearly all fossil generation in all weather conditions. The small amounts of NGOCGT deployed are in line with the assumption used here that a grid emissions intensity of ~2 gCO2/kWh is compatible with a net-zero emissions energy system. Here H2-OCGT is generally seen to dominate over H2-CCGT in capacity terms, indicating that the system benefits from the greater flexibility and lower capital costs provided by the former, while still seeing a role for both. Furthermore, Fig. 3 shows that long-term storage also acts to reduce the import dependence, even to the extent of making the UK a small net exporter annually for average weather conditions. As this system design is chosen when all technology 3.1. Net-zero compatible system designs 3.1. Net-zero compatible system designs First, we examine the cost-optimal role of nuclear and other LCD power plants for each of our 48 scenarios. Each scenario represents an adequate and secure electricity system for the weather year. Fig. 2 shows the installed capacity of all generator, storage and interconnection op tions by sensitivity case. We do not include wind and solar PV, which act to make the plots difficult to interpret owing to their total capacity of 6 Energy 262 (2023) 125450 Fig. 2. UK installed capacity by sensitivity with the three panels representing weather conditions. VREs are excluded to improve the readability of the figure due to their large installed capacities. Hydropower is also excluded as this does not change across the cases. The black dashed line shows the existing nuclear capacity in 2050 and allows the easy identification of new capacity being installed. J. Price et al. J. Price et al. Energy 262 (2023) 125450 Fig. 2. UK installed capacity by sensitivity with the three panels representing weather conditions. VREs are excluded to improve the readability of the figure due to their large installed capacities. Hydropower is also excluded as this does not change across the cases. The black dashed line shows the existing nuclear capacity in 2050 and allows the easy identification of new capacity being installed. options are available, this also highlights that a close to fossil free, highly renewable system, enabled by long-term storage, is seen to be the most cost-effective scenario explored here. This implies the added flexibility coming from BECCS (i.e. extending the annual carbon budget resulting in a greater role for NGOCGT) provides relatively greater cost benefits, and is more valuable in poorer weather conditions and when interconnector capacity is fixed. i The availability of long-term storage drives even greater cost re ductions of 9–21%, again depending on the level of each sensitivity. Once more, the smallest reductions are seen for the best weather year with the average and worst conditions leading to large system LCOE decreases of 16–21%. More substantial reductions are found when further interconnection expansion is not allowed, again demonstrating that additional forms of system flexibility are most valuable when other options are unavailable. i 3.2. UK system LCOE Again, VREs are not shown for readability. sensitivity with the three panels representing the weather condition dimension. The country’s net import position is also sho adability. Fig. 3. UK annual generation by sensitivity with the three panels representing the weather condition dimension. The co Again, VREs are not shown for readability. pronounced drop in costs associated with electricity trade, as the UK reduces its import dependence. Cost increases from the BASE scenario are largely related to the BECCS cases having greater generator variable O&M costs, driven by fuel costs for BECCS and natural gas, while the ALL cases see a rise in capex and fixed O&M costs associated with storage. Overall, as discussed above, both the BECCS and, even more so, ALL scenarios provide sizable annual cost savings compared to BASE. pronounced drop in costs associated with electricity trade, as the UK reduces its import dependence. Cost increases from the BASE scenario are largely related to the BECCS cases having greater generator variable O&M costs, driven by fuel costs for BECCS and natural gas, while the ALL cases see a rise in capex and fixed O&M costs associated with storage. Overall, as discussed above, both the BECCS and, even more so, ALL scenarios provide sizable annual cost savings compared to BASE. a small (~2%) drop in VRE share for the worst weather year while the average and best conditions modelled here see a small increase (~1%). This occurs because when interconnectors expand to leverage the geographic diversity of weather conditions across Europe the share of UK domestic VRE generation drops more than total UK domestic gen eration under the worst weather conditions. Nevertheless, these runs highlight how BECCS could support a UK power system that generates 80% or more of its electricity from VRE annually. The scenarios where long-term storage is available to the model see some of the highest VRE shares across the sensitivities at 94–95%, a finding that is essentially independent of annual weather conditions. This consistency shows that cost effective systems based on nearly 100% VRE generation, with no new build nuclear, can be robust to a variety of weather conditions when supported by long-term storage. Again, there is a small effect from further interconnector deployment resulting in a drop in VRE share, though at <1% this is very minor. 3.3. UK VRE share in annual generation Fig. 6 shows the share of VRE generation in total annual UK domestic generation by sensitivity and for each weather year. For the BASE technology scenario, VRE shares vary from 87% to over 95% depending on sensitivity, with the former being associated with, as might be ex pected, more pessimistic conditions for VREs, i.e. lower nuclear costs, worse weather and interconnection expansion not being permitted. Combining the results shown in Figs. 2 and 6 indicates that the spread in VRE penetration for the BASE-NOAK-NO case is likely driven by new nuclear capacity being deployed in the worst weather year. 3.2. UK system LCOE These results highlight that long-term storage enables the integration of some of the highest amounts of domestic VRE generation in this study. 3.2. UK system LCOE Fig. 4 shows the UK system levelised cost of electricity (LCOE), which is the total UK nodal system cost divided by the total annual electricity demand, for all 4 of the sensitivity dimensions. We use a nodal system cost approach that accounts for capital, operational and trade costs associated with the node or collection of nodes of interest. The highest system costs are found for the BASE technology scenario and range from 52 to 70 £/MWh for the best to worst weather year, respectively. Permitting the model to expand interconnection leads to an average cost reduction of 6.9% for BASE cases. The overall result of the system LCOE reductions identified above, that is greater cost reductions for the average and worst weather cases, is to compress the difference between the weather years. To understand what is driving this, in Fig. 5 we plot the average change in absolute costs when moving from the BASE to BECCS (upper panel) or ALL (lower panel) scenarios by weather year broken down into the cost components that make up the UK nodal system cost. Both panels show a reduction in annual generation capital and fixed operating and maintenance (O&M) expenditure, which is larger in the average and worst weather years compared to the best year. The ALL technology scenario also shows a The introduction of BECCS leads to a 5–15% cost reduction across the sensitivity cases. The smallest reductions occur for the best weather conditions which are found to be essentially independent of nuclear cost or interconnector expansion. Reductions of 10–15% are seen for the average and worst weather conditions with the cases where intercon nection expansion is not permitted consistently showing greater re ductions compared to their equivalent scenario with expansion allowed. 7 J. Price et al. Energy 262 (2023) 125450 Energy 262 (2023) 125450 Fig. 3. UK annual generation by sensitivity with the three panels representing the weather condition dimension. The country’s net import position is also shown. Again, VREs are not shown for readability. J. Price et al. Energy 262 (2023) 125 . 3. UK annual generation by sensitivity with the three panels representing the weather condition dimension. The country’s net import position is also sho ain, VREs are not shown for readability. rice et al. Fig. 3. UK annual generation by sensitivity with the three panels representing the weather condition dimension. The country’s net import position is also shown. 12 https://www.gov.uk/government/news/government-progresses-demonst ration-of-next-generation-nuclear-reactor. 4. Discussion For the BECCS cases, while there are differences between the different weather conditions, the share of generation from VREs is relatively consistent across the sensitivity options for a given weather year. Allowing the construction of new interconnector capacity leads to Previous UK studies have envisaged a sizable build out [24–26] or at least the replacement of existing nuclear capacity [27]. These strategies 8 J. Price et al. Energy 262 (2023) 125450 Fig. 4. UK system LCOE by sensitivity. The weather dimension is shown using marker style and colour such as Best is red, Average is green and Worst is black. ty. The weather dimension is shown using marker style and colour such as Best is red, Average is green and Worst is black. Fig. 4. UK system LCOE by sensitivity. The weather dimension is shown using marker style and colour such as Best is reflect comments in July 2021 from the UK Minister of State for Energy that “while renewables like wind and solar will become an integral part of where our electricity will come from by 2050, they will always require a stable low-carbon baseload from nuclear.“12 We have shown that new nuclear power generation is not necessary to provide electricity system adequacy and security. Even with challenging weather condi tions, VRE generation could supply over 90% of total generation annually if coupled with technologies that provide or support system flexibility such as long-term storage, batteries, interconnector expansion and synchronous condensers. There are no substantial barriers to the use of these technologies in the UK. Fig. 5. Average UK total system cost change from the BASE technology sce nario by weather year. Average is across the nuclear and interconnection di mensions. Cost categories are “gen” for generation including SC, “store” for storage, “trade” for interconnection and “trans” for within country trans mission. Cost terms are “capex” for capital cost, “fom” for fixed operating and maintenance cost, “varom” for variable operating and maintenance cost, “crent” for congestion rent and “trade_cost” the net of import costs – export revenues. Net change is denoted by black marker. We have not modelled demand-side measures in this study, which would offer another source of flexibility to the system and would help support the integration of VREs, due to significant uncertainty around the scale of their utilisation. We therefore consider our results a pessi mistic case for VREs. 4.1. Cost assumptions We assume an overnight capital cost for nuclear of 3927 £2010/kW (FOAK) and 3520 £2010/kW (NOAK). Assuming a 9% discount rate for the private sector [40], similar to Hinkley Point C [41], these are equivalent to LCOEs of 86 £2010/MWh and 68 £2010/MWh, respectively. These include interest during construction based on 10 and 6 year construction times for FOAK and NOAK respectively, where the latter should be regarded as very optimistic given construction lead times in the recent past [42]. Fig. 5. Average UK total system cost change from the BASE technology sce nario by weather year. Average is across the nuclear and interconnection di mensions. Cost categories are “gen” for generation including SC, “store” for storage, “trade” for interconnection and “trans” for within country trans mission. Cost terms are “capex” for capital cost, “fom” for fixed operating and maintenance cost, “varom” for variable operating and maintenance cost, “crent” for congestion rent and “trade_cost” the net of import costs – export revenues. Net change is denoted by black marker. However, it is not clear that NOAK costs would be lower than FOAK costs. Negative learning-by-doing has been measured for the French and USA nuclear programmes (i.e. capital costs increased rather than reducing as the programme progressed). At best, costs have been reduced only very slightly in major programmes, for example in South 9 J. Price et al. Energy 262 (2023) 125450 Fig. 6. UK annual share of domestic generation from VRE by sensitivity. The weather dimension is again expressed using marker colour and style with Best as red circles, Average as green crosses and Worst as black dashes. Fig. 6. UK annual share of domestic generation from VRE by sensitivity. The weather dimension is again expressed using marker colour and style with Best as red circles, Average as green crosses and Worst as black dashes. Fig. 6. UK annual share of domestic generation from VRE by sensitivity. The weather dimension is again expressed using circles, Average as green crosses and Worst as black dashes. Korea [43]. An assessment of US LCOEs concluded that cost estimates tend to be overly low and that historical analogues provide a better indication of likely costs [44]; one reason is that nuclear reactors are the most prone to cost overruns as a percentage of budget and frequency [45]. In contrast, solar PV and wind generation technology costs have reduced substantially over the last decade. 14 https://www.gov.uk/government/news/government-progresses-demonst ration-of-next-generation-nuclear-reactor. 13 https://www.gov.uk/government/consultations/regulated-asset-base-ra b-model-for-nuclear. 4.3. Role of generation IV reactors The UK Government has funded a £170 million Advanced Modular Reactor Demonstration Programme for Fourth Generation high tem perature gas reactors (HTGRs).14 They have identified markets including hydrogen production and high temperature heat production to decar bonise heavy industry. These reactors are much smaller than Third Generation reactors and are designed to be modular in nature in order to reduce capital costs through learning-by-doing. Existing nuclear reactor sizes have broadly increased over time to benefit from economies of scale [47], as smaller reactors need similar investments in safety systems as large reactors. While HTGRs are designed with inherent safety features, it is not clear that they could be operated safely without a similar range of safety systems. Hence while individual reactors would be smaller and cheaper than Third Generation reactors, it is not clear that the LCOE would be lower. As such plants have not yet been developed, there is no credible cost data and we have not considered them in this study. There are, however, questions about both the cost and the deliverability of these reactors for a net-zero system. The UK Government has proposed to fund new nuclear plants using a Regulated Asset Base (RAB)13 model that would substantially reduce the discount rate and hence the strike price by making UK consumers liable for cost overruns and the risk of plants not being completed [46]. Given that this study has shown that a lower-cost low-carbon electricity system could be built using alternative technologies, it is questionable whether such favourable treatment can still be justified for nuclear generation, even before considering issues such as safely storing nuclear waste and hazards from low-probably but high-impact accidents. 4.1. Cost assumptions Offshore wind strike prices for UK farms being built now are substantially lower (40 £/MWh) than the costs of new nuclear generation, even before accounting for nuclear cost overruns. system, in large part because of the necessary trade-offs between tech nical, temporal and spatial detail and time horizon needed to maintain computational tractability. Renewable generation capacity has increased substantially over the last decade in Europe and there is confidence that the high penetration in our scenarios could be deployed. In contrast, new nuclear reactors have construction lead times of 6–12 years and a large specialised construction industry does not exist in the UK. Through not considering the pathway to a net-zero system, we have again most likely over estimated the potential role of nuclear generation. We have largely neglected socio-political considerations, for example public acceptance around nuclear, CCS and renewables. For example, the visual impact of onshore wind and solar PV can increase the cost of highly renewable systems (see e.g. Ref. [31]. For nuclear, we have not included the cost of regulatory guarantees such as implicit public liability insurance in the case of an accident. We have also not included the cost of safely disposing spent nuclear fuel, which is also a political issue as the UK currently does not have a permanent safe storage facility. 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https://openalex.org/W3037588010	https://www.nature.com/articles/s41598-019-57337-3.pdf	English	null	Cavity-enhanced metallic metalens with improved Efficiency	Scientific reports	2,020	cc-by	5,170	Cavity-enhanced metallic metalens with improved Efficiency OPEN Hanmeng Li, Bin Fang, Chen Chen, Shining Zhu & Tao Li* Metasurfaces are made of subwavelength nanoantennas with a flat, ultrathin architecture, and strong capability in manipulating the propagation of light by flexible modulations on its phase, amplitude, and polarization. Conventional metallic metalenses always suffer from its low efficiencies due to large intrinsic loss. Here, we demonstrate a cavity enhanced bilayer metalens composed of aluminum nanobars and its complementary structures. The focusing and imaging experiments definitely show an improved efficiency of such kind of bilayer metalens compared with its single layer counterpart. Detailed theoretical analyses based on full-wave simulations are carried out with respect to different cavity lengthes and working wavelengths, which reveals that the improvement rightly attributes to enhanced cavity mode. Our design will not only improve the working efficiency for metalens with simplified manufacturing procedure, but also indicates more possibilities by employing the metal as electrodes. Metasurface is a kind of two-dimensional (2D) arrayed ultrathin structure with subwavelength unit cells1, which is of strong capability to manipulate the phase, amplitude, polarization of light within a ultrathin film and thus arrested remarkable attentions in recent years2–16. Plenty of functionalities based on metasurfaces have been demonstrated, such as optical vortex plates2,7, holograms16–22, color filters23, and so on. By utilizing a conical phase profile, metasurface can work as a lens (i.e., metalens), which is capable of focusing incident light within a very compact dimension. Metallic metalens has shown its strong capability in beam focusing with a very thin layer5,13. However, the high intrinsic losses of metal in optical wavelengths (especially in the visible range) greatly limit the metalens working efficiency and prevented its real applications. Although this challenge can be circum- vented by using reflective-type metasurfaces18, transmission-type devices have a much wider range of applica- tions. More recently, people have developed all-dielectric metasurfaces to obtain high efficiency in transmission scheme, and important progresses have been achieved in metalens imaging24, efficient holography25, achromatic design26–31, and so on. Nevertheless, there are still challenges in nanofabrication for large scale devices according to requirement of high-aspect-ratio nanostructures, which are essential in dielectric metasurface designs. In addi- tion, all-dielectric design somewhat restricts applying external electric field, which is a common means in tun- able devices with electro-optical effect. Therefore, it would be applausive to develop metallic metasurfaces with improved transmission efficiency, where the metallic layer would possibly work as electrodes as well. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Cavity-enhanced metallic metalens with improved Efficiency OPEN However, there are only few studies on improving the efficiency of metallic metasurfaces and most of them are limited in reflection-type devices18,19,32. l yp In this letter, we propose a transmission-type metallic metalens with a cavity inside to improve the working efficiency significantly both in simulation and experiments. It is clearly observed in the improvement of diffrac- tive efficiency with strong focusing intensity and enhanced imaging contrast despite of the decrease in total trans- mission. The cavity plays an important role with local field enhancement that gives rise to the ultimate improved efficiency. Moreover, this bilayer metalens can further reduce the processing in nanofabrications compared with the conventional lift-off approach, and hold the advantage for massive production. Results and Discussions To function like a focusing lens, the phase profile ϕ (x, y) of the metalens is designed as φ π λ = − + + . x y f x y f ( , ) 2 ( ) (1) 0 2 2 2 (1) Here, λ0 is the design wavelength and in this work we choose the 632.8 nm, x and y are the coordinates of each nanobar, and f is the focal length. This phase profile is imparted via rotation of each nanobar at a given coordinate (x, y) by an angle θ (x, y). In the case of right-handed circularly polarized incident light, these rotations yield a phase shift as ϕ (x, y) = 2θ (x, y), accompanied by polarization conversion to left-handed circularly polarized light. Thus, each nanobar at (x, y) is rotated by an angle Here, λ0 is the design wavelength and in this work we choose the 632.8 nm, x and y are the coordinates of each nanobar, and f is the focal length. This phase profile is imparted via rotation of each nanobar at a given coordinate (x, y) by an angle θ (x, y). In the case of right-handed circularly polarized incident light, these rotations yield a phase shift as ϕ (x, y) = 2θ (x, y), accompanied by polarization conversion to left-handed circularly polarized light. Thus, each nanobar at (x, y) is rotated by an angle θ π λ = − + + . x y f x y f ( , ) ( ) (2) 0 2 2 2 (2) Both metalenses with the same diameter of 80 μm and focal length of 120 μm (NA = 0.32) were fabricated by electron-beam lithography (EBL), where the PB phase designed metalens patterns of the PMMA were spin-coated on an ITO coated quartz substrate. The thickness of the PMMA layer is 100 nm (as t1 in Fig. 1(c)), and electron beam evaporation is subsequently used to deposit aluminum onto the developed resist to form the aluminum nanobars. The thickness of aluminum is 30 nm (referred as t2), and so does the nanobar thickness. The width W, and length L of the nanobar here we choose are 120 nm and 300 nm respectively with the periodically arranged unit cell dimensions (S = 400 nm). Aluminum is chosen for its relatively low loss in the visible region. Results and Discussions Figure 1(a) schematically shows the bilayer metalens (BLM) with a circular polarized illumination, where a polym- ethyl methacrylate (PMMA) spacer is designed with nanohole array on a silica substrate, and aluminum rectan- gular nanobars is filled inside nanoholes and their complementary structure on the top surface. These Al nanobars are rotationally arranged according to the geometric phase (i.e., Pancharatnam–Berry (PB) phase) design2. To more clearly show the structural parameters, the side section of the unit cell is shown in Fig. 1(c). Compared with the conventional metallic metalens (Fig. 1(b)) only with metal nanobar/nanodisc on a dielectric substrate, the National Laboratory of Solid State Microstructures, Key Laboratory of Intelligent Optical Sensing and Integration, Jiangsu Key Laboratory of Artificial Functional Materials, College of Engineering and Applied Sciences, Nanjing University, Nanjing, 210093, China. *email: taoli@nju.edu.cn Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3 www.nature.com/scientificreports/ Figure 1. (a) Schematics of part of the bilayer metalens (BLM) under circular polarized illumination with cross-polarization analysis after transmission. (b) Side section view of the unit cell of the (b) BLM and (c) conventional metalens only composed of aluminum. (d,e) SEM micrographs of the two kinds of metalens corresponding to (b,c), respectively (scale bars = 300 nm). Figure 1. (a) Schematics of part of the bilayer metalens (BLM) under circular polarized illumination with cross-polarization analysis after transmission. (b) Side section view of the unit cell of the (b) BLM and (c) conventional metalens only composed of aluminum. (d,e) SEM micrographs of the two kinds of metalens corresponding to (b,c), respectively (scale bars = 300 nm). proposed BML can be regarded as a staggered cavity. The corresponding scanning electron microscope (SEM) images of two samples are shown in the Fig. 1(d,e), which are fabricated by e-beam lithography (ZEISS ULTRA 55). In fact, this bilayer sample (Fig. 1(c,e)) can be regarded as partially prepared product compared with the con- ventional metasurfaces (Fig. 1(b,d)), since it lacks the final lift-off procedure. To function like a focusing lens, the phase profile ϕ (x, y) of the metalens is designed as proposed BML can be regarded as a staggered cavity. The corresponding scanning electron microscope (SEM) images of two samples are shown in the Fig. 1(d,e), which are fabricated by e-beam lithography (ZEISS ULTRA 55). In fact, this bilayer sample (Fig. 1(c,e)) can be regarded as partially prepared product compared with the con- ventional metasurfaces (Fig. 1(b,d)), since it lacks the final lift-off procedure. Results and Discussions The two inserts are the corresponding images of focal spot captured by CCD. (c,d) Images of 1951 USAF resolution test chart taken from the conventional metalens and the BLM with white light illumination (scale bar = 7 μm). The two inserts are the corresponding results of monochromatic images after 630 nm- wavelength filter, respectively. BLM keeps clear observation, as shown in the inset figures in Fig. 2(c,d). The details in optical setups and meas- urements are provided in Methods. To quantitively analyze the performance we defined a diffractive efficiency as LM keeps clear observation, as shown in the inset figures in Fig. 2(c,d). The details in optical setups and meas- rements are provided in Methods.iffi p To quantitively analyze the performance, we defined a diffractive efficiency as p To quantitively analyze the performance, we defined a diffractive efficiency as η = + σ σ σ − + − I I I , (3) d η = + σ σ σ − + − I I I , d (3) where Iσ + and Iσ − represent the intensity of the detected beam with the same and cross circularly polarization states, respectively. Then, we obtain the total focusing efficiency as where Iσ + and Iσ − represent the intensity of the detected beam with the same and cross circularly polarization states, respectively. Then, we obtain the total focusing efficiency as η η = ⋅T, (4) t d η η = ⋅T, t d (4) where T is the total transmittance of the metalens. Obviously, the total focusing efficiency of our new designed structure is much higher than the conventional one. In the BLM structure, there are several parameters affect the focusing efficiency. Among them, the separation thickness between the two metal layers is considered as a major contribution to the cavity effect that strongly affect the diffractive efficiency and the transmittance. To figure out the cavity effect on the efficiency of the BLM, we employed a commercial software (Lumerical FDTD Solutions) for the full-wave simulations. In simulations, we choose the same parameter as obtained in experiments, say, the thicknesses of the two aluminum layers (t2) are both 30 nm, the period of the unit cell (S) is 400 nm and each nanobar dimension of W = 120 nm and L = 300 nm. Results and Discussions For the conventional metallic metalens, a lift-off step is needed to strip the PMMA surroundings with the Al nanobars left only. But in the BLM design, we do not need to conduct the lift-off, which means the deposition of metal is the last step of our fabrication to obtain the bilayer structure. By cutting off the lift-off step, we can not only save much time in fabrication but also avoid the structural fluctuations induced in the last process. ( b) h h f h f l h b d f h l l d l Figure 2(a,b) show the cross-sections of the focal spots that are obtained for the conventional metalens and BLM at the design wavelength (λ0 = 632.8 nm) in our experiments, from which we obtained the full-widths at half-maximum (FWHM) of focal spots are 1.1 μm and 1.05 μm, respectively. The corresponding focal spots images through two metalenses are shown in the insert figures of Fig. 2(a,b). Besides smaller and more sym- metric focal spot, the BLM sample gives rise to a much stronger intensity (~2.63 times enhancement) than the conventional one at the same exposure time of the camera with the same illumination. Moreover, the 1951 United State Air Force (USAF) resolution test chart was used as the imaging target, and was illuminated by a halogen white-light source. Figure 2(c,d) show the results from the conventional metalens and BLM. It is clearly shown that image contrast and signal-to-noise ratio (SNR) via BLM is much better than the conventional one, thought it has higher total transmittance. It is further confirmed by the monochromatic image quality as filtered at 630 nm wavelength. The image via conventional metalens even disappear according to its low efficiency, while that of Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3 www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 2. (a,b) Measured cross-sections of the focal spots of the conventional metalens and the BLM, respectively. The two inserts are the corresponding images of focal spot captured by CCD. (c,d) Images of 1951 USAF resolution test chart taken from the conventional metalens and the BLM with white light illumination (scale bar = 7 μm). The two inserts are the corresponding results of monochromatic images after 630 nm- wavelength filter, respectively. Figure 2. (a,b) Measured cross-sections of the focal spots of the conventional metalens and the BLM, respectively. Results and Discussions We calculated the diffractive efficiency ηd, transmittance T, and the total efficiency ηt with respect to different thicknesses of PMMA (t1), as the results shown in Fig. 3. It is seen that though the transmittance is considerable low (<18%), the diffractive efficiency is relatively high (>45%) in the whole range. As the thickness of PMMA increases from 50 nm to 200 nm in our simulation, both the trans- mittance T and the total efficiency ηt exhibit a maximum when the t1 ≈ 140 nm. While the ηd undergoes a nearly inverse trend, which changes very little when t1 < 140 nm.ffi g y Compared with the conventional metalens whose diffractive efficiency ηd is only at the level of 5% in our simu- lation (not shown in Fig. 3), BLM has a much higher diffractive efficiency ηd (the lowest one is 46%). Although the structure of BLM may decrease the transmittance T, the improvement of the diffractive efficiency ηd can make up for it and exceed. The red horizontal dotted line represents the value of the total efficiency ηt of the conventional Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3 www.nature.com/scientificreports/ Figure 3. Diffractive efficiency, transmittance (left label), and total efficiency (right label) of the BLM with respect to different thickness of PMMA (related to the cavity length). Here, the small symbols with connection lines are the simulation results, and the larger symbols with error bars are the experimental data. The red dashed horizontal line represents the simulated total efficiency of the conventional single layer metalens. Figure 3. Diffractive efficiency, transmittance (left label), and total efficiency (right label) of the BLM with respect to different thickness of PMMA (related to the cavity length). Here, the small symbols with connection lines are the simulation results, and the larger symbols with error bars are the experimental data. The red dashed horizontal line represents the simulated total efficiency of the conventional single layer metalens. Figure 4. (a,b) Simulated E-field distribution in the BLM with a period of 400 nm and a separation thickness of 140 nm (optimal) in x-z and y-z planes, respectively, as excited by a circular polarized illumination at λ0 = 632.8 nm. (c,d) The corresponding simulated results of the un-optimized cavity (t1 = 80 nm), where the E-field is much weaker than that in optimized case. All the electrical field are normalized in same scale. Figure 4. Results and Discussions (a,b) Simulated E-field distribution in the BLM with a period of 400 nm and a separation thickness of 140 nm (optimal) in x-z and y-z planes, respectively, as excited by a circular polarized illumination at λ0 = 632.8 nm. (c,d) The corresponding simulated results of the un-optimized cavity (t1 = 80 nm), where the E-field is much weaker than that in optimized case. All the electrical field are normalized in same scale. metalens (ηt = 3.2%) while that of BLM is higher when the thickness of PMMA ranges from 70 nm to 190 nm. The maximum efficiency at t1 ≈ 140 nm (ηt = 8.8%) is about 2.75 times improvement. Our experimental data is also consistent with calculation, as the symbols of red solid circles shown in Fig. 3, where the total efficiency is not as high as the simulation but still reaches an improved level (~6%). According to our experiments with same condition, the conventional single-layer metalens only has 2.31% in total efficiency. Thus, this cavity-enhanced double-layer metalens definitely has a considerable improvement in the focusing efficiency. ifi To understand the cavity enhancement mechanism of the BLM, we choose the optimized parameter (t1 ≈ 140 nm) case with another un-optimized one (t1 ≈ 80 nm) for detailed investigations. Figure 4 displays their field dis- tributions in x-z and y-z planes, revealing the characteristics of cavity mode. It is clearly shown that optimized struc- ture exhibits a much stronger field excited at the long-edges of the upper aluminum hole (see Fig. 4(a)) compared Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3 www.nature.com/scientificreports/ www.nature.com/scientificreport tificreports/ Figure 5. The optimized cavity lengths (with maximum efficiency) in BLM with respect to the working wavelengths. Here, the black line is theoretical prediction by cavity mode and the red square symbols are the simulation results. Two inset figures display the field distributions of optimized cavity at λ = 532 nm and 582 nm. Figure 5. The optimized cavity lengths (with maximum efficiency) in BLM with respect to the working wavelengths. Here, the black line is theoretical prediction by cavity mode and the red square symbols are the simulation results. Two inset figures display the field distributions of optimized cavity at λ = 532 nm and 582 nm. to the un-optimized one (see Fig. 4(c)), which gives rise to the improved diffractive efficiency. Results and Discussions Indeed, the proper separation thickness (t1 ≈ 140 nm) should account for a complete cavity mode inside the hole between two metal layers. Figure 4(b) clearly show the cavity mode property in x-z dimension, where we can find a “standing-wave” appearance centered at the upper aluminum layer that should accommodate a resonant mode with wavelength of fourfold the cavity length. In our simulation and experiments, the light wavelength is 632.8 nm, which would correspond to the cavity length of 158.2 nm. Considering our structure, the cavity length of the optimized case is t1 + t2/2 = 155 nm, agreeing extremely well with the prediction of cavity model.if To further confirm this model, we performed more simulations with respect to different wavelengths search- ing for the optimized cavity length. The obtained simulation data together with the theoretical prediction are plotted in Fig. 5, which shows quite good agreement with each other. As examples, the field distributions of two optimized cavity modes at λ = 532 nm and 582 nm are displayed as inset figures in Fig. 5, which show good con- sistent with the cavity theory as well as the previous λ = 632.8 nm case (see Fig. 4). Thus, we have confidence that the existence of the cavity in the BLM rightly contribute to the improvement of the working efficiencies. It should be mentioned that in a recently published work33, a similar type of bilayer plasmonic metasurface was proposed and show a greatly improved efficiency. However, it is recognized from the viewpoint of interference scattering of multipolar meta-atom without any discussion on the cavity effect. We do believe that multipolar scattering would also make contribution in the similar system. However, our work provides a new perspective to understand the enhancement mechanism with a cavity mode, which was well validated by convincing experimental and simu- lation data. Conclusions In summary, we have demonstrated a bilayer metallic metalens with improved efficiency both in simulation and experiments. The focusing efficiencies can be improved considerably (~2.6 times) compared with conventional single-layer metallic metalens, in which the cavity length can be optimized to reach a maximum improvement in efficiency. Our approach also can simplify the nanofabrication procedure, which is helpful in massive and efficient production. The cavity design enriches the mechanism for designing metasurfaces and is promising for possible dynamic tuning with the external electric field applied on electro-responses inclusions inside the cavity as the double metallic layers can work as the electrodes properly. References GaN Metalens for Pixel-Level Full-Color Routing at Visible Light. Nano Lett. 17, 6345–6352, https://doi. org/10.1021/acs.nanolett.7b03135 (2017). g 3. Ellenbogen, T., Seo, K. & Crozier, K. B. Chromatic Plasmonic Polarizers for Active Visible Color Filtering and Polarimetry. Nano Lett. 12, 1026–1031, https://doi.org/10.1021/nl204257g (2012).f 24. Khorasaninejad, M. et al. Metalenses at visible wavelengths: Diffraction-limited focusing and subwavelength resolution imaging. Science 352, 1190 (2016). 25. Wang, L. et al. Grayscale transparent metasurface holograms. 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An objective (50× mag- nification) is used to image the light focused by metalens for measuring the size of the focal spot and a lens (f = 200 mm) is used to project the image on a CCD camera. Another group of a polarizer and a quarter-wave- plate is arranged between the lens and the camera for circular polarization check. In the imaging experiment, the He-Ne laser is replaced by a halogen white-light source and the 1951 United State Air Force (USAF) resolu- tion test chart is used as the imaging target. The beam is focused by the objective onto the target object and the metalens is placed a focal length away from the object and paired with an objective and a lens to form an image on a CCD camera. When measuring the monochromatic image quality, a filter at 630 nm wavelength is inserted before the camera. inite-difference time-domain numerical simulations. Finite-difference time-domain (FDTD) simu- ations (Lumerical FDTD) are performed to analysis the cavity enhancement mechanism of the BLM. In modeling Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3 www.nature.com/scientificreports/ the unit cells, periodic boundary conditions are defined at the x and y boundaries and perfectly matched layers (PML) at the z boundaries. The refractive indices of Al, SiO2 and PMMA are taken from the software and the unit cells are illuminated by a normalized-incident plane wave source with circular polarization in full 3D simulations. The electric-field distributions are obtained via the surface-detectors. the unit cells, periodic boundary conditions are defined at the x and y boundaries and perfectly matched layers (PML) at the z boundaries. The refractive indices of Al, SiO2 and PMMA are taken from the software and the unit cells are illuminated by a normalized-incident plane wave source with circular polarization in full 3D simulations. The electric-field distributions are obtained via the surface-detectors. Received: 6 November 2019; Accepted: 28 December 2019; Published: xx xx xxxx Received: 6 November 2019; Accepted: 28 December 2019; Published: xx xx xxxx References Express 26, 1351–1360, https://doi.org/10.1364/OE.26.001351 (2018 g p p p g 8. Ratni, B., de Lustrac, A., Piau, G.-P. & Burokur, S. N. Reconfigurable meta-mirror for wavefronts co antennas. Opt. Express 26, 2613–2624, https://doi.org/10.1364/OE.26.002613 (2018). 8. Ratni, B., de Lustrac, A., Piau, G.-P. & Burokur, S. N. Reconfigurable meta-mirror for wavefronts control: applications to microwave antennas. Opt. Express 26, 2613–2624, https://doi.org/10.1364/OE.26.002613 (2018). p p g 9. Feng, R. et al. 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Phase-engineered metalenses to generate converging and non-diffractive vortex beam carrying orbital angular momentum in microwave region Opt Express 26 1351–1360 https://doi org/10 1364/OE 26 001351 (2018) f se-engineered metalenses to generate converging and non-diffra 7. Zhang, K. et al. Phase-engineered metalenses to generate converging and non-diffractive vortex beam ca momentum in microwave region. Opt. Acknowledgements g This work was jointly supported by the National Key R&D Program of China (2016YFA0202103, 2017YFA0303701); National Natural Science Foundation of China (Nos. 91850204, 11674167, 11621091). Tao Li is thankful for the support from Dengfeng Project B of Nanjing University. Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3 www.nature.com/scientificreports/ Author contributions T.L. supervised the study. T.L. and H.L. proposed the idea and design. H.L. performed the numerical simulations. H.L. fabricated the sample and performed the optical analyses with the assistance from B.F. and C.C, T.L. and H.L. analyzed the results and wrote the paper. S.Z. and T.L. directed the project. All authors contributed to the discussions. Competing interestsh p g The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to T.L. 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. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports \| (2020) 10:417 \| https://doi.org/10.1038/s41598-019-57337-3
https://openalex.org/W2083210872	https://www.scielo.br/j/gmb/a/vz3VmnjCDSmWG5SmrWw4Brq/?lang=en&format=pdf	English	null	Comparison of base substitutions in response to nitrogen ion implantation and 60Co-gamma ray irradiation in Escherichia coli	Genetics and Molecular Biology	2,004	cc-by	4,975	Introduction small deletions are based upon Lac+ reversion of a specific mutation located within the lacZ gene in the F’ plasmid. A number of lacZ constructs and strains were developed in this experimental system (Cupples et al., 1990; Cupples and Miller 1989; Ohta et al., 1999). In particular, the lacZ system has been widely used in mutation spectrum evi- dence for specific mutations from defined constructs. Yang et al. (1997) reported some in vitro mutational spectra of low-energy nitrogen ion beam implantation by using the lacZ constructs. However, it should be noted that the DNA molecules interact in vivo with many molecules and chemi- cals such as proteins and lipids, indicating that the structure of DNA molecules is more complex than naked DNA. The interaction between an ion beam and DNA is different in vi- tro and in vivo. Therefore, it is necessary to establish an ex- perimental system to study how the low-energy nitrogen ion beam induces a mutation in vivo. The mutational spectrum induced by ionizing radia- tion has been an issue of long-standing interest in radiation biology (Grosowsky et al., 1988). Southern blotting analy- sis permits the partitioning of ionizing radiation-induced mutagenesis into detectable deletions and major genomic rearrangements and into point mutations (Grosowsky et al., 1988; (Grosowski et al., 1986). Methods based on specific locus PCR were established to determine the mutational spectrum of fairly large fragments (Hei et al., 1997; Wu et al., 1999). The molecular nature of the point mutations, however, has been left unresolved (Grosowsky et al., 1988). Point mutations comprise base substitutions (transi- tions and transversions), frameshifts, small deletions and insertions. Among them, base substitutions represent ap- proximately 2/3 of the point mutations analyzed induced by ionizing radiation (Grosowsky et al., 1988). The mecha- nism by which ionizing radiation produces mutagenicity is not entirely understood at present, which is partially due to little evidence from the mutational spectrum. Assays for transitions, transversions, frameshifts at specific sites and Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase, which is encoded by the rpoB gene, can confer resistance to rifampicin (Jin et al., 1988; Jin and Gross 1988). Increased mutagenesis to rifampicin resistance reveals that base substitutions in rpoB confer E. coli cells this capacity (Jin et al., 1988; Matic et al., 1997). Send correspondence to Zeng-Liang Yu. Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences, Institute of Plasma Physics, Hefei P.O.Box 1126, Anhui, Chin. Email: cxxie@ipp.ac.cn. Genetics and Molecular Biology, 27, 2, 284-290 (2004) Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br Research Article Abstract To identify the specificity of base substitutions, a novel experimental system was established based on rifampicin- resistant (Rif r) mutant screening and sequencing of the defined region of the rpoB gene in E. coli. We focused on comparing mutational spectra of base substitutions induced by either low energy nitrogen ion beam implantation or 60Co-gamma rays. The most significant difference in the frequency of specific kinds of mutations induced by low en- ergy nitrogen ion beam was that CG →TA transitions were significantly increased from 32 to 46, AT → TA transversions were doubled from 7 to 15 in 50 mutants, respectively. The preferential base substitutions induced by nitrogen ion beam implantation were CG →TA transitions, AT →GC transitions, AT →TA transversions, which ac- count for 92.13% (82/89) of the total. The mutations induced by 60Co-gamma rays were preferentially GC →AT and AT →GC transitions, which totaled 84.31% (43/51). Key words: base substitution, low-energy nitrogen ion beam implantation, 60Co-gamma rays, Escherichia coli. Received: July 25, 2003; Accepted: November 14, 2003. Key words: base substitution, low-energy nitrogen ion beam implantation, 60Co-gamma rays, Escherichia coli. Chuan-Xiao Xie1, An Xu1, Li-Jun Wu1, Jian-Min Yao1, Jian-Bo Yang2, Zeng-Liang Yu1* Chuan-Xiao Xie1, An Xu1, Li-Jun Wu1, Jian-Min Yao1, Jian-Bo Yang2, Zeng-Liang Yu1* 1Key Laboratory of Ion Beam Bioengineering Chinese Academy of Sciences, Institute of Plasma Physics, Anhui, China. Chuan-Xiao Xie1, An Xu1, Li-Jun Wu1, Jian-Min Yao1, Jian-Bo Yang2, Zeng-Liang Yu1* 1Key Laboratory of Ion Beam Bioengineering Chinese Academy of Sciences, Institute of Plasma Physics, Anhui, China. 1Key Laboratory of Ion Beam Bioengineering Chinese Academy of Sciences, Institute of Plasma Physics, Anhui, China. nstitute of Atomic Energy, Anhui Academy of Agricultural Science, Hefei, Anhui Province, China Primer design, oligomer synthesis, PCR amplification and sequencing were PCR-amplified and sequenced for analyzing the base substitutions. We compared the base substitution mutations induced by nitrogen ion beam implantation with those in- duced by gamma radiation, in order to analyze the mutagenicity of this procedure. The primer pairs for amplification of the regions were designed by the program WEBPRIMER (Stanford Univer- sity) and reevaluated by the program PRIMER DESIGN. All primers were synthesized in the ExpediteTM Nucleic Acid Synthesis System (workstation) in TaKaRa (Dalian Corporation). Media and strain After confirmation by streak culture on LB-rif plates, the mutant clones were toothpicked from the plates and sus- pended in 200 µL of 1Pfu PCR buffer, boiled for 10 min in the PCR apparatus (Perkin-Elmer 9600) for lysing the bac- teria. Then, the content of each tube was divided into two tubes for two different rpoB region amplifications. The PCR mixture was then completed, and 40 cycles (30 s at 94 °C, 30 s at 58 °C, and 45 s at 72 °C) were performed. The final composition of the PCR mixture was 1Pfu PCR buffer with Mg2+, 75 pmol of both primers, 200 µM each dNTP (TakaRa, Dalian), and 3 U of cloned Pfu polymerase (Ta- kaRa, Dalian). The PCR products were purified with a DNA purification Kit (Wizard® SV Gel and PCR Clean- Up System, Promega) with low-melting agarose gel and sent to sequencing (TaKaRa, Dalian). DNA sequencings were all performed in both directions, using a Perkin-Elmer Applied Biosystems Model 377 DNA Sequencer in TaKa- Ra (Dalian) Corporation. Culture media LB (1% tryptone, 0.5% yeast extract, 1% NaCl) or MM (minimal medium: 0.05% L-asparagine, 0.02% MgSO4, 0.001% FeSO4, 1% glucose) were used, so- lidified when required with 1.5% agar, and supplemented with rifampicin (150 µg/mL) and streptomycin (30µg/mL), respectively, as appropriate when needed. The E. coli K-12 strain AB1157 was kindly provided by M.G. Marinus from the University of Massachusetts Medical School. Its geno- type is F- thr-1 ara-14 leuB6 (gpt-proA)62 lacY1 tsx-33 supE44(AS) galK2(Oc) hisG4(Oc) rfbD1 mgl-51 rpoS396(Am) rpsL31(StrR) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1. Survival fraction and mutant frequency determination Figure 1 shows the surviving fraction and mutant fre- quencies of E. coli cells irradiated by either nitrogen ion beam with various dosages or 60Co-gamma rays. As com- pared to 60Co-gamma rays (Figure 1 B), the 10 keV low- energy nitrogen ion beam induced a different pattern of cytoxicity (Figure 1 A). The moderate decreased pattern of the survival curve showed that low-energy nitrogen ion beam implantation may have a moderate cytotoxic effect on E. coli cells. At the dosage of 50% cell killing, the mu- tant frequency of nitrogen ion beam implantation (Figure 1 C: 6.5×1014 ions/cm2) reached 9.5×10-7, which is ~10-fold higher than that of 60Co-gamma rays (Figure 1 D: 10 Gy mutant frequency = 9.3×10-8). This suggests that low- energy nitrogen ion beam implantation might generate Determination of base substitutions The base substitutions were determined by a run of blastn (blast 2 sequences) between the sense strand se- quence of the wild type and mutants on line on the website (http://www.ncbi.nih.gov/blast/). The subject sequence is the sequence of the same region as in E. coli K-12 strain MG1655 (GenBank ACCESSION: U00096). Nitrogen ion beam implantation, 60Co-gamma ray irradiation Overnight cultures were diluted ~100-fold and grown in LB medium until early log phase (optical density at 600 nm [OD600] = ~0.3). Aliquots of 10 mL were pelleted and resuspended in 10 mL of MM salt solution with 10% glycerol for nitrogen ion treament. The liquid was spread onto the surface of plates. The ion implanted was nitrogen, with an energy of 10 keV, and the dosages (fluence) used were 1.3×1014, 2.6×1014, 3.9×1014, 5.2×1014, 6.5×1014, 7.8×1014, 9.1×1014, 10.4×1014, 11.7×1014 ions/cm2. During implantation, the pressure of the target chamber was ~10-3 Pa, and the temperature of the implantation environment was estimated to be approximately 0 °C. The cell- containing liquid was irradiated with 60Co-gamma rays at doses of 0.5 Gy, 1 Gy, 2 Gy, 5 Gy, 10 Gy, 20 Gy, 50 Gy, 80 Gy, 100 Gy, and 120 Gy, respectively. Introduction Here, we designed two pairs of primers for spe- cific regions of the rpoB gene. The corresponding regions Xie et al. 285 Primer design, oligomer synthesis, PCR amplification and sequencing Optimization of PCR amplification higher frequency of mutagenicity, specially base substitu- tions, as they were identified by Rifr mutant screening. Since there is no single set of working conditions ful- filling the requirements of all PCR amplifications, the fac- tors related to these reactions, including reaction component concentrations and procedures (time, tempera- ture parameters and cycles), need to be adjusted within the- oretically suggested ranges for efficient amplification of specific targets. In the present study, the bacterial lysate was used directly as PCR template. Briefly, mutant bacteriolysis was achieved in 1pfu PCR buffer at 95 °C for 10 min in the PCR apparatus. The content of each tube was divided into two tubes for two different rpoB region ampli- fications in the same mutant. 50 Rifr mutant clones induced by the 10 keV nitrogen ion beam (three of them shown in Mutant screening, mutant frequency, and determination of survivors The treated and control cells were washed down with 10 mL MM salt solution, and centrifuged for 1 min at 9,000 rpm. The precipitates were resuspended with 5 mL LB medium and incubated at 37 °C for 30 min at 220 rpm. After resuspension in MM salt solution, 50 µL of appropri- ate dilutions (104 ~ 106 fold) were spread onto MM plates to determine the total count, and 100 µL of them, with an ap- propriate cell density, were spread onto LB-rif plates to screen the mutant. The mutant frequencies were calculated by dividing the mean number of mutants by the average number of total cells. 60Co-gamma ray irradiation in Escherichia coli 286 rpoB region A is a sensitive region for determination of base substitutions After amplification and purification, the PCR prod- ucts were sequenced in both directions. The sequences were compared with the corresponding wild-type sequence by the blastn program (blast the two sequences http://www. nibi.nlm.nih.gov/BLAST.html) to find the mutations. Each mutation site was also verified by comparing the sense strand with its complementary sequence. The distribution and types of mutations are summarized in Table 2. Ninety- seven out of a hundred of the mutants had at least one base substitution mutation in region A. Ninety-nine percent of the mutants had no base changes in region B. This finding suggests that most of the mutations (99%) are derived from the selection of rifampicin resistance, and that region A of the rpoB gene is the sensitive region for determining the specificity of base substitutions. The treatment was done with double-stranded DNA, and, thus, it was impossible to discriminate from which strand the position of the damage was derived. The types of base substitutions were grouped as CG →TA transitions, AT → GC transitions, AT → TA transversions, and GC →CG transversions. For instance, a CG →TA transi- tion can also be derived from a GC →AT transition on the complementary strand. The specificity of base substitutions derived from low-energy nitrogen ion beam implantation and 60Co-gamma ray-irradiated cells is also summarized in Table 2. The most significant difference in the frequency of specific kinds of mutations induced by low-energy nitrogen ion beam was that CG →TA transitions were significantly increased from 32 to 46, and AT →TA transversions were doubled from 7 to 15, as compared to the frequencies of gamma ray-induced mutations. In summary, the frequen- cies of nitrogen ion beam implantation-induced mutations In addition, our observation is consistent with the se- quence studies of a number of other laboratories. Severinov et al. (1993) analyzed mutations in rpoB leading to Rifr. They found that most of the base substitutions responsible for the Rifr phenotype lied in two clusters. Even though the full length of this gene is 4029 bp, the Rifr responsible re- gion was only 177 bp. Previous studies (Jin and Gross 1988; Miller et al., 2002; Phanchaisri et al., 2002) identi- fied 47 single-base substitutions at 29 sites and distributed among 21 coding positions. Primers To analyze the Rifr selection mutation and non- selective mutation spectrum, we here chose two regions for determination of the mutation (Figure 2). Region A (492 bp in size) is the highly conserved region, which confers the E. coli cell the capacity of Rifr, when an appropriate base sub- stitution is induced. Region B (530 bp in size) covers a non-conserved region of the rpoB coding sequence (419 bp, from 3611 to 4029), 77 bp of non-coding sequence between rpoB and rpoC genes, 34 bp coding sequence of rpoC (from 1 to 34). The primer sequences are listed in Table 1. 1 to 34). The primer sequences are listed in Table 1. by the 10 keV nitrogen ion beam (three of them shown in Figure 1 - Cytotoxicity (A, B) and mutagenicity (C, D) induced by low-energy nitrogen ion beam implantation and 60Co-gamma rays. Figure 2 - Sketch map showing the high-fidelity PCR amplification regions. Figure 1 - Cytotoxicity (A, B) and mutagenicity (C, D) induced by low-energy nitrogen ion beam implantation and 60Co-gamma rays. Figure 2 - Sketch map showing the high-fidelity PCR amplification regions. Figure 2 - Sketch map showing the high-fidelity PCR amplification regions. 287 Xie et al. Table 1 - Primer pairs for amplification of the rpoB gene. Forward primer Reverse primer 5’-AGGGACGCACCGACGGATACCA-3’ 5’-AGAGTTCTTCGGTTCCAGCCAG-3’ 5’-TGCCGACTTCCGGTCAGAT-3’ 5’-GTCTGCGCTTTCAGAAACTT-3’ Comparison of the specificity of base substitutions Figure 3 A) and by 10 Gy 60Co-gamma rays (three of them shown in Figure 3 B) were picked respectively to amplify the two defined regions. The optimized PCR components and procedures are described in Materials and Methods. To compare the mutagenicity of the two mutagens, a total of 100 independent mutants (50 each) in rpoB genes were sequenced. The frequency of total detected nitrogen ion-induced mutation reaches 89, while the 60Co-gamma ray-induced mutation frequency is only 51 (Table 2). The transitions prominently account for 75.28% and 84.31% of base substitutions induced by nitrogen ion beam and 60Co- gamma rays, respectively. On the other hand, the types of base substitutions are different between the two mutagens. Two types of base substitutions (GC →CG transversions, AT →GC transitions) were induced by low-energy nitro- gen ion beam, but were not found in cells treated with 60Co-gamma rays, whereas AT →CG transversions were not found in low-energy nitrogen ion beam implantation, but were found in cells treated with 60Co-gamma rays (Ta- ble 2). rpoB region A is a sensitive region for determination of base substitutions rpoB region A is a sensitive region for determination of base substitutions Most of them (46/47) lie in the region A defined in this research, whereas the mutations of the base substitution type leading to Rifr in rpoB covered all kinds of transitions and transversions. Figure 3 - High-fidelity PCR amplification of two regions of rpoB of Rifr mutants induced by either 10 keV nitrogen ion beam (A) or 60Co-gamma ray (B). M: marker (GeneRulerTM 100bp DNA ladder plus, MBI). Mutant numbers (Mut. no.) are shown above the electrophoresis bands (A) (B) 1,3,5: region A of the corresponding mutant (492 bp in size); (A) (B) 2,4,6: region B of the corresponding mutant (530 bp in size). Figure 3 - High-fidelity PCR amplification of two regions of rpoB of Rifr mutants induced by either 10 keV nitrogen ion beam (A) or 60Co-gamma ray (B). M: marker (GeneRulerTM 100bp DNA ladder plus, MBI). Mutant numbers (Mut. no.) are shown above the electrophoresis bands (A) (B) 1,3,5: region A of the corresponding mutant (492 bp in size); (A) (B) 2,4,6: region B of the corresponding mutant (530 bp in size). 60Co-gamma ray irradiation in Escherichia coli 288 Table 2 - Distribution of specific types of base substitutions and relative information. Table 2 - Distribution of specific types of base substitutions and relative information. Type of base substitution Position Target sequence (5’ →3’) Amino acid change Occurrence Total I. 10 keV N+ 89 CG →TA transitions 1592 GTATCTCCGCACT Ser531 Phe 12 1576 ATTACGCACAAAC His526 Tyr 8 1565 CGCTGTCTGAGAT Ser522 Phe 5 1692* AAACCCCTGAAGG Pro564 Leu 2 3921* TAAAAACATCGTG No change 1 1546 TTTATGGACCAGA Asp516 Asn 10 1600 GCACTCGGCCCAG Gly534 Ser 7 1586 AACGTCGTATCTC Arg529 His 1 AT →GC transitions 1552 GACCAGAACAACC Asn518 Asp 9 1547 TTATGGACCAGAA Asp516 Gly 7 1598 CCGCACTCGGCCC Leu533 Pro 2 1598 CCGCACTCGGCCC Leu533 Pro 3 AT →TA transversions 1547 TTATGGACCAGAA Asp516 Val 8 1577 TTACGCACAAACG His526 Leu 5 1714 GGTCTGATCAACT Ile572 Phe 1 1598 CCGCACTCGGCCC Leu533 His 1 GC →CG Transversions 1551 GGACCAGAACAAC Gln517 His 6 One T insertion 1983_1984 GTGGTA+TCCGTC 1 II. 60Co-γ ray 51 GC →AT transitions 1546 TTTATGGACCAGA Asp516 Asn 8 1600 GCACTCGGCCCAG Gly534 Ser 6 1586 AACGTCGTATCTC Arg529 His 3 1576 ATTACGCACAAAC His526 Tyr 7 1565 CGCTGTCTGAGAT Ser522 Phe 5 1692* AAACCCCTGAAGG Pro564 Leu 3 AT →GC transitions 1547 TTATGGACCAGAA Asp516 Gly 5 1552 GACCAGAACAACC Asn518 Asp 3 1598 CCGCACTCGGCCC Leu533 Pro 2 1538 TGTCTCAGTTTAT Gln513 Arg 1 AT →TA transversions 1577 TTACGCACAAACG His526 Leu 3 1598 CCGCACTCGGCCC Leu533 His 3 1714 GGTCTGATCAACT Ile572 Phe 1 AT →CG transversions 1714 GGTCTGATCAACT Ile572 Leu 1 showed that the preference of base substitutions induced by nitrogen ion beam were GC →AT transitions, AT →GC transitions, and AT →TA transversions which account for 92.13% (82/89) of the total, while the mutations induced by 60Co-gamma ray were mainly GC →AT transitions, and AT →GC transitions, 84.31% (43/51). and the temperature was as low as 0 °C in the target cham- ber. The working conditions of the ion implantation were not suitable for living cells. Even though they were pro- tected with 10% glycerol, 15% of them died during treat- ment, as compared to a complete control (data not shown). Phanchaisri et al. (2002) reported a similar cell-killing ef- fect on E. coli cells, when treated with Ar+ at a working condition of 10-4 Pa and approximately 0 °C. We may esti- mate that the E. coli cell has a side surface of about 0.5 to 1 µm2. Therefore, around 105 ~ 106 ions per cell were bom- barded on the cell surface when the cells were spread flat. Yu et al. (2002) had found evidence that most of Ar+ with energy of 30 keV can only penetrate the cell wall into ~100 Experimental system It has been deduced that mismatch DNA damages were the most important source of mutations induced by ni- trogen ion beam implantation in naked DNA (see review by Yu, 2000). We here constructed a novel experimental sys- tem through which the specificity of base substitutions in living cells can be detected and analyzed. The E. coli chro- mosome rpoB gene region A contains the two clusters re- sponsible for Rifr. Previous studies (Jin and Gross 1988; Miller et al., 2002; Severinov et al., 1993) have identified 47 single-base substitutions which cover all kinds of transi- tions and transversions in region A. Our experiment was time and cost-saving because it was based on a single E. coli strain and did not require preparation of chromosome or plasmid DNA. Moreover, it was sensitive and efficient for determining the specificity of base substitutions. We also sequenced the region B of the rpoB gene, in order to obtain the nonselective mutation. However, only one T in- sertion mutation induced by low energy nitrogen ion beam implantation was identified through this screening system, suggesting that it is very hard to identify nonselective muta- tions based on this system. Since mutational spectra convey only the end point of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult to assess the mutational specificity of muta- gens directly from them. Exposure to ionizing radiation can damage DNA directly, but the predominant pathway arises from radiolysis of H2O, which results in the formation of re- active species such as OH.. There is evidence indicating that ROS can react with DNA (Wang et al., 1998). The gamma radiation-induced mutations were derived from these direct and/or indirect DNA damages. 8-OH-dG and 5-OH-dC might offer some explanations for the preference of GC →AT transition, according to the summary of poten- tial correlation between mutations observed in oxidant- induced mutational spectra (Hirano et al., 2001; Wang et al., 1998). The greater number of types and higher frequen- cies of nitrogen ion beam-induced mutations suggest that the mechanisms of nitrogen ion mutagenesis are more com- plicated than those of gamma-ray radiation. This complex- ity might result from the complex interactions between nitrogen ions and the target molecules. Experimental system Low-energy nitro- gen ion beams could not only generate ionizing radiation effects similar to gamma-ray radiation, but also the ions themselves could play a role in the formation of adducts. Some in vitro studies performed in our laboratory seem to support this point. New amino acids were synthesized in component solutions, and the nitrogen ion itself might also provide a nitrogen group in this reaction (Shi et al., 2001b). As for nucleotides, nitrogen ion implantation might pro- duce an effect of damage and form some adducts (Shi et al., 2001a). As a heavy ion, the high LET irradiation effect (Hendry 1999) might be considered, even though the mu- tant screening system was aimed at determining base sub- stitution. The cascade effects of high LET irradiation might also play a role in forming the base substitution mutations. We also compared the results with our former naked DNA irradiation studies, in which the transitions were mainly from CG to TA and from AT to GC, and the transversions were mainly from CG to AT and from CG to GC (Yang et al., 1997). This simple mutational spectrum indicated that the interaction between naked DNA in vitro and nitrogen ions was also likely to be simple. Therefore, more types and species of reactive adducts could be generated in the pro- cess of implantation than in that of gamma ray irradiation and naked DNAs. Apparently, a reactive accelerated nitro- Generally, E. coli strains have an average mutation rate to rifampicin resistance of about 1×10-8, also called spontaneous mutation rate. Some strains have a spontane- ous mutation rate of about 2.0×10-9(Matic et al., 1997). We here found a lower spontaneous mutation rate of about 1×10-9 (Figure 1 C and Figure 1 D, dosage = 0). The differ- ence might be due to our protocols, since the cell concentra- tion for mutant screening was around ~109 fold higher than the survival-determining one. A different loss in the pro- cess of manipulation might lead to this fairly lower sponta- neous mutation rate. Theoretically, after irradiated or implanted, the mutant cells have a physiological delay to stabilize the mutant phenotype of up to 4 ~ 5 generations. Routinely, the treated cells are grown overnight (Cupples et al., 1990; Cupples and Miller 1989). Here, the treated cells were grown in an enriched culture medium for only 30 min, and a little longer to wait for spreading. Nitrogen ion beam implantation and mutation specificity During the treatment of E. coli cells with 10 keV ni- trogen ion beam implantation, the air pressure was 10-3 Pa Xie et al. 289 to 200 nm, at a fluence of 1.5×1015 ions/cm2. Accidentally, rare ions could reach a depth of 10 µm in the plant cell wall (Yu et al., 2002). In the present study, 10 keV nitrogen ions at the fluence rank of 1014 to 1015 were used to implant the E. coli cells. We presumed that the cytoplasmic effect might play the most important role in the induction of the mutation, as in the study of Hei et al. (1997) in a mamma- lian system, using exact numbers of alpha particles for the irradiation of cytoplasm. gen ion group and its series products could act as compo- nent of adducts. Studies about the roles of heavy ions in the formation of the reactive DNA adducts are underway, to explain the detailed mechanisms of the specificity of base substitutions. New sites leading to Rif r Jin DJ, Cashel M, Friedman DI, Nakamura Y, Walter WA and Gross CA (1988) Effects of rifampicin resistant rpoB muta- tions on antitermination and interaction with nusA in Esche- richia coli. J Mol Biol 204:247-261. Previous studies (Jin and Gross 1988; Miller et al., 2002; Severinov et al., 1993) have identified 47 single-base substitutions at 29 sites and distributed among 21 coding positions leading to Rifr in E. coli. Here, we identified two new Rifr-determining sites (Table 2). The complete new site determined in this study is located at nucleotide site 1551 and amino acid site 517. GC →CG transversion at 1551 caused a histidine substitute, Gln517, and led to a fairly high Rifr capacity. The other new site determined is located at 1692. When dC1692 was replaced by a dT, it re- sulted in a change of Pro564 into leucine. The synonymous mutation of the second site had been reported previously (Miller et al., 2002)[11], but the nucleotide substitution, dC1692 →dT1692, had not yet been identified. This find- ing might supplement the Rifr analysis in E. coli and some other pathogenic bacteria. Jin DJ and Gross CA (1988) Mapping and sequencing of muta- tions in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol 202:45-58. Matic I, Radman M, Taddei F, Picard B, Doit C, Bingen E, Denamur E and Elion J (1997) Highly variable mutation rates in commensal and pathogenic Escherichia coli. Sci- ence 277:1833-1834. Miller JH, Funchain P, Clendenin W, Huang T, Nguyen A, Wolff E, Yeung A, Chiang JH, Garibyan L, Slupska MM and Yang HJ (2002) Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitu- tions and frameshifts in mismatch-repair-deficient strains. Genetics 162:5-13. Ohta T, Watanabe-Akanuma M, Tokishita S and Yamagata H (1999) Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P- WP3106P tester strains. Mutat Res 440(1):59-74 Acknowledgements Phanchaisri B, Yu LD, Anuntalabhochai S, Chandej R, Apavatjrut P, Vilaithong T and Brown IG (2002) Characteristics of heavy ion beam-bombarded bacteria E. coli and induced di- rect DNA transfer. Surface and Coatings Technology 158- 159:624-629. This work was partly supported by grants from the National Natural Science Foundation of China (General Program n. 10375066 & n. 30170234). We thank research assistants Yu, L.X. and Liu, X.H. for their help in nitrogen ion beam implantation. Severinov K, Soushko M, Goldfarb A and Nikiforov V (1993) Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Esche- richia coli RNA polymerase. J Biol Chem 268:14820- 14825. Experimental system This time period al- lowed the cells to replicate and divide just one round. Though the mutant phenotype was not quite stable, the ho- mologous mutants could be avoided in cultures, due to cell division. The short culture time might also result in lower mutant frequencies determined in mutagens, because it may have been too short for some mutants to express their mutant phenotype. It would be necessary to have informa- tion about how long and how well the culture time was after treatment with mutagens. This work is in progress. 60Co-gamma ray irradiation in Escherichia coli 290 References Shi HB, Shao CL and Yu ZL (2001a) Dose effect of keV ions irra- diation on adenine and cytosine. Acta Biophysica Sinica 17:731-735. Cupples CG, Cabrera M, Cruz C and Miller JH (1990) A set of lacZ mutations in Escherichia coli that allow for rapid detec- tion of specific frameshif mutations. Genetics125:275-280. Shi HB, Shao CL and Yu ZL (2001b) Preliminary study on the way of formation of amino acids on primitive earth under non-reducing conditions. Radiation Physics and Chemistry 62:393-397. Cupples CG and Miller JH (1989) A set of lacZ mutations in Esch- erichia coli that allow for rapid detection of each of the six base substitutions. Proc Natl Acad Sci USA 86:5345-5349. Grosovsky AJ, de Boer JG, de Jong PJ, Drobetsky EA and Glickman BW (1988) Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc Natl Acad Sci USA 85(1):185-188. Wang D, Kreutzer DA and Essigmann JM (1998) Mutagenecity and repair of oxidative DNA damage: Insight from studies using defined lesions. Mutation Res 400:99-115. Wu LJ, Randers-Pehrson G, Xu A, Waldren CA, Yu ZL and Hei TK (1999) Targeted cytoplasmic irradiation with alpha par- ticles induces mutations in mammalian cells. Proc Natl Acad Sci USA 96:4959-4964. Grosovsky AJ, Drobetsky EA, deJong PJ and Glickman BW (1986) Southern analysis of genomic alterations in gamma- ray-induced aprt- hamster cell mutants. Genetics 113:405- 15. Yang JB, Wu LJ, Li L, Yu ZL and Xu ZH (1997) Sequence analy- sis of lacZ—mutations induced by ion beam irradiation in double-stranded M13mp18DNA. Science in China (series C) 40:107-112. Hei TK, Wu LJ, Liu SX, Diane V, Waldren CA and Randers- Pehrson G (1997) Mutagenic effects of a single and an exact number of alpha particles in mammalian cells. Proc Natl Acad Sci USA 94:3765-3770. Yu LD, Phanchaisri B, Apavatjrut P, Anuntalabhochai S, Vilaithong T and Brown IG (2002). Some investigation of ion bombardment effects on plant cell wall surfaces. Surface and Coatings Technology 158-159:146-150. Hendry JH (1999) Repair of cellular damage after high LET irra- diation. J Radiat Res 40, Suppl:60-65. Yu ZL (2000) Ion beam application in genetic modification. IEEE Transaction on Plasma Science 28:128-132. Hirano T, Hirano H, Yamaguchi R, Asami S, Tsurudome Y and Kasai H (2001) Sequence specificity of the 8-hydroxygua- nine repair activity in rat organs. J Radiat Res 42:247-254. EditorAssociado: Carlos F.M. Menck
https://openalex.org/W1979765051	https://www.frontiersin.org/articles/10.3389/fimmu.2012.00055/pdf	English	null	The Complement System at the Embryo Implantation Site: Friend or Foe?	Frontiers in immunology	2,012	cc-by	9,146	INTRODUCTION cells. Unlike villous trophoblasts, EVTs express MHC molecules, but these are restricted to class I classical HLA-C and non-classical HLA-E, HLA-F, and HLA-G proteins that are not usually used to stimulate T cells. During the early phase of pregnancy the endometrium of the uterus undergoes profound changes and transforms into decidua, anewlyformedtissuethatplaysacriticalroleforsuccessfulembryo implantation and regular fetal growth. The decidua provides a physical anchorage for the implanted embryo tightly attached to the maternal tissue by means of anchoring villi surrounded by an outer layer of trophoblasts that form cell columns. Some of these cells leave the villi and invade the decidua as extravillous trophoblasts (EVTs) reaching the inner third of the myometrium where they form multinucleated giant cells (Plaisier, 2011). A dis- tinct group of EVTs penetrates the uterine spiral arteries and migrates upward against the blood ﬂow partially replacing the endothelial cells (ECs) to form mosaic vessels (Bulla et al., 2005; Figure 1). The mother adopts a different strategy to protect the fetus organizing a protective environment at site of embryo implan- tation with the aim to neutralize harmful attack of the fetus by the maternal immune response. Maternal tolerance of the fetus is maintained primarily by CD4+ CD25+ regulatory T cells (Aluvi- hare et al., 2004) with the contribution of CD8+ T cells (Tilburgs et al., 2010) and NK cells (Hanna et al., 2006), which have recently been shown to induce formation of T regulatory cells as a result of physical interaction with decidual myelomonocytic CD14+ (Vacca et al., 2010). Embryo implantation is associated with an inﬂammatory process that develops in decidua in response to trophoblast cells that make their way through the stroma by means of the pro- teases of the matrix metalloproteinase and plasminogen activa- tor/plasmin systems (Salamonsen, 1999; Pepper, 2001) to invade the maternal tissue. Trophoblast invasion of decidua results in recruitment and activation of leukocytes and precursors of ECs and in the release of various cytokines, chemokines, growth fac- tors that promote tissue remodeling (Plaisier, 2011). Hormonal changes contribute to the development of inﬂammation in mater- nal decidua in the early pregnancy as suggested by the observation that a similar process, though less intense, occurs in non-pregnant Being of fetal origin, villous trophoblasts exposed to mater- nal blood circulating in the intervillous space and EVTs present in human decidua represent a real challenge for the maternal immune system. Keywords: complement, C1q, C7, inﬂammation, placenta, pregnancy disorders R. Bulla, F. Bossi and F.Tedesco An inﬂammatory-like process and vascular remodeling represent the main changes that occur in decidua in the early phase of pregnancy. These changes are partly induced by trophoblast cells that colonize the decidua and are also contributed by the complement system, which can easily be activated as a result of tissue remodeling. Local control by several complement regulators including surface-bound and soluble molecules is critical to prevent complement-mediated tissue damage in normal pregnancy. C7 expressed on the endothelial cells (ECs) surface has been recognized as a novel complement regulator involved in the control of the proinﬂammatory effect of the terminal complement complex. The protective role of placental complement regulators in pregnancy is underscored by the recent ﬁnding of an association of preeclampsia with mutations in the genes encoding for some of these proteins. Complement components produced at feto-maternal inter- face serve an important function in placental development. C1q synthesized by decidual ECs and expressed on the cell surface is particularly important in this regard because it acts as a molecular bridge between endovascular trophoblast and ECs. C1q is also produced by extravillous trophoblast and is used to favor trophoblast migration through the decidua. Defective expression of C1q by trophoblast is associated with impaired tro- phoblast invasion of decidua and may have important implications in pregnancy disorders such as preeclampsia characterized by reduced vascular remodeling. Edited by: Berhane Ghebrehiwet, Stonybrook University, USA Reviewed by: Berhane Ghebrehiwet, Stonybrook University, USA Min Wu, University of North Dakota, USA Correspondence: R. Bulla, Department of Life Sciences, University of Trieste, Via Valerio 28, 34127 Trieste, Italy. e-mail: rbulla@units.it Correspondence: Correspondence: R. Bulla, Department of Life Sciences, University of Trieste, Via Valerio 28, 34127 Trieste, Italy. e-mail: rbulla@units.it REVIEW ARTICLE published: 19 March 2012 doi: 10.3389/ﬁmmu.2012.00055 REVIEW ARTICLE published: 19 March 2012 doi: 10.3389/ﬁmmu.2012.00055 Edited by: Edited by: Berhane Ghebrehiwet, Stonybrook University, USA Reviewed by: Berhane Ghebrehiwet, Stonybrook University, USA Reviewed by: Berhane Ghebrehiwet, Stonybrook University, USA Min Wu, University of North Dakota, USA University, USA Min Wu, University of North Dakota, USA INTRODUCTION As a result, a vigorous response may be mounted against paternal antigens expressed on trophoblasts leading to potential deleterious effects on fetal survival. To avoid these unde- sired consequences,different strategies are developed to prevent an immunologic attack of the mother against the fetus while keeping intact the protective function against harmful pathogens. Failure to express classical polymorphic MHC class I and class II mol- ecules is used by villous trophoblasts to evade recognition by T March 2012 \| Volume 3 \| Article 55 \| 1 www.frontiersin.org www.frontiersin.org The complement system in pregnancy Bulla et al. These cells are stimulated by trophoblasts to remove apoptotic cells in order to prevent the release of harmful proinﬂammatory and pro-immunogenic intracellular contents (Abrahams et al., 2004) and are also educated to secrete cytokines and chemokines that support trophoblast growth and survival (Fest et al., 2007). FIGURE 1 \| Interstitial and endovascular invasion of trophoblast cells. (A) Histological section of ﬁrst trimester placenta stained for cytokeratin 7, showing an anchoring villi (AV). (B)Transmission electron microscope image of a remodeled vessel in which a trophoblast cell (TC) lines the vascular wall together with endothelial cells (ECs). Vascular changes represent an important feature of the decid- ual tissue in the early phase of pregnancy and are characterized by active angiogenesis and vascular remodeling involving the spi- ral arteries. These changes ensure a regular supply of maternal blood to the developing placenta at an optimal ﬂow rate and are made possible by the transformation of the spiral arteries into low- resistance dilated thin-walled vessels as a result of loss of vascular smooth muscle cells and partial replacement of ECs by endovas- cular trophoblasts (Lyall et al., 2001; Pijnenborg et al., 2006). Data collected both in mice (Guimond et al., 1998; Greenwood et al., 2000), and humans (Smith et al., 2009) suggest that decidual NK cells clustered around spiral arteries in early pregnancy play a role in vessel remodeling. Perivascular trophoblasts have also been shown to contribute to this process inducing degradation of the internal elastic lamina and medial elastin ﬁbers of the vessel wall through the action of metalloproteinases MMP-12 (Harris et al., 2010). The original idea that pregnancy represents an anti- inﬂammatory state is no longer tenable in the light of evidence supporting the concept that a proinﬂammatory environment is required for embryo implantation and trophoblast invasion (Mor et al., 2011). INTRODUCTION The data so far available indicate that the inﬂamma- tory state is sustained by cytokines and chemokines released by the cells that accumulate in the decidua. Less attention has been paid to the contribution of the complement (C) system to the success of embryo implantation and also to the regular development of the fetus. In this review we shall examine the double role that C plays in the pathophysiology of pregnancy focusing in particular on the local effect of C components and C activation products in placenta. FIGURE 1 \| Interstitial and endovascular invasion of trophoblast cells. (A) Histological section of ﬁrst trimester placenta stained for cytokeratin 7, showing an anchoring villi (AV). (B)Transmission electron microscope image of a remodeled vessel in which a trophoblast cell (TC) lines the vascular wall together with endothelial cells (ECs). uterusduringthemid-lutealphaseof themenstrualcycle.Itshould be pointed out, however, that this process rather than having dele- terious effects helps to promote fetal survival and somehow resem- bles the inﬂammation developing within the tumor microenviron- ment that stimulates tumor progression, tissue remodeling and angiogenesis, and down-modulates adaptive immune response (Sica and Bronte, 2007; Le Bitoux and Stamenkovic, 2008). Frontiers in Immunology \| Molecular Innate Immunity NON-CANONICAL ROLE OF THE C SYSTEM IN HUMAN PLACENTA l h b d ff h For a long time C has been viewed as an effector system that, once recognized the target to neutralize, acts by promoting the inﬂam- matory process or by inducing cell cytotoxicity. Data accumulated over the last few years indicate that C components and C acti- vation products may exhibit alternative functions. The increased expression of C1q and C4 mRNA in some areas of the rat brain during the embryonic life is suggestive of a novel role of C in brain development (Johnson et al., 1994). C3 and C5 have been implicated in lens regeneration (Kimura et al., 2003), while C3a and C5a were shown to promote liver regeneration through the intermediate action of IL-6 and TNFα (Strey et al., 2003). SeveralCcomponentsof bothclassicalandalternativepathways and C regulators are secreted by chorionic tissue and chorion- derived cells and their synthesis is differently regulated by various cytokines (Goldberg et al., 2007). The ability of some of these cytokines to up-regulate local production of C components and regulators in normal pregnancy is critical to neutralize invading pathogens and also to protect the fetus from attack by the maternal immune system. The decidua is another tissue where C has been found to con- tribute to tissue remodeling and vascular changes (Bulla et al., 2008). The recognition that the C system serves non-canonical functions in human placenta stems from studies showing that C1q is involved in placental development (Bulla et al., 2008). More speciﬁcally, C1q was found to play an important role in the replacement of DECs by endovascular trophoblasts migrating along the decidual spiral arteries and in trophoblast invasion of maternal decidua. The original observation that prompted these studies was the detection of C1q on the surface of DECs in normal pregnancy in the absence of immunoglobulins and C4. This ﬁnd- ing was surprising because deposits of C1q are usually observed in pathologic conditions associated with C activation. Interest- ingly, C1q was found to be localized at the contact site between endovascular trophoblasts and DECs suggesting its implication in the physical interaction between the two cell types. In vitro exper- iments of adhesion assay conﬁrmed this hypothesis showing that the adhesion of puriﬁed trophoblasts to a conﬂuent monolayer of y Trophoblasts constitute a major component of decidual cells and contribute to local synthesis of C components in maternal decidua representing the primary source of C3 and C4 at feto- maternal interface (Bulla et al., 2009). PRODUCTION OF C COMPONENTS AND REGULATORS AT THE FETO-MATERNAL INTERFACE Although the liver represents the major source of circulating C components (Morris et al., 1982), evidence collected over the last three decades indicates that they can also be synthesized at extra- hepatic sites in various tissues and organs (Morgan and Gasque, 1997). Several cell types have been found to produce C com- ponents including macrophages (Johnson and Hetland, 1988), ﬁbroblasts (Garred et al., 1990), ECs (Langeggen et al., 2000) as well as tissue speciﬁc cells (Brooimans et al., 1991; Dovezenski et al., 1992; Khirwadkar et al., 1993; Gulati et al., 1994; Andoh et al., 1998; Grifﬁths et al., 2009; Pattrick et al., 2009). As a result of the contribution of different cell types to local synthesis of C components, an entire C system can be organized at tissue sites that can be readily available for local needs. The C proteins are secreted locally in limited amount under physiologic conditions, but their level is expected to meet the special requirements elicited by pathologic situations that regulate their biosynthesis through the action of cytokines and other stimuli (Colten and Garnier, 1998). The cell inﬁltrate present in decidua during normal pregnancy includes leukocytes that constitute 30% of stromal cells and com- prise approximately 70% NK cells, 20–25% macrophages, and 1.7% dendritic cells, while T lymphocytes represent about 3–10% of the decidual immune cells (Vacca et al., 2010). These cells com- municate with each other and talk to EVTs contributing in this way to the important changes that occur in decidua. Thus, decid- ual NK cells have been found to regulate trophoblast invasion (Hanna et al., 2006) while EVTs recruit decidual NK through the release of CXCL12 (Hanna et al., 2003; Wu et al., 2005). A cross-talk is also established between EVTs and other cells of both innate and adaptive immunity leading to the differenti- ation of immune cells into a trophoblast-supporting phenotype through the action of HLA-G expressed by invading trophoblasts (Shakhawat et al., 2010; Mor et al., 2011). A special interac- tion occurs between trophoblasts and macrophages that represent approximately 20–25% of the leukocyte population in decidua. Frontiers in Immunology \| Molecular Innate Immunity March 2012 \| Volume 3 \| Article 55 \| 2 The complement system in pregnancy Bulla et al. PRODUCTION OF C COMPONENTS AND REGULATORS AT THE FETO-MATERNAL INTERFACE Although locally synthesized C components may contribute to tissue damage, as is the case of C3 produced by renal tubular epithelium involved in the development of inﬂammation leading to transplant rejection (Pratt et al., 2002), they play a critical role in host defense at tissue and organ level (Morgan and Gasque, 1997; Laufer et al., 2001). The protective function of the C system is particularly important in the female reproductive tract during pregnancy since the implanted embryo is vulnerable to attack by pathogens that colonize the cervico-vaginal cavity. It is therefore not surprising that a local protection is needed to prevent the access of infectious agents to the feto-placental unit. The recogni- tion molecules of the C system MBL, C1q, and C3 are present in the cervico-vaginal cavity where they provide a ﬁrst line of defense against infectious agents (Pellis et al.,2005). Interestingly,the vagi- nal epithelium is the main local source of MBL which is secreted under hormonal control reaching the highest value in the post- ovulatory phase of the menstrual cycle in close correlation with the increased level of progesterone. also detected on the membrane of DECs (Figure 2; Bossi et al., 2009). However, the expression of C7 on the cell surface, unlike that of C1q, is not restricted to DECs but it is observed also on the vessel endothelium of several tissues including endometrium of non-pregnant uterus, skin, kidney, and brain. The expression level of membrane bound C7 is up-regulated by the proinﬂamma- tory cytokines I-1α, TNF-α, and IFN-γ. In contrast with bound C1q, surface expressed C7 cannot be removed by treatment with heparinases and is associated with vimentin. REGULATORY FUNCTIONS OF THE C SYSTEM AT THE FETO-MATERNAL INTERFACE Extensive remodeling of decidua favors C activation that may lead to tissue damage and adverse pregnancy outcome. C-dependent damage of trophoblasts may promote cell destruction or more likely increased permeability of the barrier, opening the way to bacteria, virus, and other toxic molecules that compromise fetal survival. No wonder if decidual cells have developed strategies to prevent C-mediated damage consisting in the expression of C regulatory proteins (CRPs) DAF or CD55, MCP or CD46, and CD59. Syncytiotrophoblasts that form the outer layer of villous tro- phoblasts require a particular protection against C activation products because they are directly exposed to maternal blood and are therefore at special risk of being attacked by activated C. These cells express all three regulatory proteins (Holmes et al., 1990, 1992) and are further protected by the binding of vit- ronectin or S protein, a soluble inhibitor of the assembling mem- brane attack complex (MAC) of the C system acquired from the maternal blood (Tedesco et al., 1990). Villous cytotrophoblasts form a continuous layer of cells adhering to the basal mem- brane of the villi below the syncytiotrophoblasts and express MCP and CD59, while controversy exists on the expression of DAF (Holmes et al., 1990, 1992; Hsi et al., 1991). EVTs also express CRPs on their surface, in particular CD59, while DAF and MCP are mainly detected on decidual giant cells (Hsi et al., 1991; Bulla et al., 2003). Endovascular trophoblast cells that colonize decidual spiral arteries are similarly exposed to maternal blood and are a potential C target. Damage may also involve ECs and other cells of maternal origin in decidua, since locally released C effector molecules do not distinguish between foreign agents and self components and cause tissue alterations through the destructive effect of MAC or via promotion of the inﬂammatory process. The importance of C regulation at the embryo implantation site is emphasized by the data obtained in murine models of preg- nancy failure. Xu et al. (2000) made the original observation that Crry deﬁciency in utero leads to progressive embryonic lethality and Crry−/−embryos are surrounded by activated C3 fragments and PMN. Crry is expressed on fetal trophoblast cells and mater- nal decidua, where it controls deposition of C3 and C4 on the cell surface, preventing formation of the cytolytic MAC (Weigle et al., 1983; Molina et al., 1992; Kim et al., 1995). NON-CANONICAL ROLE OF THE C SYSTEM IN HUMAN PLACENTA l h b d ff h This cell bound C com- ponent acts as a bridge between endovascular trophoblasts and decidual endothelium by virtue of its interaction with a receptor for the globular head of C1q (gC1qR) expressed on trophoblasts. Binding of trophoblasts to DECs is a rather complex process pro- moted by several molecules including C1q andVE-cadherin (Bulla et al., 2005) and is followed by transendothelial migration of tro- phoblasts that eventually reach the basal membrane and partially replace ECs. Evidencefortheprotectivefunctionof theregulatorymolecules expressed on the surface of syncytiotrophoblasts was obtained in studies analyzing the susceptibility of these cells to C-dependent killing induced by antibodies directed against steroid producing cell that recognize syncytiotrophoblasts (Tedesco et al., 1993). Antibody-mediated neutralization of CD46 and CD59 expressed on syncytiotrophoblasts led to a substantial increase in cell killing emphasizing the important role of the two C regulators in tro- phoblast protection while CD55 seems to be less critical being expressed in only 30% of these cells (Tedesco et al., 1993). DECs is prevented by antibodies to C1q. This cell bound C com- ponent acts as a bridge between endovascular trophoblasts and decidual endothelium by virtue of its interaction with a receptor for the globular head of C1q (gC1qR) expressed on trophoblasts. Binding of trophoblasts to DECs is a rather complex process pro- moted by several molecules including C1q andVE-cadherin (Bulla et al., 2005) and is followed by transendothelial migration of tro- phoblasts that eventually reach the basal membrane and partially replace ECs. The ﬁnding that C1q is actively synthesized by EVTs and is widely distributed in decidual stroma led to the recognition of an additional function of the early C component in promoting tro- phoblast migration through the decidua. Trophoblast cells were found to adhere to C1q and to migrate through an insert of a transwell coated with C1q that recognizes cell-expressed gC1qR, as well as a4 and β1 integrins and triggers activation of the MAP kinases pathway. The in vivo relevance of these ﬁndings is sup- ported by the observation of impaired labyrinth development and decreased remodeling of decidual vessel associated with increased fetal resorption rate, reduced fetal weight, and smaller litter size observedinC1qKOmiceascomparedwithWTanimals(Agostinis et al., 2010). p y ( , ) Endothelial cells are another potential target of C attack in decidua and are protected by several CRPs expressed on the cell surface or acquired from the ﬂuid phase (Fischetti and Tedesco, 2006). NON-CANONICAL ROLE OF THE C SYSTEM IN HUMAN PLACENTA l h b d ff h Evidence has been collected supporting an additional pro- tective mechanism used by ECs to control the proinﬂammatory effects of the terminal complex SC5b-9. This complex is assembled in the ﬂuid phase as a result of C activation and can be detected both in plasma and in the extravascular ﬂuids as a cytolytically inactive complex (Bossi et al., 2009). We have previously shown that this inactive complex, despite its failure to cause cell dam- age, is still able to bind to ECs and stimulate the cells to express adhesion molecules and tissue factor (TF; Tedesco et al., 1997), to release chemokines (Dobrina et al.,2002),to induce transendothe- lial migration of PMN (Dobrina et al., 2002), and to promote vascular leakage (Bossi et al., 2004). Protection against the dam- aging effect of the assembling terminal C complex is provided by EC-bound C7, that acts as a trapping molecule for excess C5b6 complex released in the ﬂuid phase under conditions of unre- stricted C activation during acute phase response. Subsequent binding of C8 and C9 leads to the assembly of a cytolytically inac- tive membrane bound terminal complex (mTCC), that contains also S protein and clusterin and is cytolytically inactive. How- ever, unlike soluble SC5b-9, mTCC fails to activate ECs, and in addition down-regulates cell activation induced by SC5b-9 mod- ulating the expression of adhesion molecules, the release of IL-8, and EC permeability (Bossi et al.,2009). ECs normally utilize GPI- anchored molecule CD59 to neutralize the C-mediated cell lysis caused by the assembling MAC, but this C regulator has no effect on the damaging effect of the soluble complex SC5b-9 which can only be controlled by mTCC. Tight control of C activation by several regulators is particularly important at feto-placental site where direct C-mediated damage and indirect effect associated with excessive inﬂammation may impair fetal growth and possibly lead to miscarriage (Christiansen et al., 2006). NON-CANONICAL ROLE OF THE C SYSTEM IN HUMAN PLACENTA l h b d ff h The expression of these C components by trophoblast cells is not modulated by IL-1α and TNF-α but is up-regulated by IFNγ. The latter cytokine pro- duced at placental level mainly by decidual NK (Lash et al., 2006) controls trophoblast activation (Gagioti et al., 2000) and inva- sion into the decidua (Hu et al., 2006; Lash et al., 2006), and plays a critical role in remodeling spiral arteries in the mouse (Ashkar et al., 2000). The ﬁnding that trophoblast cells secrete C3 and C4 involved in opsonophagocytosis further supports previ- ous observations suggesting that these cells help to promote the innate immune response at the placental level during pregnancy and potentially contribute to placental immune defense against pathogens (Amarante-Paffaro et al., 2004). In addition to C3 and C4, trophoblast cells have been found to secrete C1q (Agostinis et al., 2010), which is relatively abundant in maternal decidua being synthesized by other cells including macrophages, ﬁbrob- last, and ECs. It is important to note that the decidua is the only tissue where ECs are able to produce C1q in physiologic condi- tions (Bulla et al., 2008). Interestingly, the molecule synthesized by decidual ECs (DECs) is expressed to a large extent on the cell sur- face and only marginally secreted. The high avidity binding of C1q to the cell membrane is not due to its interaction with the receptors for the collagen tail or the globular head of the molecule identiﬁed on ECs (Peerschke et al., 1996). Removal of a substantial portion of bound C1q from the cell membrane by treatment with hepari- nases rather suggests that surface expressed heparan sulfates offer the binding site for the molecule (Bulla et al., 2008). C7 is another C component synthesized by ECs (Langeggen et al., 2000) and is FIGURE 2 \| C7 is synthesized and expressed by DECs. (A) Light microscopy image of a decidual vessel from human ﬁrst trimester placenta. (B) Confocal analysis of the same decidual vessel stained for C7. FIGURE 2 \| C7 is synthesized and expressed by DECs. (A) Light microscopy image of a decidual vessel from human ﬁrst trimester placenta. (B) Confocal analysis of the same decidual vessel stained for C7. March 2012 \| Volume 3 \| Article 55 \| 3 www.frontiersin.org Bulla et al. The complement system in pregnancy DECs is prevented by antibodies to C1q. Frontiers in Immunology \| Molecular Innate Immunity REGULATORY FUNCTIONS OF THE C SYSTEM AT THE FETO-MATERNAL INTERFACE The critical role played by C activation products in causing fetal loss in Crry−/−mice is supported by the ﬁnding that the embryos are completely rescued from lethality and live pups are born at a normal Mendelian fre- quency when Crry+/−parents are intercrossed with C3−/−mice Frontiers in Immunology \| Molecular Innate Immunity March 2012 \| Volume 3 \| Article 55 \| 4 The complement system in pregnancy Bulla et al. revealed the involvement of C in the onset of the two main clinical manifestations of the syndrome represented by fetal loss and vas- cular thrombosis (Holers et al., 2002; Fischetti et al., 2005; Girardi et al., 2006). Pregnancy failure in the mouse model of APS has been attributed to the release of C5a and recruitment of PMN fol- lowed by TF-dependent promotion of coagulation as a result of C activation induced by APL (Girardi et al., 2003; Ritis et al., 2006; Redechaetal.,2007).Theserumproteinβ2-glycoproteinI(β2GPI) represents the main target of these antibodies once bound to the cell surface of trophoblasts and ECs (Pierangeli et al.,2008; Meroni et al., 2011). Binding of β2GPI to trophoblasts and ECs in placenta occurs spontaneously, but its interaction with the endothelium of other tissues such as brain and gut requires priming with LPS (Agostinis et al., 2011a). to generate C3−/−, Crry−/−embryos (Xu et al., 2000). This out- come is a genetic proof that Crry−/−embryos die in utero due to their inability to suppress C activation and tissue damage medi- ated by C3. The C regulator Crry has also been shown to control fetal resorption in a murine model of antibody-mediated anti- phospholipid syndrome (APS; Holers et al., 2002), as suggested by the ability of human IgG containing anti-phospholipid anti- bodies (aPL) injected into pregnant mice to cause fetal resorption in C sufﬁcient animal, but not in C3 deﬁcient mice. Protection against fetal loss was also observed in mice that received recombi- nant Crry-Ig to prevent C activation through the inhibition of C3 convertase. There is some indication that C regulators contribute to protect the feto-placental unit also in human APS. Francis et al. (2006) reported an impaired endometrial expression of DAF/CD55 in patients with recurrent pregnancy loss associated with APS sug- gesting that the abnormal expression of this C regulator before conception may compromise implantation and predisposes to C-mediated pregnancy failure. REGULATORY FUNCTIONS OF THE C SYSTEM AT THE FETO-MATERNAL INTERFACE While the contribution of the C system to fetal loss and throm- bosis in animal models is well established, the role played by C in mediating tissue damage in human APS is less clear, though this is suggested by the ﬁnding of C deposition in placental tissue from patients with aPL (Girardi et al., 2006). Analysis of placentae obtained from a large number of APS patients by Shamonki et al. (2007) revealed increased deposition of C4d in the cytoplasm of villous trophoblasts correlated with the histologic alterations of APS placentae. An essentially similar conclusion was reached by Cohenetal.(2011),whofounddiffuseC4ddepositsonvilloussyn- cytiotrophoblasts of placentae from patients with SLE and/or APS suggestive of adverse pregnancy outcome. The relevance of C5b-9 deposits in APS patients is somewhat controversial since the local level of this complex was found to be lower in APS patients com- pared with that of the control group (Shamonki et al., 2007). In a recent prospective study on full-term placentae from patients with primary APS and normal control we were able to reveal marked C5b-9 deposits that were only weak or undetectable in the con- trol group, although the presence of the complex was not always related to pregnancy outcome or therapy (Meroni et al., 2011). A protective role for C regulators has also been established in animal models of PE developed in CBA/J females mated to DBA/J males. These mice manifest many features of human PE, such as primigravidity, albuminuria, glomerular endotheliosis, increased sensitivity to angiotensin II and increased plasma leptin levels, correlated with bad pregnancy outcome (Ahmed et al., 2010). Administration of Crry to pregnant DBA/2 CBA/J mice helped to reduce the level of proteinuria, and blood urea nitrogen, ﬁb- rin deposition, and glomerular endotheliosis (Qing et al., 2011), supporting the concept that local C activation is deleterious to the developing fetus. The importance of the C regulators in patients with PE has recently been underscored by Salmon et al. (2011),who performed a prospective study of 250 pregnant patients with SLE and/or APL antibodies to identify predictors of pregnancy outcome. They sequenced the genes encoding three C control proteins, MCP, factor H (CFH),and Factor I (CFI) and found heterozygous muta- tions in 7 out of 40 patients who developed PE. ROLE OF C IN PATHOLOGIC PREGNANCY It is commonly believed that C contributes to tissue damage in pathologic conditions through the destructive activity of MAC or the proinﬂammatory effect of C5a and other activation products including the non-cytolytic complex SC5b-9. Placenta is partic- ularly susceptible to C-dependent damage because trophoblast exposure to maternal blood and extensive tissue remodeling in maternal decidua promote C activation during physiologic preg- nancy. Unrestricted local activation of the system may over- come the control of CRPs resulting in pregnancy failure or poor pregnancy outcome. PREECLAMPSIA Thi i l if This is a multifactorial pathological condition of pregnancy that affects 3%–5% of all pregnancies and is characterized by hyper- tension and proteinuria. The C system has been implicated in the development of PE placental alterations together with other pathogenic factors. Activation products of both early and late C components have been detected in the circulation of PE patients and the split product Bb has been proposed as a marker for high risk of PE (Lynch et al., 2008), though there is no general agree- ment on this issue (Derzsy et al., 2010). Deposits of C components including late components and their activation products respon- sible for promotion of inﬂammation and tissue damage have been documented in PE placenta, in particular in chorionic villi, in inter- and peri-villus ﬁbrin surrounding syncytiotrophoblasts,and in maternal decidua (Sinha et al.,1984; Tedesco et al.,1990; Girardi et al., 2006). Despite the failure to detect increased activity of the MBL–MASP2 complex in the PE patients (Csuka et al., 2010), two activators of the lectin pathway (LP) H-ﬁcolin and L-ﬁcolin have been detected on syncytiotrophoblasts of PE placentae (Wang et al., 2007) suggesting an association of the placental abnormali- ties with the activation of this pathway. The involvement of LP of C activation is further supported by the ﬁnding that MBL-A, one ROLE OF C IN PATHOLOGIC PREGNANCY REGULATORY FUNCTIONS OF THE C SYSTEM AT THE FETO-MATERNAL INTERFACE Based on these ﬁndings, they concluded that mutations in MCP and CFI predis- pose to the development of PE, as already observed in patients with atypical hemolytic uremic syndrome, and that dysregulation of C activation represents a risk factor for PE. ACKNOWLEDGMENTS h k d The contribution of the C system to host defense and tissue damage mediated through the inﬂammatory process and cell cytotoxicity is well established. More recently, alternative func- tions on cells and tissues have been identiﬁed involving both native components and C activation products. The C system has been shown to contribute to tissue regeneration and brain This work was supported by the European NoE “EMBIC” within FP6 (contract number LSHN-CT-2004-512040) and by a research grant of the Ministry of Health (Ricerca Finalizzata RC41/08 e RC 01/09), Italian Association of Cancer Research (AIRC), Fon- dazione Casali – Trieste, and Italian Ministry of University and Research (PRIN2009 MFXE7L_004). REFERENCES tissue remodeling: contribution to trophoblast invasion, and placen- tal development. J. Immunol. 185, 4420–4429. Bulla, R., Agostinis, C., Bossi, F., Rizzi, L., Debeus, A., Tripodo, C., Radillo, O., De Seta, F., Ghebrehiwet, B., and Tedesco, F. (2008). Decidual endothelial cells express surface- bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium. Mol. 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C1q is emerging as one of the C components able to exert functions independent on C activation. Several cell tar- gets have been found to respond to C1q including cell of innate and acquired immunity as well as other cells not directly involved in the immune response. Most of the information available on this issue have been obtained in in vitro studies using puriﬁed C1q. The pitfall of these studies is that C1q detected in plasma and in extravascular ﬂuid is bound to C1r and C1s to form a complex. Cells synthesizing C1q at tissue level represent a source of free C component that may exert alternative functions. This is the case of trophoblasts and DECs that secrete C1q at the embryo implantation site and contribute to its widespread dis- tribution in decidual stroma. C1q has been shown to play a critical role in placental development favoring interaction between trophoblasts and DECs and promoting EVT migration. 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This is an open-access article distributed under the terms of the Cre- ative Commons Attribution Non Com- mercial License, which permits non- commercial use, distribution, and repro- duction in other forums, provided the original authors and source are credited. Shamonki, J. M., Salmon, J. E., Hyjek, E., and Baergen, R. N. (2007). Excessive complement activation is associated with placental injury in patients with antiphospholipid anti- bodies. Am. J. Obstet. Gynecol. 196, e161–e165. Pijnenborg, R., Vercruysse, L., and Hanssens, M. (2006). ACKNOWLEDGMENTS h k d The uterine spiral arteries in human pregnancy: Vacca, P., Cantoni, C., Vitale, M., Prato, C., Canegallo, F., Fenoglio, D., Ragni, N., Moretta, L., and Mingari, M. March 2012 \| Volume 3 \| Article 55 \| 8 Frontiers in Immunology \| Molecular Innate Immunity
https://openalex.org/W4287888479	https://aclanthology.org/2022.naacl-tutorials.3.pdf	English	null	New Frontiers of Information Extraction	null	2,022	cc-by	7,900	Proceedings of the 2022 Conference of the North American Chapter of the Association for Computational Linguistics Human Language Technologies: Tutorial Abstracts, pages 14 - 25 July 10-15, 2022 ©2022 Association for Computational Linguistics Abstract assists machines with narrative prediction (Zhang et al., 2021b; Chaturvedi et al., 2017) and summa- rization tasks (Liu et al., 2018; Chen et al., 2019b). Medical IE also beneﬁts important but expensive clinical tasks such as drug discovery and repurpos- ing (Sosa et al., 2019; Munkhdalai et al., 2018). Despite the importance, frontier research in IE still faces several key challenges. The ﬁrst challenge is that existing dominant methods using language modeling representation cannot sufﬁciently capture the essential knowledge and structures required for IE tasks. The second challenge is on the develop- ment of extraction models for ﬁne-grained infor- mation with less supervision, considering that ob- taining structural annotation on unlabeled data has been very costly. The third challenge is to extend the reliability and generalizability of IE systems in real-world scenarios, where data sources often con- tain incorrect, invalid or unrecognizable inputs, as well as inputs containing unseen labels and mixture of modalities. By tackling those critical challenges, recent literature is leading to transformative ad- vancement in principles and methodologies of IE system development. We believe it is necessary to present a timely tutorial to comprehensively sum- marize the new frontiers in IE research and point out the emerging challenges that deserve further investigation. This tutorial targets researchers and practition- ers who are interested in AI and ML technolo- gies for structural information extraction (IE) from unstructured textual sources. In particu- lar, this tutorial will provide audience with a systematic introduction to recent advances in IE, by addressing several important research questions. These questions include (i) how to develop a robust IE system from a small amount of noisy training data, while ensur- ing the reliability of its prediction? (ii) how to foster the generalizability of IE through enhancing the system’s cross-lingual, cross- domain, cross-task and cross-modal transfer- ability? (iii) how to support extracting struc- tural information with extremely ﬁne-grained and diverse labels? (iv) how to further im- prove IE by leveraging indirect supervision from other NLP tasks, such as Natural Lan- guage Generation (NLG), Natural Language Inference (NLI), Question Answering (QA) or summarization, and pre-trained language mod- els? (v) how to acquire knowledge to guide inference in IE systems? We will discuss sev- eral lines of frontier research that tackle those challenges, and will conclude the tutorial by outlining directions for further investigation. 1 Introduction Information extraction (IE) is the process of au- tomatically extracting structural information from unstructured or semi-structured data. It provides the essential support for natural language under- standing by recognizing and resolving the concepts, entities, events described in text, and inferring the relations among them. In various application do- mains, IE automates the costly acquisition process of domain-speciﬁc knowledge representations that have been the backbone of any knowledge-driven AI systems. For example, automated knowledge base construction has relied on technologies for entity-centric IE (Carlson et al., 2010; Lehmann et al., 2015). Extraction of events and event chains In this tutorial, we will systematically review several lines of frontier research on developing ro- bust, reliable and adaptive learning systems for ex- tracting rich structured information. Beyond intro- ducing robust learning and inference methods for unsupervised denoising, constraint capture and nov- elty detection, we will discuss recent approaches for leveraging indirect supervision from natural lan- guage inference and generation tasks to improve IE. We will also review recent minimally supervised methods for training IE models with distant super- vision from linguistic patterns, corpus statistics or language modeling objectives. In addition, we will 14 Minimally Supervised IE Indirect Supervision Weak Supervision Modeling Label Semantics QA as IE NLI as IE NLG as IE Linguistic Pattern Mining Language Model Probing Robust IE Noise-robustness Faithfulness and Fairness Logical Consistency Corpus Statistics Noise-robust Training OOD Detection Debiased Training Counterfactual Inference Constraint Learning Constrained Inference Transferable IE Cross-domain Transfer Cross-lingual Transfer Cross-type Transfer Adversarial Domain Adaptation Divergence-based Adaptation Language-invariant Representation Data projection Prototype Learning Reading Definitions Cross-modal & Knowledge-guided IE Vision-Language IE Semi-structured IE Schema-guided IE Statistics-guided IE Commonsense IE IE with Less Supervision Robust and Reliable IE Portable and Generalizable IE New Frontiers of IE Figure 1: A roadmap for new frontiers of information extraction and a graphical abstract of this tutorial. New Frontiers of IE Portable and Generalizable Robust and Reliable IE IE with Less Supervision Cross-modal & Knowledge-guided IE Figure 1: A roadmap for new frontiers of information extraction and a graphical abstract of this tutorial tems across domains, languages and tasks, and how complementary information can be extracted from data modalities other than human language. 2 Outline of Tutorial Content This half-day tutorial presents a systematic overview of recent advancement in IE technolo- gies. We will begin motivating this topic with a selection of real-world applications and emerging challenges of IE. Then, we will introduce robust learning methods and inference methods to tackle noisy supervision, prediction inconsistency and out- of-distribution (OOD) inputs. We will also discuss about indirect supervision and minimal supervision methods that further improves IE model develop- ment under limited learning resources. Based on the robust IE systems developed in close-domain settings, we will explain how transfer learning tech- nologies can adaptively extend the utility of the sys- 2.1 Background and Motivation [20min] We will deﬁne the main research problem and mo- tivate the topic by presenting several real-world NLP and knowledge-driven AI applications of IE technologies, as well as several key challenges that are at the core of frontier research in this area. 1 Introduction More- over, we will exemplify the use of aforementioned technologies in various end-user NLP applications such as misinformation detection and scientiﬁc dis- covery, and will outline emerging research chal- lenges that may catalyze further investigation on developing reliable and adaptive learning systems for IE. The detailed contents are outlined below. illustrate how a model trained on a close domain can be reliably adapted to produce extraction from data sources in different domains, languages and modalities, or acquiring global knowledge (e.g., event schemas) to guide the extraction on a highly diverse open label space. Participants will learn about recent trends and emerging challenges in this topic, representative tools and learning resources to obtain ready-to-use models, and how related technologies beneﬁt end-user NLP applications. A graphical abstract of this tutorial is provided as Fig. 1, which serves as our roadmap for new fron- tiers of information extraction. 2.2 Robust Learning and Inference for IE [35min] We will introduce methodologies that enhance the robustness of learning systems for IE in both their learning and inference phases. Those methodolo- gies involve self-supervised denoising techniques for training noise-robust IE models based on co- regularized knowledge distillation (Zhou and Chen, 2021; Liang et al., 2021), label re-weighting (Wang et al., 2019b) and label smoothing (Lukasik et al., 2020). Besides, we will also discuss about unsuper- 15 vised techniques for out-of-distribution (OOD) de- tection (Zhou et al., 2021b; Hendrycks et al., 2020), prediction with abstention (Dhamija et al., 2018; Hendrycks et al., 2018) and novelty class detection (Perera and Patel, 2019) that seek to help the IE model identify invalid inputs or inputs with seman- tic shifts during its inference phase. Speciﬁcally, to demonstrate how models can ensure the global con- sistency of the extraction, we will cover constraint learning methods that automatically capture logical constraints among relations (Wang et al., 2021a, 2022c; Pan et al., 2020), and techniques to enforce the constraints in inference (Wang et al., 2020; Li et al., 2019a; Han et al., 2019; Lin et al., 2020). To assess if the systems give faithful extracts, we will also talk about the spurious correlation problems of current IE models and how to address them with counterfactual analysis (Wang et al., 2022b; Qian et al., 2021). or languages, we have to start from scratch by creat- ing annotations and re-training the extraction mod- els. In this part of tutorial, we will cover the re- cent advances in improving the transferability of IE, including (1) cross-lingual transfer by leverag- ing adversarial training (Chen et al., 2019a; Huang et al., 2019; Zhou et al., 2019), language-invariant representations (Huang et al., 2018a; Subburathi- nam et al., 2019) and resources (Tsai et al., 2016; Pan et al., 2017), pre-trained multilingual language models (Wu and Dredze, 2019; Conneau et al., 2020) as well as data projection (Ni et al., 2017; Yarmohammadi et al., 2021), (2) cross-type trans- fer including zero-shot and few-shot IE by learning prototypes (Huang et al., 2018b; Chan et al., 2019; Huang and Ji, 2020), reading the deﬁnitions (Chen et al., 2020b; Logeswaran et al., 2019; Obeidat et al., 2019; Yu et al., 2022; Wang et al., 2022a), answering questions (Levy et al., 2017; Liu et al., 2020; Lyu et al., 2021), and (3) transfer across dif- ferent benchmark datasets (Xia and Van Durme, 2021; Wang et al., 2021b). 2.2 Robust Learning and Inference for IE [35min] Finally, we will also dis- cuss the progress on life-long learning for IE (Wang et al., 2019a; Cao et al., 2020; Yu et al., 2021; Liu et al., 2022) to enable knowledge transfer across incrementally updated models. 2.5 Cross-modal IE [20min] Cross-modal IE aims to extract structured knowl- edge from multiple modalities, including unstruc- tured and semi-structured text, images, videos, ta- bles, etc. We will start from visual event and argu- ment extraction from images (Yatskar et al., 2016; Gkioxari et al., 2018; Pratt et al., 2020; Zareian et al., 2020; Li et al., 2022b) and videos (Gu et al., 2018; Sadhu et al., 2021; Chen et al., 2021a). To extract multimedia events, the key challenge is to identify the cross-modal coreference and linking (Deng et al., 2018; Akbari et al., 2019; Zeng et al., 2019) and represent both text and vi- sual knowledge in a common semantic space (Li et al., 2020a; Chen et al., 2021b; Zhang et al., 2021a; Li et al., 2022b). We will also introduce the information extraction from semi-structured data (Katti et al., 2018; Qian et al., 2019) and tabu- lar data (Herzig et al., 2020). Cross-modal IE aims to extract structured knowl- edge from multiple modalities, including unstruc- tured and semi-structured text, images, videos, ta- bles, etc. We will start from visual event and argu- ment extraction from images (Yatskar et al., 2016; Gkioxari et al., 2018; Pratt et al., 2020; Zareian et al., 2020; Li et al., 2022b) and videos (Gu et al., 2018; Sadhu et al., 2021; Chen et al., 2021a). 2.3 Minimally and Indirectly Supervised IE [35min] We will introduce effective approaches that use al- ternative supervision sources for IE, that is, to use supervision signals from related tasks to make up for the lack of quantity and comprehensiveness in IE-speciﬁc training data. This includes indirect supervision sources such as question answering and reading comprehension (Wu et al., 2020; Lyu et al., 2021; Levy et al., 2017; Li et al., 2019b; Du and Cardie, 2020), natural language inference (Li et al., 2022a; Yin et al., 2020) and generation (Lu et al., 2021; Li et al., 2021b). We will also cover the use of weak supervision sources such as structural texts (e.g., Wikipedia) (Ji et al., 2017; Zhou et al., 2018) and global biases (Ning et al., 2018b). With the breakthrough of large-scale pre- trained language models (Devlin et al., 2019; Li et al., 2022c), methodologies have been proposed to explore the language model objective as indi- rect supervision for IE. To this end, we will cover methods includes direct probing (Feldman et al., 2019; Zhang et al., 2020c), and more recently, pre- training with distant signals acquired from linguis- tic patterns (Zhou et al., 2020, 2021a). 4 Tutorial Instructors The following are biographies of the speaker. Past tutorials given by us are listed in Appx. §A.1. Muhao Chen is an Assistant Research Professor of Computer Science at USC, where he directs the Language Understanding and Knowledge Ac- quisition (LUKA) Group. His research focuses on data-driven machine learning approaches for natural language understanding and knowledge ac- quisition. His work has been recognized with an NSF CRII Award, a Cisco Faculty Research Award, an ACM SIGBio Best Student Paper Award, and a Best Paper Nomination at CoNLL. Muhao ob- tained his B.S. in Computer Science degree from Fudan University in 2014, his PhD degree from UCLA Department of Computer Science in 2019, and was a postdoctoral researcher at UPenn prior to joining USC. Additional information is available at http://muhaochen.github.io. 2.7 Future Research Directions [30min] IE is a key component in supporting knowledge acquisition and it impacts a wide spectrum of knowledge-driven AI applications. We will con- clude the tutorial by presenting further challenges and potential research topics in identifying trust- worthiness of extracted content (Zhang et al., 2019, 2020b), IE with quantitative reasoning (Elazar et al., 2019; Zhang et al., 2020a), cross-document IE (Caciularu et al., 2021), incorporating domain- speciﬁc knowledge (Lai et al., 2021; Zhang et al., 2021c), extension to knowledge reasoning and pre- diction, modeling of label semantics (Huang et al., 2022; Mueller et al., 2022; Ma et al., 2022; Chen et al., 2020a), and challenges for acquiring implicit but essential information from corpora that poten- tially involve reporting bias (Sap et al., 2020). Lifu Huang is an Assistant Professor at the Com- puter Science department of Virginia Tech. He obtained a PhD in Computer Science from UIUC. He has a wide range of research interests in NLP, including extracting structured knowledge with lim- ited supervision, natural language understanding and reasoning with external knowledge and com- monsense, natural language generation, represen- tation learning for cross-lingual and cross-domain transfer, and multi-modality learning. He is a recip- ient of the 2019 AI2 Fellowship and 2021 Amazon Research Award. Additional information is avail- able at https://wilburone.github.io/. 2.6 Knowledge-guided IE [15min] One important challenge of developing IE sys- tems lies in the limited coverage of predeﬁned schemas (e.g., predeﬁned types of entities, rela- tions or events) and the heavy reliance on human annotations. When moving to new types, domains Global knowledge representation induced from large-scale corpora can guide the inference about the complicated connections between knowledge elements and help ﬁx the extraction errors. We will 16 Open Access All the materials are openly avail- able at https://cogcomp.seas.upenn. edu/page/tutorial.202207. introduce cross-task and cross-instance statistical constraint knowledge (Lin et al., 2020; Van Nguyen et al., 2021), commonsense knowledge (Ning et al., 2018a), and global event schema knowledge (Li et al., 2020b; Wen et al., 2021; Li et al., 2021a; Jin et al., 2022) that help jointly extract entities, relations, and events. Ethical Considerations Innovations in technology often face the ethical dilemma of dual use: the same advance may of- fer potential beneﬁts and harms. For the IE tech- nologies introduced in this tutorial, the distinction between beneﬁcial use and harmful use depends mainly on the data. Proper use of the technology requires that input text corpora, as well as other modalities of inputs, are legally and ethically ob- tained. Regulation and standards provide a legal framework for ensuring that such data is properly used and that any individual whose data is used has the right to request its removal. In the absence of such regulation, society relies on those who apply technology to ensure that data is used in an ethical way. Besides, training and assessment data may be biased in ways that limit system accuracy on less well represented populations and in new domains, for example causing disparity of performance for different sub-populations based on ethnic, racial, gender, and other attributes. Furthermore, trained systems degrade when used on new data that is distant from their training data. Thus questions concerning generalizability and fairness need to be carefully considered when applying the IE tech- nologies to speciﬁc datasets. Dan Roth is the Eduardo D. Glandt Distinguished Professor at the Department of Computer and In- formation Science, UPenn, the NLP Lead at AWS AI Labs, and a Fellow of the AAAS, ACM, AAAI, and ACL. In 2017 Roth was awarded the John Mc- Carthy Award, the highest award the AI commu- nity gives to mid-career AI researchers. Roth was recognized “for major conceptual and theoretical advances in the modeling of natural language under- standing, machine learning, and reasoning.” Roth has published broadly in machine learning, NLP, KRR, and learning theory, and has given keynote talks and tutorials in all ACL and AAAI major con- ferences. Roth was the Editor-in-Chief of JAIR until 2017, and was the program chair of AAAI’11, ACL’03 and CoNLL’02; he serves regularly as an area chair and senior program committee mem- ber in the major conferences in his research areas. Prof. Roth received his B.A Summa cum laude in Mathematics from the Technion, and his Ph.D. in Computer Science from Harvard University in 1995. Additional information is available at http: //www.cis.upenn.edu/~danroth/. 3 Speciﬁcation of the Tutorial The proposed tutorial is considered a cutting-edge tutorial that introduces new frontiers in IE re- search. The presented topic has not been covered by ACL/EMNLP/NAACL/EACL/COLING tutori- als in the past 4 years. One exception is the ACL 2020 tutorial “Multi-modal Information Extraction from Text, Semi-structured, and Tabular Data on the Web” that is partly relevant to one of our tech- nical sections (§2.5). That particular section of our talk will focus on IE from visual and multi-media data in addition to semi-structured data, being dif- ferent from the aforementioned ACL 2020 tutorial that has mainly covered topics on semi-structured data. Manling Li is a fourth-year Ph.D. student at the Computer Science Department of UIUC. Manling has won the Best Demo Paper Award at ACL’20, the Best Demo Paper Award at NAACL’21, C.L. Manling Li is a fourth-year Ph.D. student at the Computer Science Department of UIUC. Manling has won the Best Demo Paper Award at ACL’20, the Best Demo Paper Award at NAACL’21, C.L. Dave and Jane W.S. Liu Award, and has been selected as Mavis Future Faculty Fellow. She is a recipient of Microsoft Research PhD Fel- lowship. She has more than 30 publications on knowledge extraction and reasoning from multi- media data. Additional information is available at https://limanling.github.io. Dave and Jane W.S. Liu Award, and has been selected as Mavis Future Faculty Fellow. She is a recipient of Microsoft Research PhD Fel- lowship. She has more than 30 publications on knowledge extraction and reasoning from multi- media data. Additional information is available at https://limanling.github.io. Audience and Prerequisites Based on the level of interest in this topic, we expect around 150 partic- ipants. While no speciﬁc background knowledge is assumed of the audience, it would be the best for the attendees to know about basic deep learning technologies, pre-trained word embeddings (e.g. Word2Vec) and language models (e.g. BERT). A reading list that could help provide background knowledge to the audience before attending this tutorial is given in Appx. §A.2. Ben Zhou is a third-year Ph.D. student at the De- partment of Computer and Information Science, University of Pennsylvania. He obtained his B.S. from UIUC in 2019. Ben’s research interests 17 Acknowledgement are distant supervision extraction and experiential knowledge reasoning, and he has more than 5 re- cent papers on related topics. He is a recipient of the ENIAC fellowship from the University of Pennsylvania, and a ﬁnalist of the CRA outstanding undergraduate researcher award. Additional infor- mation is available at http://xuanyu.me/. are distant supervision extraction and experiential knowledge reasoning, and he has more than 5 re- cent papers on related topics. He is a recipient of the ENIAC fellowship from the University of Pennsylvania, and a ﬁnalist of the CRA outstanding undergraduate researcher award. Additional infor- mation is available at http://xuanyu.me/. This presenters’ research is supported in part by U.S. DARPA KAIROS Program No. FA8750-19-2- 1004, DARPA AIDA Program No. FA8750-18-2- 0014, DARPA MCS program No. N660011924033, and by the NSF of United States Grant IIS 2105329. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the ofﬁcial policies, either expressed or implied, of DARPA, or the U.S. Gov- ernment. The U.S. Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. Heng Ji is a Professor at Computer Science Depart- ment of University of Illinois Urbana-Champaign, and an Amazon Scholar. She received her B.A. and M. A. in Computational Linguistics from Ts- inghua University, and her M.S. and Ph.D. in Computer Science from New York University. Her research interests focus on NLP, especially on Multimedia Multilingual Information Extrac- tion, Knowledge Base Population and Knowledge- driven Generation. She was selected as “Young Sci- entist” and a member of the Global Future Council on the Future of Computing by the World Eco- nomic Forum. The awards she received include “AI’s 10 to Watch” Award, NSF CAREER award, Google Research Award, IBM Watson Faculty Award, Bosch Research Award, and Amazon AWS Award, ACL2020 Best Demo Paper Award, and NAACL2021 Best Demo Paper Award. Additional information is available at https://blender. cs.illinois.edu/hengji.html. 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Contrastive out-of-distribution detection for pre- trained transformers. In EMNLP. Linhai Zhang, Deyu Zhou, Yulan He, and Zeng Yang. 2021a. Merl: Multimodal event representation learning in heterogeneous embedding spaces. In Proceedings of the AAAI Conference on Artiﬁcial In- telligence, volume 35, pages 14420–14427. • Muhao Chen: A.1 Past Tutorials by the Instructors The presenters of this tutorial have given the follow- ing tutorials at leading international conferences and venues in the past: Xikun Zhang, Deepak Ramachandran, Ian Tenney, Yanai Elazar, and Dan Roth. 2020a. Do language embeddings capture scales? In Proceedings of the 2020 Conference on Empirical Methods in Natural Language Processing: Findings, pages 4889–4896. 23 – ACL’21: Event-Centric Natural Language Pro- cessing. – ACL’14: Wikiﬁcation and Beyond: The Chal- lenges of Entity and Concept Grounding. – ACL’21: Event-Centric Natural Language Pro- cessing. – AAAI’21: Event-Centric Natural Language Un- derstanding. – AAAI’21: Event-Centric Natural Language Un- derstanding. – NLPCC’14: Wikiﬁcation and Beyond: The Chal- lenges of Entity and Concept Grounding. – NLPCC’14: Wikiﬁcation and Beyond: The Chal- lenges of Entity and Concept Grounding. – KDD’21: From Tables to Knowledge: Recent Advances in Table Understanding. – KDD’21: From Tables to Knowledge: Recent Advances in Table Understanding. AAAI’20 R Ad f T f bl R – COLING’12: Temporal Information Extraction and Shallow Temporal Reasoning. – COLING’12: Temporal Information Extraction and Shallow Temporal Reasoning. – AAAI’20: Recent Advances of Transferable Rep- resentation Learning. • • Dan Roth: • Dan Roth: – ACL’21: Event-Centric Natural Language Pro- cessing. • Manling Li: – ACL’21: Event-Centric Natural Language Pro- cessing. – AAAI’21: Event-Centric Natural Language Un- derstanding. – AAAI’21: Event-Centric Natural Language Un- derstanding. – ACL’20: Commonsense Reasoning for Natural Language Processing. • Heng Ji: – AAAI’20: Recent Advances of Transferable Rep- resentation Learning. – KDD’22: The Battlefront of Combating Misinfor- mation and Coping with Media Bias. – ACL’18: A tutorial on Multi-lingual Entity Dis- covery and Linking. – AACL’22: The Battlefront of Combating Misin- formation and Coping with Media Bias. – EACL’17: A tutorial on Integer Linear Program- ming Formulations in Natural Language Process- ing. – KDD’22: New Frontiers of Scientiﬁc Text Min- ing: Tasks, Data, and Tools. – AAAI’16: A tutorial on Structured Prediction. – WWW’22: Modern Natural Language Process- ing Techniques for Scientiﬁc Web Mining: Tasks, Data, and Tools. – ACL’14: A tutorial on Wikiﬁcation and Entity Linking. – AAAI’13: Information Trustworthiness. – AAAI’22: Deep Learning on Graphs for Natural Language Processing. – COLING’12: A Tutorial on Temporal Informa- tion Extraction and Shallow Temporal Reasoning. – KDD’21: Deep Learning on Graphs for Natural Language Processing. – NAACL’12: A Tutorial on Constrained Condi- tional Models: Structured Predictions in NLP. – IJCAI’21: Deep Learning on Graphs for Natural Language Processing. A.1 Past Tutorials by the Instructors – NAACL’10: A Tutorial on Integer Linear Pro- gramming Methods in NLP. – NAACL’10: A Tutorial on Integer Linear Pro- gramming Methods in NLP. – SIGIR’21: Deep Learning on Graphs for Natural Language Processing. – EACL’09: A Tutorial on Constrained Conditional Models. – EACL’09: A Tutorial on Constrained Conditional Models. – EMNLP’21: Knowledge-Enriched Natural Lan- guage Generation. – ACL’07: A Tutorial on Textual Entailment. A.2 Recommended Paper List – ACL’21: Event-Centric Natural Language Pro- cessing. The following is a reading list that could help pro- vide background knowledge to the audience before attending this tutorial: – NAACL’21: Deep Learning on Graphs for Natu- ral Language Processing. – AAAI’21: Event-Centric Natural Language Un- derstanding. • Wenxuan Zhou, Muhao Chen. Learning from Noisy Labels for Entity-Centric Information Ex- traction. EMNLP, 2021. – CCL’18 and NLP-NADB’18: Multi-lingual Entity Discovery and Linking. • Wenxuan Zhou, Fanyu Liu, Muhao Chen. Con- trastive Out-of-Distribution Detection for Pre- trained Transformers. EMNLP, 2021. – ACL’18: Multi-lingual Entity Discovery and Link- ing. – SIGMOD’16: Automatic Entity Recognition and Typing in Massive Text Data. • Xingyuan Pan, Maitrey Mehta, Vivek Srikumar. Learning Constraints for Structured Prediction Us- ing Rectiﬁer Networks. ACL, 2020. – ACL’15: Successful Data Mining Methods for NLP. 24 • Hangfeng He, Mingyuan Zhang, Qiang Ning, Dan Roth. Foreseeing the Beneﬁts of Incidental Super- vision. EMNLP, 2021. • Hangfeng He, Mingyuan Zhang, Qiang Ning, Dan Roth. Foreseeing the Beneﬁts of Incidental Super- vision. EMNLP, 2021. • Ben Zhou, Qiang Ning, Daniel Khashabi, Dan Roth. Temporal Common Sense Acquisition with Minimal Supervision. ACL, 2020. • Wenpeng Yin, Nazneen Fatema Rajani, Dragomir Radev, Richard Socher, Caiming Xiong. Universal natural language processing with limited annota- tions: Try few-shot textual entailment as astart. EMNLP, 2020. • Bangzheng Li, Wenpeng Yin, Muhao Chen. Ultra- ﬁne Entity Typing with Indirect Supervision from Natural Language Inference. TACL, 2022. • Bangzheng Li, Wenpeng Yin, Muhao Chen. Ultra- ﬁne Entity Typing with Indirect Supervision from Natural Language Inference. TACL, 2022. • Lifu Huang, Heng Ji, Kyunghyun Cho, Ido Dagan, Sebastian Riedel, Clare Voss. Zero-shot transfer learning for event extraction. ACL, 2018. • Ananya Subburathinam, Di Lu, Heng Ji, Jonathan May, Shih-Fu Chang, Avirup Sil, Clare Voss. Cross-lingual structure transfer for relation and event extraction. EMNLP, 2019. • Ananya Subburathinam, Di Lu, Heng Ji, Jonathan May, Shih-Fu Chang, Avirup Sil, Clare Voss. Cross-lingual structure transfer for relation and event extraction. EMNLP, 2019. • Hong Wang, Wenhan Xiong, Mo Yu, Xiaoxiao Guo, Shiyu Chang, William Yang Wang. Sentence Embedding Alignment for Lifelong Relation Ex- traction. NAACL, 2019. • Hassan Akbari, Svebor Karaman, Surabhi Bhar- gava, Brian Chen, Carl Vondrick, and Shih-Fu Chang. Multi-level multimodal common semantic space for image-phrase grounding. CVPR, 2019. • Manling Li, Alireza Zareian, Qi Zeng, Spencer Whitehead, Di Lu, Heng Ji, Shih-Fu Chang. Cross- media structured common space for multimedia event extraction. A.2 Recommended Paper List ACL, 2020. 25
https://openalex.org/W2591801370	https://europepmc.org/articles/pmc5362264?pdf=render	English	null	Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane	eLife	2,017	cc-by	11,277	Electrostatic anchoring precedes stable membrane attachment of SNAP25/ SNAP23 to the plasma membrane Pascal Weber1, Helena Batoulis1, Kerstin M Rink2, Stefan Dahlhoff1, Kerstin Pinkwart1, Thomas H So¨ llner2, Thorsten Lang1* Pascal Weber1, Helena Batoulis1, Kerstin M Rink2, Stefan Dahlhoff1, Kerstin Pinkwart1, Thomas H So¨ llner2, Thorsten Lang1* 1Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany; 2Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany Abstract The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane. / f RESEARCH ARTICLE Electrostatic anchoring precedes stable membrane attachment of SNAP25/ SNAP23 to the plasma membrane Introduction For example, when a neuron is stimulated, vesicles merge with its cell membrane and release their content into a gap between itself and other neurons. This complicated process involves many steps and molecules, including proteins called SNAREs. Some SNARE proteins reside at the inner side of the cell membrane and help vesicles to fuse with this membrane. Two SNARE proteins called SNAP25 and SNAP23 are produced in the liquid inside the cell and initially float freely. Eventually, these proteins become directly anchored to the cell membrane, however, not much is known about what happens to these proteins in between these stages, or how they first attach to the membrane before anchoring to it. Electrostatic forces between oppositely charged molecules are known to be important for them to bind with each other. Here, electrostatic forces are less likely to occur because SNAP25 and SNAP23 are both mostly negatively charged, and should therefore be repelled from the cell membrane, which also typically has a negative charge. However, both SNAP25 and SNAP23 have a small cluster of positively charged amino acids (the building blocks of proteins) near the attachment site, and Weber et al. have now tested whether this charge is sufficient to overcome the predicted repulsion. The approach involved making mutant proteins with either more or less positively charged attachment regions. Mutant SNAP25 or SNAP23 proteins with more positive charges may stick more tightly but not necessarily more permanently to the membrane. However, when the number of positive charges was lowered, more of the proteins remained floating freely in the liquid inside the cell. These results suggest that even a small number of positively charged amino acids is sufficient to help a protein bind to a cell membrane for further processing. The findings of Weber et al. reveal an early step in the life cycle of SNAP25 and SNAP23 before they anchor to the cell membrane. They suggest that finely tuned protein electrostatics can regulate how long a protein spends at a specific site and thereby indirectly determine its fate. Such fine- tuned protein electrostatics are difficult to recognize and could represent an underestimated regulatory mechanism in all types of cells. (Gonzalo et al., 1999; Greaves et al., 2010). For DHHC interactions to occur, proximity to the membrane is an important factor and could even be rate limiting in the attachment process. Introduction Palmitoylation is a post-translational modification of a protein which causes its stable attachment to a cellular membrane. Examples of proteins that follow this paradigm are the homologous SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins SNAP25 and SNAP23, which after translation are initially cytosolic proteins. In order to function in vesicle fusion, they relocate to the plasma membrane. SNAP23 is ubiquitously expressed, whereas the neuronal SNAP25 is highly abundant in the synapse and in the plasma membrane of neuroendocrine cells (Jahn and Fasshauer, 2012; Wilhelm et al., 2014; Knowles et al., 2010). Stable attachment to membranes is achieved after palmitoylation of a cysteine cluster, which is most probably catalyzed by the plasma membrane resident palmitoyl acyltransferase DHHC2. DHHC2 is characterized by the presence of a conserved DH(H/Y)C motif and can palmitoylate SNAP25 and SNAP23 (Greaves et al., 2010). Competing interests: The authors declare that no competing interests exist. Funding: See page 16 Received: 05 July 2016 Accepted: 26 February 2017 Published: 27 February 2017 Reviewing editor: Axel T Brunger, Stanford University Medical Center, United States Reviewing editor: Axel T Brunger, Stanford University Medical Center, United States Reviewing editor: Axel T Brunger, Stanford University Medical Center, United States The majority of SNAP25 molecules reside in the plasma membrane, while 20% are located in a perinuclear recycling endosome-trans-Golgi network (Aikawa et al., 2006). A two-compartment model for SNAP25 trafficking has been proposed which speculates that the endocytic recycling of SNAP25 might be coupled to its depalmitoylation, followed by its repalmitoylation and recycling back to the plasma membrane (Aikawa et al., 2006). In any case, in steady-state, the large majority of SNAP25 molecules are stably attached to the cell membrane. Copyright Weber et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. The minimal domain necessary for SNAP25 plasma membrane targeting has been mapped to amino acids 85–120 (Gonzalo et al., 1999), comprising the cysteine cluster at the N-terminus (for relevant SNAP25 region see Figure 1), while the C-terminus interacts with DHHC proteins 1 of 18 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Research article Research article Biophysics and Structural Biology Cell Biology eLife digest Cells often communicate with each other by releasing chemicals that normally are stored in small membrane-bound compartments called vesicles. Introduction SNAP25 and SNAP23 are not modified in the cytosol by isoprenyl- or myristoyl groups, which would increase their membrane affinity and facilitate initial membrane contact; neither receptors nor membrane-targeting motifs have been identified previously and thus there has been some debate over how this initial contact may be mediated. Polybasic amino acid patches are known to mediate non-specific interactions with anionic lipids. For instance, plasma membrane targeting of myristoylated K-Ras requires an N-terminal polybasic domain (Cadwallader et al., 1994; Wright and Philips, 2006), which localizes G protein a subunits to the plasma membrane, although this region is not required for subunit palmitoylation (Pedone and Hepler, 2007; Crouthamel et al., 2008). Other examples suggest that phosphoryla- tion of basic residues located upstream of palmitoylated cysteines regulates the palmitoylation of a potassium channel through an electrostatic switch (Jeffries et al., 2012). In some instances, polyba- sic patches are the main driving force for protein attachment to the negatively charged plasma membrane (Cho and Stahelin, 2005). SNAP25 has a net negative charge: of its 206 amino acids, 21% are negatively and 14% are posi- tively charged. Still, a modest excess of three positive charges around the cysteine cluster might be available for non-specific interactions with anionic lipids. Nevertheless, this charge accumulation appears very small when compared to the number of positive charges that mediate electrostatic contacts in other cellular processes (Heo et al., 2006). Here, we set out to investigate whether the subcellular distribution of SNAP25 and SNAP23 can be regulated through such a small accumulation of positive charges, despite the proteins’ overall negative net charge. Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 2 of 18 Research article Research article Figure 1. SNAP25 plasma membrane targeting in live cells. (a) Diagram of the amino acid sequences from position 51 to 125 for wild type S or several constructs in which the net charge of the cysteine rich region (CRR; box) is increased or decreased. Numbers associated with the names refer to the net charge of the CRR. Cysteines are highlighted in yellow; negatively and positively charged amino acids are highlighted blue, respectively. A net charge of +3 is found in all mammalian SNAP25 proteins whose encoding genes are available in the UniProtKB/Swis Figure 1 continued on next page Research article Biophysics and Structural Biology C Biophysics and Structural Biology Cell Biology Figure 1. SNAP25 plasma membrane targeting in live cells. Analysis of SNAP25 targeting in live cells Analysis of SNAP25 targeting in live cells We wondered whether the above mentioned small charge accumulation close to the cysteine cluster is exclusively found in rat SNAP25. Comparison of the cysteine-rich region of the SNAP25 protein (UniProtKB/Swiss-Prot-data bank) in a variety of species revealed that this region carries a net posi- tive charge in all species except Drosophila (Figure 1—figure supplement 1). The distribution of charged amino acids adds up to +3 in all seven mammalian species, and also in chicken, zebrafish and goldfish (although for zebrafish and goldfish there is also an isoform with a charge of +1). The cysteine-rich region has four cysteines for palmitoylation in the centre, which are flanked by four lysines on each side (Figure 1a). The sequence around the cysteines also contains five acidic amino acids: four upstream and one downstream of the cysteines. Hence, eight basic and five acidic amino acids yield a net charge of +3 located downstream of the cysteine cluster. If positive charges close to the cysteine cluster (Figure 1a) constitute the main driving force for initial plasma membrane association, their elimination should diminish plasma membrane targeting. To test this hypothesis, we overexpressed GFP-tagged SNAP25 in neuroendocrine PC12 cells and analysed how plasma membrane targeting depends on these charges. Equatorial optical sections were imaged in live cells (Figure 1b). Linescans perpendicular to the plasma membrane reveal the GFP-SNAP25 distribution at the cell periphery (plasma membrane + cytosol that optically cannot be resolved) and the cytosol (Figure 1b and c). Relating these values to each other yields a ratio >1 if there is a plasma membrane-associated fraction. A variety of SNAP25 mutants with increased or decreased charge in the cysteine-rich region were analysed. As control for non-targetable protein, cysteine palmitoylation sites were substituted by glycines (Figure 1d). As expected, SNAP25 proteins that lacked cysteines (construct SNAP25(C-to-G)) did not locate to the plasma membrane (Figure 1d). We then reduced the charge from wt-SNAP25 (+3) to 1 by substituting four lysines with alanines (for construct details, see Figure 1a). In SNAP25-1distal, the outer four lysines up- and downstream of the cysteine cluster are exchanged, and in SNAP25-1proximal the four inner ones are exchanged. Both of these constructs showed strongly diminished plasma membrane targeting (Figure 1e). Further reducing positive charges to a net charge of 5, by substituting all eight lysines with alanines (SNAP25-5; see Figure 1a), abolishes targeting almost completely (Figure 1e). Figure 1 continued Figure 1 continued bank (see Figure 1—figure supplement 1). (b) Confocal micrographs from live PC12 cells expressing wt-SNAP25 (+3), SNAP25–5 and SNAP25+10 as N-terminally GFP-tagged constructs. Also shown are two more constructs, based on wt-SNAP25 (+3) and SNAP25+10, in which the four cysteines for palmitoylation are exchanged for glycines (SNAP25(C-to-G) and SNAP25+10(C-to-G)). Red elongated boxes mark the regions of interest (ROIs) in which the fluorescence distribution at the cell periphery was analysed by linescans. White graphs illustrate the corresponding fluorescence traces. (c) For one experiment several traces were averaged. (d–f) Ratio between cell periphery and cytosol signal for (d) the variants lacking cysteines for palmitoylation, (e) constructs with an altered charge around the cysteine cluster, and (f) a construct with an eliminated polybasic cluster located at the C-terminus of SNAP25. Values are given as means ± S.E.M. (n = 3–19; t-test p<0.05, p<0.01, *p<0.001, ns = not significant). For the constructs exhibiting weakest and strongest targeting in (c), Figure 1—figure supplement 2 shows that the ratio between cell periphery and cytosol signal is independent of the expression level. DOI: 10.7554/eLife.19394.003 e following figure supplements are available for figure 1 Figure supplement 1. Cysteine-rich regions from different species. DOI: 10.7554/eLife.19394.004 Figure supplement 1. Cysteine-rich regions from different species. DOI: 10.7554/eLife.19394.004 Figure supplement 2. No correlation between the periphery/cytosol signal ratio and the expression level. DOI: 10.7554/eLife.19394.005 Figure supplement 2. No correlation between the periphery/cytosol signal ratio and the expression level. DOI: 10.7554/eLife.19394.005 Research article Research article Biophysics and Structural Biology Cell Biology Introduction (a) Diagram of the amino acid sequences from position 51 to 125 for wild type SNAP25 and for several constructs in which the net charge of the cysteine rich region (CRR; box) is increased or decreased. Numbers associated with the constructs’ names refer to the net charge of the CRR. Cysteines are highlighted in yellow; negatively and positively charged amino acids are highlighted in red and blue, respectively. A net charge of +3 is found in all mammalian SNAP25 proteins whose encoding genes are available in the UniProtKB/Swiss-Prot-data Figure 1 continued on next page Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 3 of 18 Research article R Analysis of SNAP25 targeting in live cells We also tested a construct termed SNAP25-5hydrophob which is identical to SNAP25-5 except that the lysines were not replaced by alanines but instead by more hydrophobic leucines (Figure 1a). The reasoning for increasing the hydrophobicity is a previous report suggesting that the hydrophobicity of this domain plays a role in initial membrane association (Greaves et al., 2009). Compared to SNAP25-5, SNAP25-5hydrophob showed no increase in targeting; instead, targeting was further reduced and hardly detectable. Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 4 of 18 Research article Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology We next tested whether more positive charges would increase targeting efficiency. Here, the find- ing was ambiguous, as increasing the charge to +7 (SNAP25+7) or +10 (SNAP25+10; for constructs see Figure 1a) diminishes or promotes targeting, respectively (Figure 1e). Substituting the cysteines by glycines in the SNAP25+10 mutant (SNAP25+10(C-to-G)) causes a cytosolic distribution (Figure 1d). Hence, an increase in positive charges cannot substitute for attachment by palmitoylation. There is another small cluster of positive charges downstream of the cysteine-rich region in the C-terminal part of the SNAP25 linker. Elimination of these charges by introducing the mutations R191A, R198A and K201A has no effect on membrane targeting (Figure 1f). This indicates that the mere presence of positive charges is not sufficient for membrane targeting. Rather, the position of charged residues within the protein structure determines their effect. SNAP23 is 60% identical to SNAP25. Like SNAP25, it carries three positive charges in the cyste- ine-rich region (Figure 2a). Reduction of the positive charges by six units in SNAP23-3 diminishes membrane association (Figure 2). As observed for SNAP25, increase of positive charges can pro- mote (see SNAP23+10 in Figure 2 and SNAP23+16 in Figure 2—figure supplement 1) or diminish (see SNAP23+11a and SNAP23+11b in Figure 2—figure supplement 1) membrane targeting. Finally, exchange of cysteines for glycines (SNAP23(C-to-G)) yields a cytosolic distribution (Figure 2), showing that palmitoylation is also required for stable attachment of SNAP23. Figure 2. SNAP23 plasma membrane targeting. (a) Amino acid sequences from positions 45 to 119 shown for SNAP23+10, wt-SNAP23 (+3) and SNAP23-3. The box indicates the cysteine-rich region (CRR) in which mutations were introduced. Red, blue and yellow, respectively, highlight negatively charged amino acids, positively charged amino acids and cysteines. (b, c) The periphery/cytosol signal ratio from confocal micrographs was analysed as described in Figure 1. Research article Biophysics and Structural Biology Cell Biology DOI: 10.7554/eLife.19394.008 h f ll f l l bl f f Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology In conclusion, the analysis of SNAP25/SNAP23 constructs shows that membrane targeting is dependent on positive charges located close to the cysteine cluster. Charges > +3 can increase or decrease targeting, suggesting that primary structure is not the only determinant for stronger targeting and that secondary structural elements are also important. Structural features may define whether the charges can be exposed to the membrane environment. Alternatively, they may deter- mine the geometry of the electrostatic contact (and hence the accessibility of the cysteines) that plays a role in the palmitoylation reaction. g y ( y y ) plays a role in the palmitoylation reaction. SNAP25-association with isolated membranes In addition to immunofluorescence imaging, we employed an independent method to analyse the subcellular distribution. SNAP25 constructs that showed strong effects in live cells and the constructs without palmitoylation sites were studied by cell fractionation (Figure 3). In contrast to the imaging analysis, the biochemical cell fractionation experiment makes it possible to relate the absolute amounts of the membrane and cytosol fraction to each other. In line with the imaging experiments, we observed that SNAP25-5 has a weaker and SNAP25+10 a stronger membrane association than wt-SNAP25 (+3). In the imaging experiment, no membrane-associated fraction of SNAP25(C-to-G) or SNAP25+10(C-to-G) was visible. This is different in the membrane fractionation assay where we detected a small membrane-associated fraction for SNAP25+10(C-to-G) (Figure 3). This fraction might have been overlooked by confocal microscopy, which is unable to resolve small plasma membrane- associated pools in the presence of a strong cytosolic background. For a better microscopic analysis, it was necessary to eliminate the cytosolic background. We therefore turned to ‘unroofed cells’ Figure 3. Subcellular distribution of SNAP25 constructs analysed by cell fractionation. PC12 cells expressing the indicated constructs were mechanically homogenised followed by centrifugation, yielding supernatant and pellet that contain the cytosolic and the membrane fraction, respectively. Fractions were analysed by Western blotting using an antibody against GFP. Left: immunoblots of one representative experiment. For each construct, the respective cytosol and membrane fractions are shown at arbitrary scaling (for the entire blot see Figure 3—figure supplement 1; for the average expression levels see Figure 3—figure supplement 2). Right: the ratio between membrane-associated and cytosolic protein was quantified from the band intensities. Values are given as means ± S.E.M. (n = 4; t-test p<0.05, p<0.01, p<0.001). Research article Analysis of SNAP25 targeting in live cells Values are given as means ± S.E.M. (n = 3–8; t-test p<0.05, p<0.01, p<0.001). For more SNAP23 constructs, see Figure 2— figure supplement 1. DOI: 10 7554/eLife 19394 006 Figure 2. SNAP23 plasma membrane targeting. (a) Amino acid sequences from positions 45 to 119 shown for SNAP23+10, wt-SNAP23 (+3) and SNAP23-3. The box indicates the cysteine-rich region (CRR) in which mutations were introduced. Red, blue and yellow, respectively, highlight negatively charged amino acids, positively charged amino acids and cysteines. (b, c) The periphery/cytosol signal ratio from confocal micrographs was analysed as described in Figure 1. Values are given as means ± S.E.M. (n = 3–8; t-test p<0.05, p<0.01, p<0.001). For more SNAP23 constructs, see Figure 2— figure supplement 1. DOI: 10.7554/eLife.19394.006 The following figure supplement is available for figure 2: Figure supplement 1. Correlation between SNAP23 targeting and charge of the cysteine-rich region. DOI: 10 7554/eLife 19394 007 Figure 2. SNAP23 plasma membrane targeting. (a) Amino acid sequences from positions 45 to 119 shown for SNAP23+10, wt-SNAP23 (+3) and SNAP23-3. The box indicates the cysteine-rich region (CRR) in which mutations were introduced. Red, blue and yellow, respectively, highlight negatively charged amino acids, positively charged amino acids and cysteines. (b, c) The periphery/cytosol signal ratio from confocal micrographs was analysed as described in Figure 1. Values are given as means ± S.E.M. (n = 3–8; t-test p<0.05, p<0.01, p<0.001). For more SNAP23 constructs, see Figure 2— figure supplement 1. DOI: 10.7554/eLife.19394.006 5 of 18 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Research article Research article Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 SNAP25-association with isolated membranes In addition to immunofluorescence imaging, we employed an independent method to analyse the subcellular distribution. SNAP25 constructs that showed strong effects in live cells and the constructs without palmitoylation sites were studied by cell fractionation (Figure 3). In contrast to the imaging analysis, the biochemical cell fractionation experiment makes it possible to relate the absolute amounts of the membrane and cytosol fraction to each other. In line with the imaging experiments, we observed that SNAP25-5 has a weaker and SNAP25+10 a stronger membrane association than wt-SNAP25 (+3). In the imaging experiment, no membrane-associated fraction of SNAP25(C-to-G) or SNAP25+10(C-to-G) was visible. This is different in the membrane fractionation assay where we detected a small membrane-associated fraction for SNAP25+10(C-to-G) (Figure 3). This fraction might have been overlooked by confocal microscopy, which is unable to resolve small plasma membrane- associated pools in the presence of a strong cytosolic background. For a better microscopic analysis, it was necessary to eliminate the cytosolic background. We therefore turned to ‘unroofed cells’ Figure 3. Subcellular distribution of SNAP25 constructs analysed by cell fractionation. PC12 cells expressing the indicated constructs were mechanically homogenised followed by centrifugation, yielding supernatant and pellet that contain the cytosolic and the membrane fraction, respectively. Fractions were analysed by Western blotting using an antibody against GFP. Left: immunoblots of one representative experiment. For each construct, the respective cytosol and membrane fractions are shown at arbitrary scaling (for the entire blot see Figure 3—figure supplement 1; for the average expression levels see Figure 3—figure supplement 2). Right: the ratio between membrane-associated and cytosolic protein was quantified from the band intensities. Values are given as means ± S.E.M. (n = 4; t-test p<0.05, p<0.01, p<0.001). DOI 10 7554/ Lif 19394 008 Figure 3. Subcellular distribution of SNAP25 constructs analysed by cell fractionation. PC12 cells expressing the indicated constructs were mechanically homogenised followed by centrifugation, yielding supernatant and pellet that contain the cytosolic and the membrane fraction, respectively. Fractions were analysed by Western blotting using an antibody against GFP. Left: immunoblots of one representative experiment. For each construct, the respective cytosol and membrane fractions are shown at arbitrary scaling (for the entire blot see Figure 3—figure supplement 1; for the average expression levels see Figure 3—figure supplement 2). Right: the ratio between membrane-associated and cytosolic protein was quantified from the band intensities. Values are given as means ± S.E.M. (n = 4; t-test p<0.05, p<0.01, p<0.001). Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology (Heuser, 2000), also called plasma membrane sheets. To achieve this, cells were exposed to a brief ultrasound pulse that exerts a shearing force to the upper cellular parts, leaving behind the basal plasma membranes. Then, the membrane sheets were imaged to quantify the recruited protein in the absence of the cytosolic background. Wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were readily detected on the membrane sheets (Figure 4) in amounts that would be expected from the confocal microscopic subcellular distribution analysis. SNAP25(C-to-G) was not detectable, either because it is present at undetectable amounts after membrane sheet generation (see also the hardly detectable membrane fraction of SNAP25(C-to-G) in the cell fractionation assay in Figure 3), or because it binds weakly and washes off during sample mounting and imaging (which takes up to » 35 min in total). By contrast, SNAP25 +10(C-to-G) was clearly present, despite the absence of palmitoylation sites, doc- umenting a higher plasma membrane affinity when compared to SNAP25(C-to-G). (Heuser, 2000), also called plasma membrane sheets. To achieve this, cells were exposed to a brief ultrasound pulse that exerts a shearing force to the upper cellular parts, leaving behind the basal plasma membranes. Then, the membrane sheets were imaged to quantify the recruited protein in the absence of the cytosolic background. Wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were readily detected on the membrane sheets (Figure 4) in amounts that would be expected from the confocal microscopic subcellular distribution analysis. SNAP25(C-to-G) was not detectable, either because it is present at undetectable amounts after membrane sheet generation (see also the hardly detectable membrane fraction of SNAP25(C-to-G) in the cell fractionation assay in Figure 3), or because it binds weakly and washes off during sample mounting and imaging (which takes up to » 35 min in total). By contrast, SNAP25 +10(C-to-G) was clearly present, despite the absence of palmitoylation sites, doc- umenting a higher plasma membrane affinity when compared to SNAP25(C-to-G). Assessment of palmitoylation Cell fractionation and membrane sheet experiments both show that an increase in positive charge produces a membrane-associated fraction even in the absence of palmitoylation. This poses the question of whether the small increase in membrane targeting of SNAP25+10 is, in part, achieved independently from stable membrane-attachment through palmitoylation. To clarify this issue, we examined whether the extent of plasma membrane targeting correlates with the degree of palmitoylation for wt-SNAP25 (+3), SNAP25-5 and SNAP25+10, using SNAP25(C- to-G) as a negative control. Freshly transfected cells were incubated with a palmitate analogue carry- ing an alkyne-group to allow click-labelling with a fluorophore. Hence, the newly synthesized SNAP25 constructs should be palmitoylated by the clickable palmitate. Cells were lysed and the con- structs were immunoprecipitated using their GFP-tags. A Cy5-fluorophore was covalently attached to the alkyne group through click-chemistry and the immunoprecipitate was subjected to western blot analysis. The membrane was immunostained for GFP, and the palmitate-Cy5 fluorescence was related to the GFP signal. As expected, the data show that SNAP25-5 is much less palmitoylated than wt-SNAP25 (+3), and SNAP25(C-to-G) shows no detectable traces of clickable-palmitates (Fig- ure 5). SNAP25+10 is not significantly more palmitoylated than wt-SNAP25 (+3) (Figure 5). The outcome of this experiment is well in line with (i) the linescan analysis (Figure 1), (ii) the bio- chemical cell fractionation assay (Figure 3) and (iii) the membrane sheet association assay (Figure 4). The four different test systems indicate that SNAP25-5 is less targeted to the plasma membrane and less palmitoylated than wt-SNAP25 (+3). Moreover, SNAP25(C-to-G) cannot associate with the mem- brane. Finally, SNAP25+10 shows a trend towards increased targeting. However, the data suggest that SNAP25+10 associates with the plasma membrane not only through palmitoylation but also by pure protein electrostatics. SNAP25-association with isolated membranes Figure supplement 1. Entire Western blot. DOI: 10.7554/eLife.19394.009 Figure supplement 2. Variation of expression levels. 6 of 18 6 of 18 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Research article Research article Effect of mutations on the interaction with syntaxin Initial plasma membrane association via protein electrostatics may not be the only mechanism affected by the introduced mutations. A disturbed interaction with palmitoyl transferases is unlikely as all of the substitutions are located distal to the QPARV motif which is important for the interaction of SNAP25 and DHHC palmitoyl transferases (Greaves et al., 2010). However, some mutations map to the N-terminal SNARE motif of SNAP25 that interacts with plasmalemmal syntaxin 1A. Early stud- ies on SNAP25 plasma membrane targeting proposed syntaxin as the receptor for initial contact establishment (Vogel et al., 2000; Washbourne et al., 2001), although a subsequent study pro- vided compelling evidence against this hypothesis (Loranger and Linder, 2002). To test whether our mutations affect the formation of a complex with syntaxin, we employed a fluorescence recovery after photobleaching (FRAP) assay. This assay is capable of probing interac- tions between SNAP25 and syntaxin by measuring the mobility of SNAP25 (Halemani et al., 2010). Mobility diminishes the stronger SNAP25 binds to syntaxin and the more syntaxin is present. The slow-down is dependent on a syntaxin–SNAP25 interaction, as it requires the N-terminal SNARE motif of SNAP25. Moreover, a mutant carrying introduced prolines in the N-terminal SNARE-motif of SNAP25 (prolines interfere with the alpha-helix formation, which in turn is required for SNARE- complex formation) moves almost independently of the syntaxin concentration (Halemani et al., 2010). We tested our constructs on membrane sheets with or without co-expressed syntaxin 1A- RFP. Syntaxin 1A-RFP slows down the mobility of wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 in a Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 7 of 18 Research article Research article Biophysics and Structural Biology Cell Biology Figure 4. Association of wt-SNAP25 (+3), SNAP25-5, SNAP25+10 and SNAP25+10(C-to-G) with isolated plasma membranes. (a) Plasma me were generated from cells expressing the indicated GFP-SNAP25 constructs by mechanical shearing forces, followed by direct imaging. D imaging, the sample was screened for green fluorescence and all membranes exhibiting green fluorescence were imaged in the green ch followed by imaging of the blue channel. We also analysed sheets from cells transfected with SNAP25(C-to-G), but in these samples, no gre Figure 4 continued on next page Research article Biophysics and Structural Biolog Figure 4. Association of wt-SNAP25 (+3), SNAP25-5, SNAP25+10 and SNAP25+10(C-to-G) with isolated plasma membranes. (a) Plasma membrane sheets were generated from cells expressing the indicated GFP-SNAP25 constructs by mechanical shearing forces, followed by direct imaging. Figure 4 continued fluorescence was visually detectable in the screening process. Top, blue fluorescent dye (TMA-DPH) visualizing the location and shape of the membrane sheets; bottom, GFP fluorescence of the membrane sheet associated SNAP25 variants. The same images are shown at two different lookup tables (LUT). (b) Quantification of GFP-fluorescence on membrane sheets, normalized to wt-SNAP25 (+3). Values are given as means ± S.E.M. (n = 3–7; t-test p<0.05, p<0.01, p<0.001). DOI: 10.7554/eLife.19394.011 similar fashion, arguing against an altered capability of complex formation caused by the introduced mutations (Figure 6). Therefore, we conclude that the differences in initial membrane association are not due to an altered affinity of binding to syntaxin. similar fashion, arguing against an altered capability of complex formation caused by the introduced mutations (Figure 6). Therefore, we conclude that the differences in initial membrane association are not due to an altered affinity of binding to syntaxin. Figure 5. Assessment of the palmitoylation of GFP-SNAP25 constructs. PC12 cells were transfected with wt- SNAP25 (+3), SNAP25-5, SNAP25+10, or SNAP25(C-to-G) and fed with alkyne-palmitate overnight. Cells were then lysed, and the GFP-tagged constructs were immunoprecipitated. Subsequently, a click reaction with Cy5-azide was performed to label incorporated palmitate, and the samples were subjected to SDS-PAGE and Western blotting. The amount of protein was quantified by immunolabelling (anti-GFP antibody/IRDye-labelled secondary antibody) and used for normalization of the palmitate-Cy5 signal, yielding the palmitate/GFP ratio. The panels show the fluorescence of the palmitate-Cy5 (top) and the GFP signal (bottom) of one representative immunoblot. The bar chart shows palmitate/GFP ratios of n = 5 independent experiments (mean + S.E.M.; t-test p<0.05, p<0.01, p<0.001, ns = not significant). DOI: 10.7554/eLife.19394.012 Figure 5. Assessment of the palmitoylation of GFP-SNAP25 constructs. PC12 cells were transfected with wt- SNAP25 (+3), SNAP25-5, SNAP25+10, or SNAP25(C-to-G) and fed with alkyne-palmitate overnight. Cells were then lysed, and the GFP-tagged constructs were immunoprecipitated. Subsequently, a click reaction with Cy5-azide was performed to label incorporated palmitate, and the samples were subjected to SDS-PAGE and Western blotting. The amount of protein was quantified by immunolabelling (anti-GFP antibody/IRDye-labelled secondary antibody) and used for normalization of the palmitate-Cy5 signal, yielding the palmitate/GFP ratio. The panels show the fluorescence of the palmitate-Cy5 (top) and the GFP signal (bottom) of one representative immunoblot. The bar chart shows palmitate/GFP ratios of n = 5 independent experiments (mean + S.E.M.; t-test p<0.05, p<0.01, p<0.001, ns = not significant). Effect of mutations on the interaction with syntaxin During imaging, the sample was screened for green fluorescence and all membranes exhibiting green fluorescence were imaged in the green channel, followed by imaging of the blue channel. We also analysed sheets from cells transfected with SNAP25(C-to-G), but in these samples, no green Figure 4 continued on next page Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 8 of 18 R Research article Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology Figure 4 continued DOI: 10 7554/eLife 19394 012 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 9 of 18 9 of 18 Research article Research article Biophysics and Structural Biology Cell Biology Figure 6. Probing the interaction of SNAP25 constructs with syntaxin 1A. (a) Illustration of a fluorescence recovery after photobleaching (FRAP) experiment that measures SNAP25 interactions with syntaxin on membrane sheets generated from PC12 cells expressing the respective constructs. Figure 6. Probing the interaction of SNAP25 constructs with syntaxin 1A. (a) Illustration of a fluorescence recovery after photobleaching (FRAP) experiment that measures SNAP25 interactions with syntaxin on membrane sheets generated from PC12 cells expressing the respective constructs. GFP-SNAP25 mobility was analysed in the absence (top row) or presence (bottom row) of co-expressed syntaxin 1A-RFP (for RFP fluorescence see images on the left shown at the same scaling). Right (from left to right): membrane sheets before bleaching of a square region of interest (ROI), the first image immediately after bleaching, and 5 s and 40 s after bleaching. The ROI refills with GFP-signal faster in the absence of syntaxin 1A-RFP. (b) Averaged fluorescence recovery traces from one experiment, in the absence (grey) or presence (red) of syntaxin 1A-RFP. Values are given as means ± S. D. (n = 7–12 membrane sheets). Hyperbola functions are fitted to the averaged traces yielding the half time of recovery. (c) Average half times of recovery for wt-SNAP25 (+3), SNAP25-5 and SNAP25+10, in the absence and presence of overexpressed syntaxin 1A. Values are given as means ± S.E. M. (n = 3–4; t-test p<0.05, p<0.01, p<0.001, ns = not significant). Please note that, in this experiment, large pixels were used to keep bleaching low. Therefore the spatial resolution is lower than that in the other experiments and does not allow for resolving the SNAP25 micropatterning. DOI: 10.7554/eLife.19394.013 Binding of SNAP25 constructs to liposomes Next, we tested whether membrane association of SNAP25 is directly mediated by negatively charged lipids. We used reconstituted liposomes containing distinct lipid compositions but lacking 10 of 18 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Research article Research article Biophysics and Structural Biology Cell Biology Figure 7. Binding of purified SNAP25 constructs to reconstituted liposomes. Atto647N-labeled liposomes containing either POPC or a complex lipid mixture (POPC/DOPS/POPE/cholesterol/PI) and the indicated amounts of PI(4,5)P2 or PI(3,4,5)P3 were added to immobilized GST-wtSNAP25 (+3), GST- SNAP25-5 and GST-SNAP25+10 and incubated 1 hr at 4˚C. After washing the beads, the amount of bound liposomes was measured by their Atto647N fluorescence (excitation: 639 nm, emission: 669 nm). The amounts of liposomes specifically bound to the different GST-SNAP25 constructs were determined by subtracting the values derived from the GST controls. Values are given as means ± S.E.M. (n = 3; t-test p<0.05, p<0.01, p<0.001, ns = not significant). DOI: 10.7554/eLife.19394.014 Figure 7. Binding of purified SNAP25 constructs to reconstituted liposomes. Atto647N-labeled liposomes containing either POPC or a complex lipid mixture (POPC/DOPS/POPE/cholesterol/PI) and the indicated amounts of PI(4,5)P2 or PI(3,4,5)P3 were added to immobilized GST-wtSNAP25 (+3), GST- SNAP25-5 and GST-SNAP25+10 and incubated 1 hr at 4˚C. After washing the beads, the amount of bound liposomes was measured by their Atto647N fluorescence (excitation: 639 nm, emission: 669 nm). The amounts of liposomes specifically bound to the different GST-SNAP25 constructs were determined by subtracting the values derived from the GST controls. Values are given as means ± S.E.M. (n = 3; t-test p<0.05, p<0.01, *p<0.001, ns = not significant). O 0 / f 939 0 DOI: 10.7554/eLife.19394.014 any proteins (thus eliminating the role of potential SNAP25 binding partners such as syntaxin 1) (Fig- ure 7). This assay also clarifies whether the few amino acids located in the relatively small SNAP25 segment do indeed affect lipid binding. GST-tagged wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were expressed in bacteria and thus lack the palmitoylation modification. The constructs were puri- fied, immobilized on glutathione beads and incubated with Atto647N-PE-labelled liposomes con- taining either PC/PS or PC/PS/PE/PI/cholesterol in the absence or the presence of distinct phosphoinositides. SNAP25-bound liposomes were quantified by their Atto647N-PE fluorescence and the results were corrected for unspecific binding to GST-beads. All SNAP25 constructs show only weak liposome binding in the absence of phosphoinositides, which excludes phosphatidylserine (PS) as a major/sole binding partner (Figure 7). Binding of SNAP25 constructs to liposomes The presence of 4% PI(4,5)P2 significantly enhanced SNAP25 binding in the PC samples and in the complex lipid mixture. As in the cellular assays, the reduction or addition of positive charges reduced or increased the SNAP25 interaction, respectively. Replacing PI(4,5)P2 by PI(3,4,5)P3 pro- foundly increased binding, but largely diminished the differences between SNAP25 constructs. This suggests that the presence of additional negative charges creates additional binding contacts, which are probably outside of the cysteine-rich region. In order to keep the number of negative charges comparable to those provided by 4% PI(4,5)P2, PI(3,4,5)P3 was reduced to 2.8%. This condition Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 11 of 18 Research article Research article Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology yielded a stronger difference in binding in the range between the cell fractionation (Figure 3) and the membrane sheet assay (Figure 4). Although the magnitudes of the effects are difficult to com- pare because of the different assay systems (the complex composition of the plasma membrane ver- sus the simple lipid mix in liposomes, concentrations of binding partners, and the presence of SNAP25-palmitoylation in the cellular experiments may modulate the outcome), the observations point to primary interactions that occur in the cysteine-rich region. yielded a stronger difference in binding in the range between the cell fractionation (Figure 3) and the membrane sheet assay (Figure 4). Although the magnitudes of the effects are difficult to com- pare because of the different assay systems (the complex composition of the plasma membrane ver- sus the simple lipid mix in liposomes, concentrations of binding partners, and the presence of SNAP25-palmitoylation in the cellular experiments may modulate the outcome), the observations point to primary interactions that occur in the cysteine-rich region. Since PS is not capable of recruiting SNAP25, and as PI(3,4,5)P3 is much less abundant in the cel- lular membrane than PI(4,5)P2 (Balla, 2013), these findings point to PI(4,5)P2 as the most likely intra- cellular binding partner. However, the effect of PI(3,4,5)P3 on wt-SNAP25 (+3) binding was very prominent. Thus, membrane microdomains that are locally enriched in PI(3,4,5)P3 at the plasma membrane or in an endosomal compartment (Wang and Richards, 2012) could also be preferential sites for SNAP25 targeting. The liposome binding assay thus identifies phosphoinositides as primary membrane-targeting factors interacting with positive charges in the vicinity of the cysteine cluster. Discussion In conclusion, we suggest that after physically contacting the membrane, a SNAP25/SNAP23 protein increases its dwell time at the plasma membrane through an electrostatic mechanism. The basic resi- dues either directly bind to acidic lipids or produce a local positive electrostatic potential which attracts acidic PIPs. The latter mechanism has been described for MARCKS, where 13 basic residues laterally sequester three PIP2 molecules (Gambhir et al., 2004). The increased membrane binding observed after introducing additional positive charges (SNAP25+10) may reflect a MARCKS-protein- like PIP2-binding behaviour not physiologically relevant for the targeting of SNAP25/SNAP23. Once formed, such a protein-lipid aggregate seems difficult to dissolve, as SNAP25+10(C-to-G) remains attached to the membrane for more than 30 min (Figure 4). Palmitoylated cysteine residues are often preceded and/or followed by basic amino acids (Bizzozero et al., 2001). It has been speculated that these positively charged residues bind to the negatively charged acyl-coenzyme A, and thus augment the acylation rate. Our data do not argue against this hypothesis but show that these residues have a distinct function in anchoring the protein to the cell membrane. This is supported by the in vitro liposome binding assay, which shows the charge-dependent binding in the absence of palmitoylation (Figure 7). Elimination of positive charges distal from the cysteine cluster has no effect on targeting (Figure 1f; construct SNAP25(R191A, R198A, K201A)). This suggests that a random electrostatic contact is not sufficient for targeting. Rather, electrostatic anchoring needs to position the cysteine (s) in a way that facilitates palmitate attachment. The electrostatic contact thus needs to be estab- lished by amino acids close to the cysteine residues. Such a mechanism would also explain why the diminishment of positive charges is always accompanied by a loss of targeting, because fewer positive charges will decrease the binding affinity and/or change the geometry of the established contact. On the other side, not all mutants with an increased positive charge show increased mem- brane targeting. Some actually exhibit a trend towards lesser binding. Perhaps the contact can also become too tight for the attachment of palmitates. Hence, the short targeting motif seems to be optimized to mediate electrostatic anchoring, while still allowing for access to the cysteines for the attachment of palmitates. Previous reports on plasma membrane-localized, small GTPases showed that most of them con- tain two or three polybasic subclusters, each spanning about four to five amino acids (Heo et al., 2006). Binding of SNAP25 constructs to liposomes However, we cannot exclude the possibility that other sites in SNAP25 contribute to phosphoinosi- tide-dependent binding. Research article Biophysics and Structural Biology Cell Biology palmitoylation/depalmitoylation during SNAP25 recycling (see ’Introduction’). Indeed, the SNAP25 +10(C-to-G) construct remains attached to the plasma membrane without being palmitoylated. palmitoylation/depalmitoylation during SNAP25 recycling (see ’Introduction’). Indeed, the SNAP25 +10(C-to-G) construct remains attached to the plasma membrane without being palmitoylated. Our data also show that hydrophobic forces (Greaves et al., 2009) are less crucial than electro- static contacts in the plasma membrane binding of SNAP25. Although the hydrophobic amino acids in the cysteine cluster remain unchanged, the removal of charges abolishes targeting, even though lysines are exchanged for more hydrophobic residues (alanines in the constructs SNAP25-1distal, SNAP25-1proximal and SNAP25-5, and leucines in SNAP25-5hydrophob). y Hence, in a cascade of interactions, a modest modulation of the local charge regulates the effi- ciency of the cellular processes. It seems there are also other membrane contacts that could depend on charge, for instance regulation of the docking time of lipid transfer proteins. Here, the exchange of five lysines for glutamates in the protein loops that interact with the membrane leads to loss of Osh4-mediated sterol transfer (Schulz et al., 2009). In conclusion, we have identified a mechanism of initial plasma membrane association of SNAP25 and SNAP23, which precedes the stable membrane attachment mediated by palmitoylation of cys- teines. The mechanism is based on a relatively small cluster of basic amino acid residues that anchor the protein by binding to acidic lipids, in particular to polyphosphorylated phosphoinositides, with PI(4,5)P2 being the most likely candidate. The data suggest that even small changes in protein elec- trostatics can have strong effects on a cellular mechanism, merely by transiently anchoring a protein to its site of destination. Plasmids Plasmids for the expression of GFP-SNAP25 (Halemani et al., 2010) and GFP-SNAP23 are based on the expression vector pEGFP-C1 (GenBank accession No. U55763, Clontech, Mountain View, CA), which contains a monomeric variant of mEGFP fused N-terminally to the sequence of full-length rat SNAP25B (NP_112253.1) or SNAP23 (NP_073180). All fusion proteins contain a linker of five amino acids between mEGFP and the N-terminus of SNAP25B or SNAP23. Mutations in the SNAP25 or SNAP23 coding sequence were introduced via fusion PCR with purchased oligonucleoti- des (Eurofins Genomics), followed by insertion into the above-mentioned expression vector using the SacI and BamHI sites, or in the case of SNAP25(C-to-G), the XhoI and KpnI restriction sites. The construct for expression of C-terminally RFP-tagged rat Syntaxin 1A is based on the expression vec- tor pEGFP-N1 (GenBank accession No. U55762, Clontech, Mountain View, CA), into which Syntaxin- RFP is inserted using the XhoI and the NotI restriction sites. A twelve amino acid linker connects Syn- taxin 1A (NP_446240) to a monomeric RFP (Campbell et al., 2002) lacking the first amino acid. For expression of GST-tagged constructs, the coding sequences for wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were amplified with primers carrying restriction sites for BamHI (forward primer) and EcoRI (reverse primer). The sequences were first subcloned into the pGEM-T easy vector system (catalogue no. A1360, Promega) by TA cloning, and in a second step subcloned into the expression vector pGEX-6P1 (GE Healthcare Life Sciences) via the BamHI and EcoRI restriction sites. All constructs were verified by sequencing. Discussion Removal of one subcluster abolished plasma membrane targeting, leading to the proposal that association results from the additive binding energies of individual subclusters (Heo et al., 2006). Similarly, the more abundant positive charges in SNAP25+10(C-to-G) allow membrane associa- tion without palmitoylation. Why do SNAP25 and SNAP23 carry only a modest excess of positive charges instead of several charged clusters like those of the small GTPases? It is tempting to speculate that the electrostatic force, based on three positive charges in wt-SNAP25 (+3), has been optimized for mediating anchor- ing that is just sufficient to increase the dwell time at the plasma membrane, but weak enough for dissociation after depalmitoylation. Otherwise, electrostatic anchoring would interfere with SNAP25 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 12 of 18 Research article Research article Biophysics and Structural Biology Cell Biology Research article Confocal microscopy and analysis py y PC12 cells were imaged at 37˚C in Ringer solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 48 mM D(+)aGlucose, 10 mM HEPES; pH 7.4) using a FluoView1000 confocal laser scanning microscope (Olympus) and a UPLSAPO 60x oil objective (NA 1.35). Focussing on the glass–cell inter- face, all green cells were imaged provided they displayed a clear cell–glass contact area. This excludes dead or dying cells that are in the process of rounding up and detaching. In addition, we avoided large clumps of cells because for such cells it is difficult to identify a longer section of cell membrane required for analysis. For measuring membrane-to-cytosol ratios, PC12 cells expressing GFP-tagged constructs were scanned in a 256 pixel x 256 pixel field (12 bit image) at a pixel size of 137 nm using for excitation 488 nm. The equatorial plane of the cell was imaged. To facilitate subse- quent linescan analysis, the scanned field was rotated to allow for each cell placing a horizontal line- scan (length 100 pixel; five pixel width, averaged) perpendicular to the plasma membrane that records the averaged fluorescence intensities along the 100 pixel linescan (including background fluorescence). Fluorescence intensities were background corrected and normalized to the peak intensity at the plasma membrane. Traces from several cells imaged that day (ranging from 14–40 per condition) were aligned with reference to the peak intensity at the plasma membrane. The cyto- solic fluorescence level was averaged over five pixels starting at a 10 pixel distance from the peak intensity at the plasma membrane, and the cell periphery/cytosol ratio was calculated subsequently. y p , p p y y q y For mobility measurements by FRAP, the laser intensities of the 488 nm laser (for GFP) and the 543 nm (for RFP) were reduced to a minimum to prevent bleaching effects during the recording. Membrane sheets were analysed by scanning a 100 pixel x 100 pixel field with a pixel size of 0.414 mm. Recordings started with a pre-bleaching phase of three images, followed by a 500 ms bleaching step and the recording of the recovery phase. For bleaching, a region of interest (ROI) with a size of 7 pixels x 7 pixels (2.9 mm x 2.9 mm) was bleached using a 488 nm laser in combination with a 405 nm laser (both set to their maximum intensity). Confocal microscopy and analysis After bleaching, image sequences were taken at 1.2 Hz for 113 s with the scanning speed set to 40 ms per pixel. Recovery traces were background-sub- tracted and normalized to the average of the pre-bleach values. For one experiment, several normal- ized recovery traces were averaged (7–15 membrane sheets per condition). A hyperbolic curve y(t) = offset + maximal recovery x t/(t + t1/2) was fitted to the averaged recovery trace, yielding the half time (t1/2) of recovery. Epi fluorescence microscopy To measure the association of SNAP25 constructs with the plasma membrane on plasma membrane sheets, a Zeiss Axio Observer D1 epifluorescence microscope equipped with a Plan-Apochromat 100x/NA 1.4 oil immersion objective and a 12 bit CCD camera (1376 1040 pixel) was used, yield- ing a pixel size of 64.5 nm x 64.5 nm. Freshly prepared membrane sheets were imaged in sonication buffer supplemented with TMA-DPH (1-(4-tri-methyl-ammonium-phenyl)6-phenyl-1,3,5-hexatriene- p-toluenesulfonate; Thermo Fisher Scientific), up to » 35 min after membrane-sheet generation. TMA-DPH staining was applied for visualization of the shape and integrity of the membrane sheets. Pictures were taken using filter sets F11-000 (AHF Analysentechnik, Tu¨ bingen, Germany) for TMA- DPH (blue channel) and F36-525 (AHF Analysentechnik, Tu¨ bingen, Germany) for GFP (green chan- nel). From individual membrane sheets, the fluorescence intensity was measured in 30 pixel x 30 pixel ROIs and background subtracted. For each experiment and condition, the values of 21–96 membrane sheets were averaged. Cell culture and generation of membrane sheets g PC12 cells (a gift from Rolf Heumann, Bochum, Germany; similar to clone 251 (Heumann et al., 1983)) were cultured in DMEM with high (4.5 g/l) glucose (PAN biotech) supplemented with 10% horse serum (Biochrom), 5% fetal calf serum (Biochrom) and 100 U/ml penicillin/100 ng/ml strepto- mycin (PAN biotech). During the course of the project, characteristic features of the cell line — such as morphology, expression of neuronal proteins, and their responsiveness to NGF — were regularly confirmed. Cells were maintained at 37˚C and 5% CO2 in a sterile incubator and tested negative for mycoplasmic infections (GATC Biotech, Konstanz, Germany). Cells were transfected with the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA). The tip (100 ml) was loaded with 10 mg plasmid DNA of each construct. Cells were trans- fected by applying a pulse at 1410 V and 30 ms pulse width. Cells were plated onto poly-L-lysine (PLL) (Sigma, Cat. No: P-1524) coated coverslips (25 mm diameter, Menzel Gla¨ser, Braunschweig, Germany)) and maintained for at least 48 hr before imaging. 13 of 18 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Research article Research article Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology For membrane sheet generation, cells were subjected to a brief ultrasound pulse in ice-cold soni- cation buffer (120 mM KGlu, 20 mM KAc, 20 mM HEPES-KOH, 10 mM EGTA; pH 7.2). Research article Assessment of SNAP25 palmitoylation and western blot Assessment of SNAP25 palmitoylation and western blot Ten million PC12 cells were transfected with 15 mg GFP fusion constructs of wt-SNAP25 (+3), SNAP25-5, SNAP25+10 or SNAP25(C-to-G). After one hour, the medium was replaced with DMEM containing 15% delipidized FCS (PAN biotech) and 100 mM palmitate-alkyne (a kind gift from the Thiele lab, LIMES Institute, Bonn). After 15 hr of feeding, cells were harvested via trypsinization and scraping, washed once with PBS, and resuspended in lysis buffer (1% Triton, 1x cOmplete protease inhibitor cocktail, 150 mM NaCl, 5 mM MgCl2, 25 mM HEPES; pH 7.2). Lysis was promoted by vor- texing and sonication. Samples were then centrifuged for 10 min at 14,000 x g, and the supernatant was bound to a GFP-trap (Chromotek) for 2 hr at 4˚C to immunoprecipitate the GFP-SNAP25 fusion constructs. After several washing steps, incorporated palmitate alkyne was clicked to a Cy5-labelled azide (Sigma, cfinal = 100 mM) in 100 mM HEPES, pH 7.2, containing 500 mM tetrakis(acetonitrile)cop- per(I)tetrafluoroborate (Sigma) for 1 hr at 37˚C. The samples were then washed to remove non- bound Cy5, and the GFP-constructs were eluted by boiling in Laemmli buffer, and loaded onto an SDS-PA gel. Proteins were then wet blotted to a nitrocellulose membrane. The membrane was blocked with a 1:1 mixture of Odyssey blocking buffer (Li-Cor) and PBS, and then incubated with a rabbit anti-GFP antibody (RRID: AB_303395; abcam, catalog no. ab-290, diluted 1:1000 in blocking solution containing 0.1% Tween-20). After washing, the membrane was incubated with an IRDye 800CW-coupled goat-anti rabbit secondary antibody (Li-Cor, catalog no. 9263221, diluted 1:10,000 in blocking solution containing 0.1% Tween-20). The Cy5 fluorescence of the palmitate and the IRDye 800CW fluorescence of the GFP were imaged with an Odyssey infrared imaging system (Li- Cor) at 700 nm and 800 nm, respectively. The fluorescent bands were quantified using ImageJ’s Gel Analyser. Membrane fractionation and western blot About 48 hr after transfection, 9 106 PC12 cells were detached from the substrate by trypsin treat- ment for 2 min (0.05% Trypsin and 0.02% EDTA in PBS, PAN Biotech, Cat# P10-0231SP) and trypsin was inactivated by adding medium. Cells were pelleted by centrifugation at 1000 x g at room tem- perature for 3 min and the pellet was washed with PBS. Cell pellets were resuspended in 750 ml ice- cold homogenization buffer (300 mM sucrose, 5 mM Tris-HCl, 0.1 mM EDTA, 1 mM PMSF freshly added; pH 7.4). Using a Potter-Elvehjem homogenizer, cells were kept on ice and homogenized in a Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 14 of 18 Research article Research article Biophysics and Structural Biology Cell Biology Biophysics and Structural Biology Cell Biology volume of 0.75 ml by applying 100 strokes. The homogenate was centrifuged for 8 min at 800 x g at 4˚C, yielding pellet P1 (containing non-homogenized cell debris) and supernatant S1. S1 was trans- ferred into a new tube for a second centrifugation step for 120 min at 20,000 x g and 4˚C, yielding pellet P2 (containing the enriched membrane fraction, which was resuspended in 750 ml homogeni- zation buffer) and the supernatant S2 with the cytosolic fraction. Protein concentrations of P2/S2 were adjusted to the lowest concentration in the series, before adding respective amounts of 4x Laemmli buffer followed by incubation at 95˚C for 10 min. Samples of 10 mg per lane were subjected to SDS-PAGE analysis using a 12% polyacrylamide gel. Proteins were then transferred onto a Roti- NC nitrocellulose membrane (Carl Roth, Germany) applying semi-dry blotting. Nitrocellulose mem- branes were blocked with PBS-T (0.05% Tween20 in PBS) containing 5% milk powder for one hour, and incubated with primary antibody against GFP diluted in blocking solution overnight at 4˚C. Membranes were washed for 20 min with PBS-T three times. For detection a second antibody tagged with HRP (RRID:AB_631747, Cat# sc-2030, Santa Cruz Biotechnology, USA) was applied for 1 hr and, after washing three times with PBS-T, chemiluminescence was detected with Luminol Reagent (sc-2048, Santa Cruz Biotechnology, USA) using autoradiographic films. Films were scanned and band intensities were quantified from the digital images. volume of 0.75 ml by applying 100 strokes. The homogenate was centrifuged for 8 min at 800 x g at 4˚C, yielding pellet P1 (containing non-homogenized cell debris) and supernatant S1. Membrane fractionation and western blot S1 was trans- ferred into a new tube for a second centrifugation step for 120 min at 20,000 x g and 4˚C, yielding pellet P2 (containing the enriched membrane fraction, which was resuspended in 750 ml homogeni- zation buffer) and the supernatant S2 with the cytosolic fraction. Protein concentrations of P2/S2 were adjusted to the lowest concentration in the series, before adding respective amounts of 4x Laemmli buffer followed by incubation at 95˚C for 10 min. Samples of 10 mg per lane were subjected to SDS-PAGE analysis using a 12% polyacrylamide gel. Proteins were then transferred onto a Roti- NC nitrocellulose membrane (Carl Roth, Germany) applying semi-dry blotting. Nitrocellulose mem- branes were blocked with PBS-T (0.05% Tween20 in PBS) containing 5% milk powder for one hour, and incubated with primary antibody against GFP diluted in blocking solution overnight at 4˚C. Membranes were washed for 20 min with PBS-T three times. For detection a second antibody tagged with HRP (RRID:AB_631747, Cat# sc-2030, Santa Cruz Biotechnology, USA) was applied for 1 hr and, after washing three times with PBS-T, chemiluminescence was detected with Luminol Reagent (sc-2048, Santa Cruz Biotechnology, USA) using autoradiographic films. Films were scanned and band intensities were quantified from the digital images. Research article Liposome-binding studies p g Wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 pGEX-6P1 constructs were transformed into Escheri- chia coli (Rosetta (DE3)pLysS) and expression of the GST fusion proteins was induced with 1 mM IPTG. After harvesting, cells were lysed for 60 min at 4˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 7.4 and 1 mM EDTA containing Roche cOmplete protease inhibitor, 1 mM DTT, 100 mg/ml lysozyme and two units/ml DNAse I. The suspension was sonicated, centrifuged for 30 min at 20,000 x g, fro- zen in liquid nitrogen and thawed for binding of the constructs directly to glutathione beads. To monitor SNAP25 interaction with liposomes, 42 mg GST-SNAP25 constructs or a equimolar amount of GST (glutathione S-transferase) were bound to 20 ml glutathione (GSH) sepharose four fast flow beads (GE Healthcare Life Sciences) prewashed 3 x with ddH2O and 3 x with fusion buffer (25 mM HEPES/KOH pH 7.4, 135 mM KCl, 1 mM DTT, 0.1 mM EGTA, 0.5 mM MgCl2, 1 mM DDT). 160 nmol liposomes in fusion buffer were added to the beads and incubated 1 hr at 4˚C on a rota- tion wheel. The beads were washed once with 1 ml fusion buffer and resuspended in 80 ml fusion buffer. The bound liposomes were detected by measuring the Atto647N fluorescence. The amounts of liposomes specifically bound to the different GST-SNAP25 constructs were calculated by subtract- ing the values derived from the GST controls. Additional information Additional information Funding Funder Grant reference number Author Deutsche Forschungsge- meinschaft TRR83 Thomas H So¨ llner Thorsten Lang The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Funding Funder Grant reference number Author Deutsche Forschungsge- meinschaft TRR83 Thomas H So¨ llner Thorsten Lang The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Funding Funder Grant reference number Author Deutsche Forschungsge- meinschaft TRR83 Thomas H So¨ llner Thorsten Lang The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Liposome preparation Atto647N-DPPE (Att647N-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) was purchased from Atto-Tec. All other lipids were from Avanti Polar Lipids. The complex lipid mixture (5 mmol total amount of lipid) contains 34.5 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 15 mol % 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 20 mol % 1-hexadecanoyl-2-octadecenoyl- sn-glycero-3-phosphoethanolamine (POPE), 25 mol % cholesterol (from ovine wool), 5 mol % liver L-a-phosphatidylinositol (PI, from liver) and 0.5 mol % Atto647N-DPPE. For the lipid mixes contain- ing brain L-a-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or 1-stearoyl-2-arachidonoyl-sn-glyc- ero-3-phospho-(1’-myo-inositol-3’,4’,5’-trisphosphate) (PI(3,4,5)P3) the amount of PI was reduced accordingly. The lipids were dissolved in chloroform or chloroform/methanol (3:1 ratio, for PI(4,5)P2 and PI(3,4,5)P3), mixed and dried under a flow of nitrogen. The remaining chloroform was removed by vacuum for 4 hr. The lipids were dissolved in 1 ml reconstitution buffer (25 mM HEPES/KOH pH 7.4, 200 mM KCl, 1% (w/v) OG (n-Octyl-b-D glucopyranoside), 1 mM DTT (1,4-dithiothreitol)) by 30 min shaking. To form liposomes, OG was diluted below the critical micelle concentration by the addition of 2 ml buffer (25 mM HEPES/KOH; pH 7.4, 200 mM KCl, 1 mM DTT). The residual OG was Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 15 of 18 Research article Research article Biophysics and Structural Biology Cell Biology removed by flow dialyses with 4 L 25 mM HEPES/KOH pH 7.4, 135 mM KCl, 1 mM DTT overnight. Subsequently, a Nycodenz gradient centrifugation was performed to isolate the liposomes. There- fore, the dialyzed samples were mixed with an equal volume of 80% (w/v) Nycodenz and transferred into two SW60-tubes (Beckman Coulter). Layers of 750 ml 35% (w/v) Nycodenz, 150 ml 11.6% (w/v) Nycodenz and 100 ml fusion buffer were added on top of the 40% (w/v) Nycodenz/liposome solution. The gradient was spun at 55,000 rpm for 3 hr 40 min at 4˚C. The liposomes were isolated, followed by a buffer exchange (25 mM HEPES/KOH pH 7.4, 135 mM KCl, 1 mM DTT, 0.1 mM EGTA, 0.5 mM MgCl2, 1 mM DDT) using a PD MiniTrap G-25 (GE Healthcare Life Sciences). The amounts of lipids were quantified by measurement of Atto647N fluorescence (excitation 639 nm, emission 669 nm) in a Fluoroskan Ascent FL Microplate Fluorometer (Thermo Scientific). Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Acknowledgements The authors would like to thank Michael Pankratz and Nora Karnowski for comments on the manu- script. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (TRR83 to THS and TL). Author contributions PW, Conceptualization, Formal analysis, Supervision, Investigation, Writing—original draft, Writing— review and editing; HB, Conceptualization, Formal analysis, Supervision, Investigation, Writing— review and editing; KMR, Conceptualization, Formal analysis, Investigation; SD, KP, Formal analysis, Investigation; THS, Conceptualization, Supervision, Funding acquisition, Writing—review and edit- ing; TL, Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project adminis- tration, Writing—review and editing Author ORCIDs Thorsten Lang, http://orcid.org/0000-0002-9128-0137 Author ORCIDs Author ORCIDs Thorsten Lang, 16 of 18 Weber et al. eLife 2017;6:e19394. DOI: 10.7554/eLife.19394 Research article R Research article Biophysics and Structural Biology Cell Biology References Aikawa Y, Xia X, Martin TF. 2006. SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells. 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https://openalex.org/W3042313681	https://hal.inrae.fr/hal-02904081/file/2020_cells_duszka.pdf	English	null	Peroxisome Proliferator-Activated Receptors and Caloric Restriction—Common Pathways Affecting Metabolism, Health, and Longevity	Cells	2,020	cc-by	49,207	Peroxisome Proliferator-Activated Receptors and Caloric Restriction-Common Pathways Affecting Metabolism, Health, and Longevity Kalina Duszka, András Gregor, Hervé Guillou, Jürgen König, Walter Wah To cite this version: Kalina Duszka, András Gregor, Hervé Guillou, Jürgen König, Walter Wahli. Peroxisome Proliferator- Activated Receptors and Caloric Restriction-Common Pathways Affecting Metabolism, Health, and Longevity. Cells, 2020, 9, 10.3390/cells9071708. hal-02904081 Distributed under a Creative Commons Attribution 4.0 International License Peroxisome Proliferator-Activated Receptors and Caloric Restriction—Common Pathways Affecting Metabolism, Health, and Longevity Kalina Duszka 1,, András Gregor 1, Hervé Guillou 2, Jürgen König 1 and Walter Wahli 2,3,4, 1 Department of Nutritional Sciences, University of Vienna, 1090 Vienna, Austria; Andras.Gregor@univie.ac.at (A.G.); juergen.koenig@univie.ac.at (J.K.) 2 Toxalim (Research Centre in Food Toxicology) INRAE ENVT INP Purpan UMR 1331 UPS Université 1 Department of Nutritional Sciences, University of Vienna, 1090 Vienna, Austria; Andras.Gregor@univie.ac.at (A.G.); juergen.koenig@univie.ac.at (J.K.) Andras.Gregor@univie.ac.at (A.G.); juergen.koenig@univie.ac.at (J.K.) 2 Toxalim (Research Centre in Food Toxicology), INRAE, ENVT, INP-Purpan, UMR 1331, UPS, Université de Toulouse, F-31027 Toulouse, France; herve.guillou@inrae.fr 3 Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Andras.Gregor@univie.ac.at (A.G.); juergen.koenig@univie.ac.at (J.K.) 2 Toxalim (Research Centre in Food Toxicology), INRAE, ENVT, INP-Purpan, UMR 1331, UPS, Université de Toulouse, F-31027 Toulouse, France; herve.guillou@inrae.fr 2 Toxalim (Research Centre in Food Toxicology), INRAE, ENVT, INP-Purpan, UMR 1331, UPS, Université de Toulouse, F-31027 Toulouse, France; herve.guillou@inrae.fr 3 Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore 308232, Singapore 4 Center for Integrative Genomics, University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland * Correspondence: Kalina.Duszka@univie.ac.at (K.D.); Walter.Wahli@unil.ch (W.W.) 4 Center for Integrative Genomics, University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland * Correspondence: Kalina.Duszka@univie.ac.at (K.D.); Walter.Wahli@unil.ch (W.W.) Received: 24 June 2020; Accepted: 14 July 2020; Published: 16 July 2020 Abstract: Caloric restriction (CR) is a traditional but scientifically verified approach to promoting health and increasing lifespan. CR exerts its effects through multiple molecular pathways that trigger major metabolic adaptations. It influences key nutrient and energy-sensing pathways including mammalian target of rapamycin, Sirtuin 1, AMP-activated protein kinase, and insulin signaling, ultimately resulting in reductions in basic metabolic rate, inflammation, and oxidative stress, as well as increased autophagy and mitochondrial efficiency. CR shares multiple overlapping pathways with peroxisome proliferator-activated receptors (PPARs), particularly in energy metabolism and inflammation. Consequently, several lines of evidence suggest that PPARs might be indispensable for beneficial outcomes related to CR. In this review, we present the available evidence for the interconnection between CR and PPARs, highlighting their shared pathways and analyzing their interaction. We also discuss the possible contributions of PPARs to the effects of CR on whole organism outcomes. Keywords: nuclear receptors; PPARs; nutrition; caloric restriction HAL Id: hal-02904081 https://hal.inrae.fr/hal-02904081v1 Submitted on 21 Jul 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 Cells 2020, 9, 1708; doi:10.3390/cells9071708 www.mdpi.com/journal/cells 1. Introduction Caloric restriction (CR) is one of the primary interventions for weight loss and health maintenance. As early as the 16th century, Luigi Cornaro (1484–1566) described the beneficial effects of this approach in his “Discorsa della vita sobria”. Later, at the beginning of the 20th century, the first experimental evidence emerged when Osborne et al. reported that CR slowed the growth of rats but prolonged their lifespan [1]. In rats, a CR of 40% applied from weaning onward has been linked to a lifespan extension of almost two-fold [2]. In fact, CR has been associated with increases in mean and maximum lifespan, regardless of sex, in multiple species, including various rat and mouse strains, yeasts, worms, fruit flies, fishes, hamsters, dogs, cows, and owls [3]. The effects of CR in these organisms include reduced neurodegenerative disease incidence, diminished rates of age-specific mortality, and a lower incidence of cancer, diabetes, atherosclerosis, and cardiovascular disease. CR also is linked to delayed onset of age-related processes, such as immunosenescence, sarcopenia, and atrophy of the brain gray matter [3–7]. In monkeys, CR leads to diabetes suppression and a reduced incidence of neoplasia and cardiovascular diseases by up to 50% [6]. These effects have been Cells 2020, 9, 1708; doi:10.3390/cells9071708 www.mdpi.com/journal/cells 2 of 75 Cells 2020, 9, 1708 attributed to a reduction in major risk factors, including cholesterol, C-reactive protein, blood pressure, and intima-media thickness of the carotid arteries [7–9]. Beneficial outcomes of CR have been consistently reported, which supports this approach considering that distinct CR protocols are used in different publications. CR applied in diverse studies ranges from 10% to up to 50% of daily caloric intake. Furthermore, the length of CR varies from a few weeks to life-long treatment. Additionally, some protocols restrict all nutrients, whereas others limit macronutrients only and supplement micronutrients in order to investigate selectively the impact of calorie reduction and prevent malnutrition, making a distinction between “dietary restriction” and “energy restriction” [10]. As expected, the type of CR protocol influences the magnitude of outcomes [11,12]. Moreover, the results obtained from experimental models cannot be directly translated to humans [13]. Therefore, it is important to compile the results of multiple studies to identify common patterns of responses regardless of the type of CR. A comparison of the responses from different species may help to draw a more comprehensive picture of the outcomes of CR. 1. Introduction CR has been tied to a complex network of pathways implicating insulin-like growth factor 1 (IGF-1), sirtuins (SIRTs), adenosine monophosphate (AMP)-activated protein kinase (AMPK), and target of rapamycin (TOR). The sympathetic and neuroendocrine systems, as well as thyroid hormones, adipokines, and ghrelin, also have been associated with the beneficial outcomes of CR [4]. This ensemble of processes associated with CR affects the whole body, manifesting in reduced inflammation, body fat mass, resting metabolic rate, and body temperature and improved insulin sensitivity [14]. As a result of the variety of outcomes related to CR and the complexity of the contributing pathways, the exact mechanisms underlying these health benefits are still not well understood. However, the results of ongoing studies have filled in some parts of this puzzle. This review focuses on one important piece: the role of peroxisome proliferator-activated receptors (PPARs) in outcomes associated with CR. 2. PPARs In addition to ligands, PPAR transcriptional activity can be modulated by post-translational modifications including phosphorylation, ubiquitination, O- GlcNAcylation, and SUMOylation [29,37–43]. Figure 1. Activation and main functions of peroxisome proliferator-activated receptors (PPARs) in different tissues. PPARs share fatty acids (FA) as common ligands, peroxisome proliferator response elements (PPRE) as their DNA binding site, and retinoid X receptors (RXR) as their heterodimer partner. However, each PPAR shows distinct expression and function patterns. The dominant role of PPARα is connected to metabolic adjustment in the liver and brown adipose tissue (BAT). PPARβ/δ is primarily associated with muscle and white adipose tissue (WAT) metabolism, as well as with organ development. PPARγ is a master regulator of adipogenesis and WAT maintenance and plays an important anti-inflammatory role. However, this cartoon represents a schematic and simplified view of much more complex patterns. PPAR PPARγ PPARα PPARβ/δ PPRE FA Phospholipids Eicosanoids Prostaglandins… Energy storage/expenditure (feeding/fasting) Lipogenesis FA uptake and β-oxidation Ketogenesis Gluconeogenesis Inflammation reduction Energy expenditure-FA uptake and β-oxidation Placenta and gut development Inflammation reduction Energy storage-lipogenesis Glucose metabolism Inflammation reduction RXR Liver Muscle WAT WAT BAT Macrophages PPAR PPARγ PPARα PPARβ/δ PPRE FA Phospholipids Eicosanoids Prostaglandins… Energy storage/expenditure (feeding/fasting) Lipogenesis FA uptake and β-oxidation Ketogenesis Gluconeogenesis Inflammation reduction Energy expenditure-FA uptake and β-oxidation Placenta and gut development Inflammation reduction Energy storage-lipogenesis Glucose metabolism Inflammation reduction RXR Liver Muscle WAT WAT BAT Macrophages Energy storage/expenditure (feeding/fasting) Lipogenesis FA uptake and β-oxidation Ketogenesis Gluconeogenesis Inflammation reduction PPARα RXR Energy expenditure-FA uptake and β-oxidation Placenta and gut development Inflammation reduction PPARβ/δ PPARγ Energy storage-lipogenesis Glucose metabolism Inflammation reduction Figure 1. Activation and main functions of peroxisome proliferator-activated receptors (PPARs) in different tissues. PPARs share fatty acids (FA) as common ligands, peroxisome proliferator response elements (PPRE) as their DNA binding site, and retinoid X receptors (RXR) as their heterodimer partner. However, each PPAR shows distinct expression and function patterns. The dominant role of PPARα is connected to metabolic adjustment in the liver and brown adipose tissue (BAT). PPARβ/δ is primarily associated with muscle and white adipose tissue (WAT) metabolism, as well as with organ development. PPARγ is a master regulator of adipogenesis and WAT maintenance and plays an important anti-inflammatory role. However, this cartoon represents a schematic and simplified view of much more complex patterns. 2. PPARs PPARs form a subfamily of the ligand-dependent nuclear receptor superfamily, which consists of three isotypes, PPARα (NR1C1), PPARβ/δ (NR1C2), and PPARγ (NR1C3), each coded by a separate gene [15–17]. PPARs play major roles in a broad spectrum of biological processes, including cell proliferation and differentiation, fatty acid (FA) and eicosanoid signaling, bone formation, tissue repair and remodeling, insulin sensitivity, and, in particular, glucose and lipid metabolism (Figure 1). They are transcription factors that form heterodimers with retinoid X receptors (RXRs) and bind to specific peroxisome proliferator response elements (PPREs) in the transcription regulatory region of their target genes. A number of coactivators and corepressors modulate PPAR activity, either stimulating or inhibiting receptor function [18]. Two important PPAR corepressors are the nuclear corepressor 1 (NCoR1) and the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) [19–21], which are recruited to PPARs in the absence of ligands and limit PPAR transcriptional activity [22,23]. Coactivators encompass various proteins, including factors with histone acetylase activity [cAMP response element-binding (CREB) protein (CBP)/p300 and steroid receptor coactivator 1–3 complexes], helicases [PPAR A–interacting complex (Pric)285, Pric320/chromodomain helicase DNA binding protein 9], and an ATPase in the SWItch/sucrose non- fermentable (SWI/SNF) complex, and non-enzymatic activators that can be found in the active PPAR transcriptional complex [PPARγ coactivator (PGC)-1α, PGC-δ/PGC-1–related estrogen receptor coactivator, mediator of RNA polymerase II transcription subunit/TRAP220/PPAR-binding protein, PPAR-interacting protein/nuclear receptor coactivator 6, SWI/SNF-related, matrix-associated, actin- dependent regulator of chromatin, subfamily D, member 1] [18]. The characteristic feature of the PPAR ligand-binding cavity is its 3-4-fold larger size compared to other nuclear receptors. Thus, PPARs can accommodate and bind a variety of natural and synthetic lipophilic acids [24–27]. Synthetic ligands of PPARs are widely used in clinical practice to treat glucose and lipid disorders and in the prevention and treatment of cardiovascular and metabolic diseases [28–30]. Synthetic ligands can be specific for each PPAR isotype or activate two (saroglitazar, elafibranor) [31,32] or all three (bezafibrate, lanifibranor) isotypes [33,34]. Natural ligands of PPARs include various FAs, 3 of 75 Cells 2020, 9, 1708 phospholipids, prostaglandins, prostacyclins, and leukotrienes [35,36] linking the activity of PPARs to nutrition, metabolism, and inflammation. In addition to ligands, PPAR transcriptional activity can be modulated by post-translational modifications including phosphorylation, ubiquitination, O- GlcNAcylation, and SUMOylation [29,37–43]. phospholipids, prostaglandins, prostacyclins, and leukotrienes [35,36] linking the activity of PPARs to nutrition, metabolism, and inflammation. 2.1. PPARα The first cloned PPAR, now known as PPARα, was initially identified as the molecular target of xenobiotics inducing hepatic peroxisome proliferation in rodents [44]. PPARα is particularly abundant in organs with high demand for FA catabolism, such as liver and brown adipose tissue (BAT) [45]. In these tissues, this transcription factor coordinates several aspects of metabolism by modulating the expression of genes involved in peroxisomal and mitochondrial β-oxidation, FA transport and catabolism, ketogenesis, and gluconeogenesis [35]. In line with this role of PPARα, the L162V Pparα gene polymorphism, which results in a PPARα variant with lower transcriptional activity, is associated with increased serum levels of triglycerides (TGs), total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein (apo) A1 and apoB [46–50], influencing the onset and progress of type 2 diabetes (T2D) [51,52]. PPARα serves as a sensor of nutritional status. In starvation, when the organism mobilizes stored FAs, PPARα in hepatocytes increases FA uptake and β-oxidation and stimulates hepatokine gene expression [53–56]. Therefore, PPARα adapts the rates of FA catabolism and ketone body synthesis to energy depletion. Furthermore, in the mouse liver, PPARα is a key factor in the metabolic adaptation to sepsis. Deficiency in hepatic PPARα causes an impaired metabolic response, and upon bacterial infection, PPARα-null mice have a higher mortality rate. These observations suggest that activating PPARα may be considered a plausible metabolic intervention for improving sepsis outcomes. In support of this hypothesis, non-surviving critically ill patients have reduced PPARα activity in their livers [57]. The natural ligands of PPARα are FAs and their derivatives [58]. Synthetic fibrate PPARα agonists are generally used to treat patients with primary hypercholesterolemia, mixed dyslipidemia, and hypertriglyceridemia. Natural and synthetic PPARα agonists have been discussed extensively 4 of 75 Cells 2020, 9, 1708 [29,30,59,60], and their effect appears to be based mostly on the stimulation of cellular lipid trafficking and β-oxidation [61,62]. However, the need persists for better drug candidates that selectively target PPARα without the side effects of fibrates. The recent development of selective PPARα modulators may fill this gap [63]. [29,30,59,60], and their effect appears to be based mostly on the stimulation of cellular lipid trafficking and β-oxidation [61,62]. However, the need persists for better drug candidates that selectively target PPARα without the side effects of fibrates. The recent development of selective PPARα modulators may fill this gap [63]. 2.2. PPARβ/δ PPARβ/δ is ubiquitously expressed, with particularly high levels in the skin, gastrointestinal tract, liver, kidney, and various parts of the central nervous system [64,65]. PPARβ/δ is involved in cell proliferation, differentiation, and survival, and it plays a role in tissue repair [66–69]. It is necessary for placental and gut development and is involved in the control of energy homeostasis [70–74]. Metabolically, PPARβ/δ promotes FA oxidation in adipose tissue and skeletal muscle, leading to improved lipidemia, and it stimulates overall energy expenditure, protecting against diet-induced obesity and insulin resistance [75–78]. It is probably best known for stimulating energy expenditure in muscles and increasing exercise capacity [79]. Under regular exercise, PPARβ/δ induces a switch to an increased amount of type I muscle fibers, enhancing mitochondrial function and fat oxidation and boosting endurance [80]. For this reason, this receptor also is a target for doping substances, so much so that the World Anti-Doping Agency added the PPARβ/δ ligands GW501516 and other related chemicals to its prohibited list in 2009. These synthetic ligands (GW501516, GW0742, and L-165041) selectively activate PPARβ/δ at very low concentrations and are often applied in both in vivo and in vitro research models [81], but no PPARβ/δ agonist has been used in clinical practice. Nevertheless, PPARβ/δ remains a potential therapeutic target in metabolic diseases. Other synthetic and natural PPARβ/δ agonists recently have been discussed elsewhere [29]. 2.4. PPARs in CR The effects of CR on PPAR expression vary among different tissues and organs. PPAR expression either remains unchanged [PPARα, -β/δ, and -γ in the heart, PPARα in white adipose tissue (WAT), PPARγ in the liver], decreases (PPARβ/δ in the liver, PPARα in the spleen, PPARα, - β/δ, and -γ in muscle), or increases (PPARα in the liver, heart, and intestinal epithelium) [131–139]. In rat kidneys, PPAR mRNA and protein levels and DNA-binding activity decrease with age, but a 60% CR blunts this reduction [140]. PPARs can contribute to CR-related outcomes by modulating multiple pathways connected with metabolism, insulin sensitivity, inflammation, and oxidative stress, all of which we describe at the molecular level in this review. Most of the published works on the impact of PPARs in CR concern PPARα. CR induces the upregulation of PPARα expression in the heart, where it increases lipid use [139]. In fact, 19% of the hepatic genes involved in lipid metabolism, inflammation, and cell growth and whose expression is changed by CR depend on PPARα, as shown by the absence of regulation of these processes in PPARα-null mice [141]. Therefore, ligands of PPARα have been proposed to serve as mimetics of CR [141]. Although less studied so far, the two other PPAR isotypes likewise modulate multiple pathways in relation to CR, as we discuss below. 2.3. PPARγ PPARγ is recognized primarily for its insulin-sensitizing properties and its role as a master regulator of adipogenesis [82–84]. It also has been identified as a promising therapeutic target for several types of cancer because it limits tumor progression by decreasing cell proliferation [85–87], increasing cell differentiation [85,88], inducing apoptosis [85,86,89–93], and inhibiting angiogenesis [94]. In addition, PPARγ plays a role in multiple processes including inflammation, adipogenesis, FA storage, and lipid and glucose metabolism [82,95–99]. We have described novel roles for intestinal PPARγ in long-chain FA processing in the intestinal epithelium [100] and in the regulation of body adiposity through sympathetic nervous system signaling [101], and we identified it as a major regulator of mucosal defenses upon high-fat diet feeding in mice [102]. However, its effects are not exclusively beneficial. For instance, it has recently been reported that PPARγ has a carcinogenic effect in advanced brain metastases [103] and liver cancer [104]. PPARγ occurs in two isoforms, PPARγ1 and PPARγ2, with the latter distinguished by 30 and 28 extra amino acids at the N-terminus in mouse and human, respectively [105]. PPARγ2 is found at high levels in adipose tissues [106,107], whereas PPARγ1 shows a broader expression pattern. In addition to adipose tissue, PPARγ1 is expressed in the gut, brain, and vascular cells and in specific types of immune and inflammatory cells [65,105,108–110]. A plethora of factors induce PPARγ expression and activity, and not all are ligands. They can include FAs and their metabolites [25,111], eicosanoids [25], prostaglandins [112], phytanic acid [113], and multiple nutrients, along with glutamine, curcumin, capsaicin, ginsenosides, and vitamin E, all of which have been reported to exhibit anti-inflammatory properties [114]. Of note, the presence of specific bacterial strains, their metabolites, and bacterial by-products [115–120] also stimulate PPARγ expression and/or activity. y p γ p y Synthetic agonists of PPARγ can be divided into two groups: classical full agonists, which are represented by the thiazolidinediones (TZDs) [121] such as troglitazone, rosiglitazone, and pioglitazone, and partial agonists, which were developed to reduce the side effects of full agonists, including weight gain and bone loss [122,123]. PPARα/γ dual agonists exert positive influences on both lipid and glucose metabolism. They not only have antidiabetic capacity but also are hypolipemic, anti-inflammatory, hypotensive, and antiatherogenic and show anticoagulant action leading to improved endothelial function [30,31,59,124–130]. 5 of 75 Cells 2020, 9, 1708 2.4. PPARs in CR 3.1. mTOR The mammalian (m)TOR pathway is a major nutrient sensor signaling pathway known to regulate longevity (Figure 2). TOR is a well-conserved Ser/Thr protein kinase that belongs to the family of phosphatidylinositol 3 (PI3) kinase-related kinases [142,143]. It functions as an essential part of two complexes, mTORC1 and mTORC2, which have some proteins in common and some different proteins between them [142]. mTORC1 comprises the following core subunits: mTOR, mLST8 (mammalian lethal with sec-13 or GβL), DEPTOR (DEP domain-containing mTOR-interacting protein Tti1/Tel2 complex), PRAS40 (proline-rich Akt substrate of 40 kDa), and Raptor (regulatory- associated protein of mammalian target of rapamycin). mTORC2 is composed of mTOR, mLST8, DEPTOR, the Tti1/Tel2 complex, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated MAP kinase-interacting protein 1 or MAPKAP1), and protor1/2 (protein observed with Rictor 1 and 2) [144–147]. The configuration of each of these two complexes is conserved from yeast to mammals [148]. mTORC1 is sensitive to inhibition by rapamycin and plays essential roles in the regulation of mRNA translation and autophagy. Cellular energy and nutrient status regulate it directly, whereas mTORC2, which is not rapamycin sensitive, functions mainly as an important regulator of the cellular actin cytoskeleton [149,150]. Figure 2. The main signaling pathways associated with mammalian target of rapamycin (mTOR). mTOR integrates inputs from multiple sources including growth factors, insulin, stress, energy TSC1 TSC2 Akt mTORC1 Rheb Insulin receptor TNFα Amino acids Leucine Arginine DNA damage Lifespan Insulin Hypoxia MEK ERK1/2 IRS PI3K PDK4 AMPK 4E-BP PTEN starvation P38β-PRAK Wnt ERFR REDD1 REDD2 GSK3 Phosphatidic acid Redox Growth factors TNFR Energy stress p53 S6k1 Energy metabolism Nucleotide synthesis Protein synthesis Lipid synthesis Autophagy Cholesterol synthesis Cell growth Mitochondria biogenesis and function Glucose metabolism starvation Figure 2. The main signaling pathways associated with mammalian target of rapamycin (mTOR). mTOR integrates inputs from multiple sources including growth factors, insulin, stress, energy 6 of 75 Cells 2020, 9, 1708 balance, oxygen, and nutrients, and it controls many major downstream processes, including metabolism, macromolecule synthesis, mitochondria function, cell growth, and autophagy. balance, oxygen, and nutrients, and it controls many major downstream processes, including metabolism, macromolecule synthesis, mitochondria function, cell growth, and autophagy. Rheb (Ras homolog enriched in the brain) is a GTPase that in its GTP-bound form directly binds to and activates mTOR [151–154]. Rheb activity is inhibited by the heterodimer complex of tuberous sclerosis proteins 1 and 2 (TSC1 and TSC2) [155–160]. 3.1. mTOR TSC1/2 mediates for mTORC1 many of the upstream signals from growth factors, such as insulin and IGF-1, which stimulate the PI3K and Ras pathways. The effector kinases of these pathways, including Akt (or protein kinase B or PKB), extracellular-signal-regulated kinase 1/2 (ERK1/2), and ribosomal protein S6 kinase β-1 (S6K1), directly phosphorylate and inactivate the TSC1/TSC2 complex, leading to the activation of mTORC1 [154,157,161–167]. Rheb also can transmit upstream signals from the p38β–PRAK pathway, which is activated upon glucose starvation [168]. Finally, as a core component of mTORC2, mTOR functions as a tyrosine protein kinase that promotes activation of the insulin receptor and IGF-1 receptors [169]. These interactions illustrate the tightly interconnected signaling between mTOR and insulin. The mTOR pathway integrates inputs from major intracellular and extracellular physiological stimuli (growth factors, stress, energy balance, oxygen, amino acids) and controls many major downstream processes, including macromolecule synthesis, autophagy, cell cycle, growth, and metabolism [142,143,170]. For example, the canonical Wnt pathway, AMPK, some pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), and the hypoxia-inducible proteins REDD1 and REDD2 modulate mTORC1 activity via TSC1/2 [171–176]. In addition to phosphorylating TSC1/2, AMPK phosphorylates Raptor, leading to the allosteric inhibition of mTOR [177]. mTORC1 activity is further regulated by lipid-derived signaling molecules (phosphatidic acid) [178], the redox status of the cell [179], and amino acids, particularly leucine and arginine [180,181]. DNA damage also signals to mTORC1 through multiple mechanisms, all of which require p53-dependent transcription, induction of the expression of TSC2 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and AMPK activation [182–184]. Downstream signaling of mTORC1 controls autophagy and energy metabolism, including the glycolytic flux, lipid synthesis [185–188], and cholesterol synthesis via the activation of sterol regulatory element-binding protein (SREBP) 1/2 [185,189,190]. mTORC1 also promotes anabolism in the fed state by controlling lipid metabolism in the liver through the modulation of Srebp1c expression, which is a regulator of lipogenesis and lipid storage [191,192]. Under mTORC1 regulation, mitochondrial DNA content and the expression of genes involved in oxidative metabolism increase. mTORC1 exerts this effect in part by mediating the nuclear association between PPARγ coactivator 1α (PGC-1α) and the transcription factor Yin-Yang 1, which positively regulates mitochondrial biogenesis and oxidative function [193] (see the section on mitochondrial function). 3.1. mTOR The activation of mTOR also leads to the phosphorylation of many target proteins related to the translational machinery and ribosome biogenesis, such as p70 ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein (4E-BP) [170,194–199]. The regulation of protein metabolism also is a much-recognized function of mTOR. Amino acid activation of mTORC1 promotes protein synthesis via the activation of S6K and/or inhibition of 4E-BP, whereas the inactivation of mTORC1 promotes the degradation of damaged proteins and intracellular organelles via autophagy [200,201] (Figure 2). mTORC2 functions mainly as an important regulator of the actin cytoskeleton through its stimulation of F-actin stress fibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase C (PKC) α [146]. mTORC2 phosphorylates Akt [202,203] and thus affects metabolism and cell survival. mTORC2 also directly activates SGK1, which is a kinase controlling ion transport and growth [204]. Both Akt and SGK1 phosphorylate FoxO1/3a [205–207]. As a result of its role as an amino acid sensor, the TOR pathway has been proposed as a mediator of CR. The high activity of mTORC1 is a major driving force of aging, whereas the suppression of mTOR is tied to many of the benefits associated with CR, including lifespan extension [208–211], as has been demonstrated in yeast [208,212], worms [209], and flies [210]. Rapamycin treatment slightly extends the lifespan in flies subjected to CR [213]. In yeast, CR does not further extend the lifespan in 7 of 75 Cells 2020, 9, 1708 the absence of TOR1, which is one of the two TOR genes in yeast, suggesting that TOR inhibition and CR promote lifespan via a common mechanism [208]. Similarly, in C. elegans, using RNA interference against TOR or autophagy genes in eat-2 mutant worms, which have impaired feeding behavior and are used as a genetic model for CR, does not extend the lifespan [214,215]. Furthermore, the inhibition of one of the principal targets of TOR signaling, S6K, extends the lifespan of eat-2 C. elegans [216]. Of note, mTOR activation in the rat’s brain results in reduced food intake by promoting the expression of the orexigenic neuropeptide Y and agouti-related peptide in the hypothalamus [217,218]. These data suggest that CR and TOR inhibition promote lifespan via overlapping pathways. 3.2. mTOR and PPARα The various direct or indirect interactions between mTOR and PPARs have diverse effects on FA synthesis, glucose metabolism, oncogenesis, and immune system activation (Figure 3). First, mTORC1 regulates hepatic ketone body production in response to fasting [219]. mTORC1 activity is low during fasting, which correlates with increased PPARα activity. Consequently, mice with a constitutive activation of mTORC1 in the liver and with correspondingly low PPARα cannot induce ketogenesis when fasted [219]. As alluded to above, PPARα is particularly known for its role in fasting-triggered FA oxidation and lipid metabolism in the liver. The intertwined functions of mTORC1 and PPARα lie in the mTORC1 inhibition of PPARα activity during feeding and consequently blocking hepatic ketogenesis. Figure 3. Interactions between PPARs and mTOR. mTOR interacts with all PPARs, resulting in the modulation of ketogenesis, autophagy, and adipogenesis. Figure 3. Interactions between PPARs and mTOR. mTOR interacts with all PPARs, resulting in the modulation of ketogenesis, autophagy, and adipogenesis. In the fed state, the insulin-dependent PI3K pathway activates mTORC1, promoting the cytoplasmic and nuclear localization of the PPARα corepressor NCoR1 and leading to the inhibition of PPARα activity and ketogenesis [219]. Fasting leads to decreased mTORC1 and consequently S6 kinase 2 activity, which promotes the cytoplasmic localization of NCoR1. In the absence of nuclear NCoR1, the increased transcriptional activity of PPARα enhances the FA oxidation that generates substrates for ketogenesis and gluconeogenesis [219,220]. In fact, PPARα also stimulates the expression of mitochondrial hydroxymethylglutaryl-CoA synthase (HMGCS), which is a rate- limiting enzyme of ketogenesis that catalyzes the condensation of acetyl coenzyme A (acetyl-CoA) and acetoacetyl-CoA to generate 3-hydroxy-3-methylglutaryl (HMG)-CoA and CoA [37]. The CR- triggered increase in the intestinal HMG-CoA synthase 2 (HMGCS2) expression alters the regeneration and differentiation capacity of intestinal steam cells. The result is a reduction in the differentiation markers of secretory cells with the promotion of enterocytes, changing the functionality of the intestine [221]. The subsequent refeeding of CR mice leads to reduced HMGCS2 levels and an increased expression of Paneth and goblet cell markers [221]. Additionally, in the intestine, the age-related increased activity of mTOR inhibits PPARα, resulting in higher levels of Notum and decreased Wnt signaling, consequently diminishing the regenerative function of stem 8 of 75 Cells 2020, 9, 1708 cells in the Paneth cell niche [222]. 3.3. mTOR and PPARβ/δ Relatively little evidence connects mTOR and PPARβ/δ functions. In human lung carcinoma cells, nicotine activates PPARβ/δ expression through PI3K/mTOR [231], whereas the PPARβ/δ agonist GW501516 stimulates the growth of these cells through the inhibition of PTEN expression [232], indicating the interplay between the two pathways. Additionally, PPARβ/δ may modulate mTOR activity by mediating the metabolism of FAs and the production of phosphatidic acid, which is a metabolite that directly activates the mTOR complex by increasing its stability and activity. Phosphatidic acid responsiveness has been proposed as a lipid precursor sensing mechanism for the biosynthesis of cell membranes in the context of cell division and cell mass increase [233]. 3.2. mTOR and PPARα Similarly, mTORC1 activity is elevated in the livers of old mice [219], which correlates with reduced PPARα activity and hepatic ketogenesis during aging [223–225]. mTORC1 inhibition is sufficient to prevent both the aging-induced loss of PPARα activity and ketone body production [219]. Another type of functional interaction between mTOR and PPARα relates to ligand production for the latter by FA synthase (FAS). In the fed state, mTORC1 mediates insulin-dependent phosphorylation and thus the inhibition of cytoplasmic FAS, limiting ligand generation. During fasting, when mTOR is inhibited, nonphosphorylated active cytoplasmic FAS promotes the synthesis of endogenous PPARα ligands [37]. In several organs, CR induces autophagy, which is a process that integrates mTOR and PPARα. To protect the liver from acute failure, PPARα-mediated induction of autophagy attenuates a lipopolysaccharide (LPS)-induced pro-inflammatory response [226]. Furthermore, agonists of PPARα (GW7647 and WY-14,643) regulate multiple genes involved in autophagy and lysosomal biogenesis and function, such as the transcription factor EB, which is a master gene for lysosomal biogenesis [227]. Of interest, a protein called farnesoid X receptor (FXR) is activated in the fed liver and suppresses autophagy. PPARα, activated in fasted and CR livers, regulates genomic circuits that are complementary to those under FXR control. Moreover, FXR stimulates the hepatic expression of PPARα [228]. These findings highlight how the liver senses nutrient status and how these two nuclear receptors translate this status in autophagy regulation [229,230]. 3.4. mTOR and PPARγ As noted, PPARγ is a master regulator of adipogenesis. In parallel, mTORC1 senses growth factors and nutrients that drive adipose tissue accumulation. The inhibition of mTORC1 impairs adipogenesis and adipocyte maintenance in vitro [187,234–237], at least in part by modulating PPARγ expression and transcription [187,188,238,239]. mTORC1 may activate PPARγ through SREBP1, which promotes the production of endogenous PPARγ ligands [240,241]. Once activated by its natural or synthetic ligands, PPARγ stimulates mTORC1 and AMPK and upregulates TG-derived FA uptake, lipoprotein lipase activity, and accumulation in subcutaneous WAT and BAT. Chronic mTOR inhibition attenuates these processes, which leads to hyperlipidemia. These observations imply that mTOR regulates the hypolipidemic and lipogenic effects of PPARγ [239], as also suggested by the rapamycin inhibition of adipocyte differentiation [187,234,237]. Additionally, rapamycin reduces the phosphorylation of lipin-1 [242], which is a phosphatidic phosphatase that is involved in phospholipid and TG synthesis as well as the coactivation of many transcription factors linked to lipid metabolism, including PPARγ, PPARα, and PGC-1α [243–245]. A model has been proposed for nutrient and insulin signaling during adipogenesis in which the mTOR and PI3K/Akt pathways respectively act in parallel to regulate PPARγ activation [187], revealing pathway synergy in the context of metabolism. Conversely, PPARγ also affects mTOR activity. It inhibits mTOR signaling with a protective action in rats with status epilepticus [246], and after activation with rosiglitazone, it stimulates mTORC1 and AMPK [239]. The interaction between PPARγ and mTOR has been particularly well demonstrated in mouse models of breast cancer, in which the activation of PPARγ led to rapamycin 9 of 75 Cells 2020, 9, 1708 resistance, whereas rapamycin treatment triggered both the expression and activation of PPARγ [247]. Similar to PPARα, PPARγ regulates autophagy, the main cellular process targeted by mTOR. The activation of PPARγ stimulates autophagy, and its inhibition decreases it [248,249]. In addition to cell cycle arrest, the induction of autophagy is a major effect of PPARγ in cancer cells, as observed in colon [250], breast [251,252], bladder [253], and adrenocortical [254] cancer cell lines. The application of troglitazone and rosiglitazone in MDA-MB-231 breast cancer cells results in the redistribution of microtubule-associated protein 1A/1B-light chain 3 (LC3) and formation of the autophagosome [252]. Liver cells treated with 4-O-carboxymethyl ascochlorin, an agonist of PPARγ, undergo an autophagic response [249]. PPARγ activation in Caco-2 cells and other cell types affects autophagy via positive regulation of PTEN [255–259]. 3.4. mTOR and PPARγ In contrast, the deletion of PTEN results in an increased expression of PPARγ in the liver [260]. In a model of cigarette smoking extract (CSE)- induced inflammation in a human bronchial epithelial cell line (16HBE), PPARγ plays an anti- inflammatory role in an autophagy-dependent manner. In this model, PPARγ activation promotes autophagy by phosphorylating and activating AMPK (see the AMPK and PPARγ section) [261]. Accordingly, the PPARγ agonists TZDs inhibit IGF-1-induced p70S6K phosphorylation at sites specifically phosphorylated by mTOR as well as dephosphorylating a downstream factor, S6K, through AMPK activation [262]. Similarly, PPARγ induces the expression of autophagy-related proteins but reduces inflammatory molecules in aged human dental pulp cells, suggesting a mechanism by which it might maintain pulp homeostasis [263]. Conversely, PPARγ exerts anti-autophagic effects in neurons [264,265] and macrophages [266]. Rosiglitazone downregulates autophagy-related protein expression and improves locomotor function after spinal cord injury (SCI), whereas GW9662, a PPARγ inhibitor, abolishes the protective effect in SCI [265]. The PPARγ agonist 15-PGJ2 counteracts the increased expression of the autophagy-related protein in the ischemic cortex following cerebral ischemia–reperfusion (I/R) injury. Moreover, cerebral I/R injury increases levels of LC3 and cathepsin-B in neurons, whereas 15- PGJ2 decreases them. Thus, PPARγ exerts neuroprotection by inhibiting neuronal autophagy after cerebral I/R injury [264]. Finally, in human monocyte-derived macrophages, PPARγ induces cathepsin L, which is a cysteine protease that has been found to trigger apoptosis and decrease autophagy [266]. 4. AMPK CR decreases energy input, which leads to the activation of a signaling cascade to generate fuel and increase longevity. Decreased glucose intake reduces carbon flow through the glycolytic pathway and slows the conversion of ADP to ATP. As a principal cellular energy sensor, AMPK monitors the AMP:ATP and ADP:ATP ratios. Functionally, AMPK is a serine/threonine kinase comprising one catalytic subunit, α, and two regulatory subunits, β and γ. Each of the subunits occurs as different isoforms (α1, α2, β1, β2, γ1, γ2, γ3) allowing for different versions of AMPK in various tissues [267,268]. From nematodes to humans, the kinase activity of AMPK is rapidly increased by the binding of AMP or ADP to the AMPKγ subunit [269]. This binding promotes allosteric activation and the phosphorylation of AMPK by the upstream AMPK kinase and thus also inhibits its dephosphorylation [270]. An alternative activating pathway triggers AMPK in response to increases in cellular Ca2+ and involves the Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) [271]. Once activated, AMPK promotes ATP preservation by repressing energy-consuming biosynthetic pathways while enhancing the expression or activity of proteins involved in catabolism. This process results in the mobilization of deposited energy to restore the ATP supply [272]. Several downstream factors including CREB-regulated transcriptional coactivator-2 (CRTC2) [273], TBC1D1/AS160 [274,275], PGC-1α [276], and histone deacetylase (HDAC) 5 [277] mediate the impact of AMPK on metabolism. Functionally, AMPK phosphorylates acetyl-CoA carboxylase 1 (ACC1) and ACC2 [278,279], SREBP1c [280], glycerol phosphate acyl-transferase, [281], and HMG-CoA reductase [282], resulting in the inhibition of FA, cholesterol, and TG synthesis while activating FA uptake and β- oxidation. Additionally, AMPK prevents protein biosynthesis by inhibiting mTOR and TIF-IA/RRN3, 10 of 75 Cells 2020, 9, 1708 which is a transcription factor for RNA polymerase I that is responsible for ribosomal RNA synthesis [283]. AMPK also influences glucose metabolism by stimulating both nutrient-induced insulin secretion from pancreatic β-cells [284] and glucose uptake by phosphorylating Rab-GTPase- activating protein TBC1D1, which ultimately induces the fusion of glucose transporter (GLUT)4 vesicles with the plasma membrane in skeletal muscle [285]. AMPK stimulates glycolysis by the phosphorylation of 6-phosphofructo-2-kinase (fructose-2,6-bisphosphatase 2) [286], and in parallel, it inhibits glycogen synthesis through the phosphorylation of glycogen synthase [287]. In the liver, AMPK inhibits gluconeogenesis by inhibiting transcription factors including hepatocyte nuclear factor 4 and CRTC2 [288–290]. 4. AMPK AMPK also affects the energy balance by regulating circadian metabolic activities and promoting feeding through its action in the hypothalamus [291,292]. It promotes mitochondrial biogenesis via PGC-1α [276] (see the section on mitochondria) and activates antioxidant defenses. AMPK plays a major role in metabolism but is also involved in inflammation, cell growth, autophagy, and apoptosis [293]. Therefore, reducing AMPK signaling exerts a cytostatic and tumor-suppressing effect [294,295]. In C. elegans, the lifespan extension effect of CR depends on AMPK [296,297]. Similarly, in Drosophila, pathways mediating increased lifespan include AMPK activation [298]. In addition, tissue-specific overexpression of AMPK in muscle and body fat extends the lifespan in Drosophila, whereas AMPK RNA interference shortens the lifespan [299]. The link between AMPK and PPARs and their interaction in metabolism regulation in response to CR have been well documented and are discussed below. 4.1. AMPK and PPARα The contradictory information concerning the interaction between PPARα and the ligands of AMPK likely reflects tissue- and context-specific situations. AMPKα [314]. The contradictory information concerning the interaction between PPARα and the ligands of AMPK likely reflects tissue- and context-specific situations. One publication has reported that AMPK inhibits PPARα and PPARγ activity [315]. In that study, the AMPK activators, AICAR, and metformin decreased basal and WY-14,643-stimulated PPARα activity in hepatoma cells. Compound C, which is an AMPK inhibitor, increased agonist- stimulated reporter activity and partially reversed the effect of the AMPK activators. The expression of either a constitutively active or dominant-negative AMPKα subunit inhibits basal and WY-14,643- stimulated PPARα activity. The authors postulated that the AMPK inhibition of PPARα and PPARγ may allow for short-term processes to increase energy generation before the cells devote resources to increasing their capacity for FA oxidation [315]. This contradictory report may indicate further that AMPK–PPAR regulation is cell-type specific. An agonist of PPARα also can activate AMPK, suggesting that the activity regulation between AMPK and PPARα may be reciprocal. On the one hand, fenofibrate induces the phosphorylation and activation of AMPK via the induction of the small heterodimer partner (SHP; an orphan nuclear receptor) and its target genes [316]. On the other hand, WY-14,643 treatment increases the expression of AMPKα1 and α2 mRNA, leading to an increase in AMPKα subunit phosphorylation and its enzymatic activity [317]. Moreover, pterostilbene, a bioactive component of blueberries and grapes and an agonist of PPARα, activates AMPK, similarly to AICAR and metformin, and modulates several AMPK-dependent metabolic functions in the rat hepatoma cell line H4IIE [318]. The AMPK- mediated activation of PPARα reverses progressive fibrosis in steatohepatitis [316] by endothelial nitric oxide (NO) synthase (eNOS) phosphorylation in endothelial cells, which suppresses microvascular inflammation and apoptosis [319,320]. 4.2. AMPK and PPARβ/δ AMPK and PPARβ/δ, but not PPARα, interact directly and physically in muscle, leading to increased glucose oxidation via the upregulation of lactate dehydrogenase B, which is associated with improved exercise performance [310]. AICAR treatment increases endurance, and the combination of AICAR and GW0742 further potentiates it. The combination significantly increases all running parameters, which is a change that is accompanied by a significant shift to fat as the main energy source with a decline in carbohydrate use during the period near exhaustion [321]. Therefore, agonists of both AMPK and PPARβ/δ are recognized as exercise mimetics [322]. In line with these observations, the deletion of PPARβ/δ specifically in myocytes results in a reduced capacity to sustain running exercise [78]. 4.1. AMPK and PPARα AMPK and PPARα both act as sensors of intracellular energy status and adjust metabolism in response to changes. As noted, AMPK responds to intracellular ATP depletion, whereas PPARα induces the expression of genes encoding enzymes and proteins involved in increasing cellular ATP yields. Moreover, AMPK and PPARα serve as critical regulators of short-term and long-term FA oxidation, respectively, and their activity thus needs to be coordinated. Accordingly, during prolonged fasting, when glucose levels drop and FA levels rise, high intracellular AMP concentrations induce AMPK, resulting in increased mitochondrial FA uptake for β-oxidation. In parallel, the activation of PPARα elevates the maximal FA-oxidizing capacity in the liver [35,37,300,301]. Similar to AMPK, phosphorylation affects the activity of PPARα. Several kinases, including p38, ERK, protein kinase A, and PKC, and AMPK itself can phosphorylate PPARα, which modifies (mainly increasing) its transcriptional activity [302]. However, the activation of p38, which AMPK may execute [303,304], induces the activation of PPARα in some cells while reducing it in others. Additionally, the phosphorylation of PPARα by glycogen synthase kinase, also regulated by AMPK [305], leads to the degradation of PPARα [302,306]. The activation of PPARα by AMPK has been shown in multiple experimental models. In myocytes, either 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a synthetic activator of AMPK, or adiponectin, an insulin-sensitizing adipokine, increase FA oxidation gene expression via AMPK-dependent PPARα activation [307,308]. Therefore, the reduced serum levels of adiponectin in people with obesity and T2D may contribute to the observed impairment in PPARα activity [309]. Of note, in muscles, PPARα does not directly interact with AMPK [310]. Similarly, in the left atrial appendage of mixed-breed dogs, the AMPK/PPARα/VLCAD (very long-chain acyl-CoA dehydrogenase) pathway mediates the metformin-triggered reduction of lipid accumulation and increases the β-oxidation of FA [311]. In pancreatic β-cells, glucose represses PPARα gene expression via AMPK inactivation [312,313]. The mechanism of the direct interaction between AMPK and PPARα has been uncovered in hepatocytes. In this pathway, activated AMPKα subunits bind to and activate PPARα, which occurs independently of AMPK activity and is not associated with increased AMP concentration. Instead, the interaction is stimulated by increased MgATP levels. Surprisingly, treatment with AICAR decreases PPARα activity in rat hepatocytes, which is associated with translocation of the AMPKα2 isoform out of the nucleus and is independent of the kinase activity of Cells 2020, 9, 1708 11 of 75 AMPKα [314]. 4.3. AMPK and PPARγ The activation of AMPK by PPARγ agonists has been documented in many cell lines [261,323– 326], in various tissues ex vivo [327,328], and in nonhuman animals [329–331] and people [332]. In general, agonists of PPARγ act via AMPK to improve glucose and fat management. Troglitazone causes rapid increases in phosphorylated AMPK and acetyl-CoA carboxylase (ACC) within minutes after injection in rat skeletal muscle, liver, or adipose tissue. Consistently, the drug results in a two- fold increase in 2-deoxy-d-glucose uptake in skeletal muscle through AMPK activation [328]. In addition, rosiglitazone remarkably enhances AMPK-mediated glucose uptake and glycogen synthesis in muscle and adipose tissues [331]. In cardiac muscle, the impact of troglitazone on glucose uptake is triggered via AMPK and eNOS signaling [333]. Rosiglitazone increases the expression and circulating levels of adiponectin and enhances the expression of hepatic adiponectin receptors in mice, which correlates with the activation of the hepatic Sirt1/AMPK signaling system. This signaling enables rosiglitazone to attenuate alcoholic liver steatosis and nonalcoholic steatohepatitis [329,334]. Another TZD, pioglitazone, increases AMPK phosphorylation two-fold and decreases ACC activity and the concentration of malonyl-CoA by 50% in Wistar rat liver. Moreover, pre-treatment with pioglitazone prevents a 50% decrease in AMPK and ACC phosphorylation in the liver and adipose tissue, which can be triggered by a euglycemic–hyperinsulinemic clamp [330]. In endothelial cells, rosiglitazone reduces glucose-induced oxidative stress mediated by NAD(P)H oxidase hyperactivity 12 of 75 Cells 2020, 9, 1708 induced by high glucose via AMPK activation. It also uses AMPK to stimulate eNOS activity to increase NO synthesis [324,325]. induced by high glucose via AMPK activation. It also uses AMPK to stimulate eNOS activity to increase NO synthesis [324,325]. Several TZDs have been shown to reduce insulin resistance via AMPK activation [323,327,335,336]. AMPK-mediated pioglitazone signaling results in an increase in insulin-stimulated glucose disposal, enhanced expression of the genes encoding adiponectin receptors, and coding for factors connected with mitochondrial function and FA oxidation in the muscles of patients with diabetes [332]. Rosiglitazone promotes AMPK-mediated insulin secretion via the phosphorylation of the Kir6.2 subunit of the potassium ATP channel in β-cells [336]. The treatment of pancreatic β-cells with TZDs triggers the phosphorylation of AMPK and ACC and increases glucose-stimulated insulin secretion as well as the response of insulin secretion to the combined stimuli of glucose and palmitate [327]. This treatment also affects β-cell metabolism by reducing glucose oxidation, energy metabolism, and glycerolipid/FA cycling [323]. 5. Insulin Signaling Increased glucose levels in serum after food intake promote insulin secretion from pancreatic β- cells, which in turn activates insulin receptors on the surface of target cells. The tyrosine kinase activity of the insulin receptor triggers a signaling cascade starting with the activation of insulin receptor substrates (IRS 1–4) followed by the phosphorylation of PI3K, which is responsible for metabolic actions including PDK1 and Akt activation. Akt occurs in three isoforms (1–3) with Akt2 being essential for glucose homeostasis, whereas Akt1 is important for growth and Akt3 is important for brain development [338]. The Akt-driven inhibition of AS160 phosphorylation induces GLUT4 to translocate to the cell membrane, which promotes glucose transport into the intracellular compartment. Akt also phosphorylates and deactivates glycogen synthase (GS) kinase 3 (GSK3), which stimulates GS and glycogen production. In parallel, it disrupts the CBP/Torc2/CREB complex and consequently inhibits gluconeogenesis. Moreover, Akt activates mTOR, which facilitates protein synthesis, whereas mTORC2 is a critical regulator of Akt [339]. Another Akt regulator, tumor suppressor PTEN, previously mentioned in the context of mTOR, prevents Akt activation and reduces mTOR activity. In line with the above, the inhibition of IGF-1/PI3K/Akt signaling participates in the anti-cancer and DNA-repair activity of CR [340–342]. Further, Akt activation leads to the inhibitory phosphorylation of FOXO1, resulting in its nuclear exclusion [343]. Therefore, Akt functions at the crossroads of several pathways responding to CR. Among other pathways affected by insulin signaling, the most important include mitogen- activated protein kinase (MAPK), which regulates growth; SREBP-1, which promotes lipid and cholesterol synthesis; and the family of FoxO transcriptional regulators, which regulate metabolism and autophagy. In general, insulin signals an abundance of fuels and thus promotes storage and prevents the further production of energy molecules [344–347]. The beneficial effects of CR have been associated with changes in metabolism, modification of the activity of the insulin/IGF-1 pathway, reduction in fat mass, and increased stress resistance because of FoxO activation [348–350]. Insulin release and insulin action seem to play a major role in the control of aging. The modulation of longevity by insulin signaling is supported by the extended lifespan associated with mutations in the insulin/IRS/growth hormone (GH)/IGF-1/FOXO signaling pathways in humans, mice, C. elegans, and Drosophila [351–357]. Female, but not male, Igf1r+/− mice live on average 33% longer than their wild-type counterparts [355], and the fat-specific deletion of Igf1r results in an 18% increased longevity in both sexes [351]. 4.3. AMPK and PPARγ Thus, the role of TZDs in lowering serum insulin levels and in the protection of β-cells is mainly through AMPK [327]. In addition to mediating PPARγ metabolic functions, AMPK mediates the receptor’s anti- inflammatory activities. In bronchial epithelial cells, PPARγ plays a protective role in CSE-induced inflammation, as noted above (see the section on mTOR and PPARγ). CSE administration inactivates AMPK signaling, which is restored by PPARγ agonists. Consequently, the effects of PPARγ agonists on inflammation and also on autophagy can be abolished by AMPK inhibition [261], showing that AMPK is downstream of PPAR in this pathway. AMPK also mediates the anti-inflammatory effect of PPARγ in endothelial cells, in which the LPS-triggered downregulation of toll-like receptor 4 (TLR4) protein expression is inhibited by pioglitazone. LPS also reduces PPARγ expression, which can be partially restored by the knockdown of TLR4. Therefore, TLR4 and PPARγ affect each other via a negative feedback loop, and this interaction depends on the AMPK signaling pathway [326]. As discussed, agonists of PPAR exert physiological effects by modulating the activity of AMPK, which is an important cellular energy sensor. However, their action seems to be, at least in some instances, independent of the activation of the PPARs. In other words, these agonists can activate AMPK by phosphorylation independently of PPARγ or PPARα [316,317,325,328,337]. This idea is supported by a novel TZD, BLX-1002, with no PPAR affinity, which activates AMPK in β-cells and raises cytoplasmic Ca2+, thereby enhancing glucose-induced insulin secretion at a high glucose level [335]. Similarly, some agonists of PPAR likely exert some effects independently of PPAR, which is in cooperation with other cellular partners. AMPK also has been reported to feed back to PPARγ. The expression of either a constitutively active or dominant-negative AMPKα inhibits basal and rosiglitazone-stimulated PPARγ activity. AICAR and metformin inhibit PPRE reporter activity, whereas AMPK inhibitor compound C increases basal and rosiglitazone-stimulated PPARγ activity [315]. In brief, there is a very tight interaction between AMPK and PPARs (Figure 4), which involves the factors participating in the metabolic, apoptotic, and anti-inflammatory response to CR. Figure 4. The tissue-specific outcomes of the interaction between PPARs and adenosine monophosphate (AMP)-activated protein kinase (AMPK). PPARγ interacts with AMPK in multiple Figure 4. The tissue-specific outcomes of the interaction between PPARs and adenosine monophosphate (AMP)-activated protein kinase (AMPK). 4.3. AMPK and PPARγ PPARγ interacts with AMPK in multiple 13 of 75 Cells 2020, 9, 1708 tissues including blood vessels, WAT, pancreas, muscle, heart, and liver, leading to enhanced metabolism as well as reduced oxidative stress and inflammation. PPARα, in cooperation with AMPK, affects metabolism in the liver as well as reducing inflammation and apoptosis in blood vessels, whereas PPAR β/δ with AMPK affects muscle performance. 5. Insulin Signaling Accordingly, GH receptor/binding protein knockout (GHR/BP-KO) mice are characterized by a markedly extended lifespan and show severely reduced plasma IGF-1 and insulin levels, as well as low glucose levels [358,359]. Transgenic Klotho mice, which also have an increased lifespan, are insulin resistant. These findings collectively suggest that aging can be delayed by reducing insulin signaling [360]. It has even been hypothesized that insulin resistance is a physiological protective mechanism against aging and age-related disorders [361]. 5.1. Insulin Signaling and PPARα The immense impact of PPARα on glucose homeostasis and insulin signaling is particularly well illustrated by pancreas malfunction and diabetes models. PPARα directly protects pancreatic islets and their function and improves the adaptive response of the pancreas to pathological conditions. PPARα activation during the fed-to-fasted transition affects the regulation of glucose-stimulated insulin release because of the critical role of FA in insulin secretion [362]. In this condition, the activation of PPARα in β-cells increases pancreatic FA oxidation and potentiates glucose-induced insulin secretion [363,364]. In contrast, PPARα activation can oppose insulin hypersecretion elicited by high-fat feeding [365], suggesting that this activation protects pancreatic islets from lipotoxicity. 14 of 75 Cells 2020, 9, 1708 Similarly, in primary human pancreatic islets, PPARα agonist treatment prevents the FA-induced impairment of glucose-stimulated insulin secretion, apoptosis, and TG accumulation, indicating that PPARα mediates the adaptation of pancreatic β-cells to pathological conditions [366]. PPARα participates in a pathway mediating the effect of metformin on glucagon-like peptide-1 (GLP-1) receptor expression in pancreatic islets and on plasma levels of GLP-1 [367], improving glucose management. In addition, PPARα regulates hepatic glucose metabolism by upregulating glycerol-3- phosphate dehydrogenase, glycerol kinase, glycerol transport proteins [368], and pyruvate dehydrogenase kinase 4 during fasting [369], which leads to the promotion of gluconeogenesis over FA synthesis. In in vivo models of insulin resistance and diabetes, PPARα activation reverses the pregnancy- related augmentation of glucose-stimulated insulin hypersecretion by increasing insulin sensitivity [370]. Similarly, in nondiabetic patients with hypertriglyceridemia and patients with latent diabetes, the improvement in glucose metabolism observed during short-term clofibrate administration may also result from increased insulin sensitivity. Fasting plasma glucose, oral glucose tolerance test results, and immunoreactive insulin in these patients are significantly decreased, which is accompanied by enhanced glucose use and decreased serum TGs and cholesterol [371]. Furthermore, clofibrate in patients with non-insulin-dependent diabetes decreases fasting plasma glucose and insulin levels, and insulin binding to erythrocytes is enhanced because of increased insulin receptor affinity without a change in receptor number [372]. Another study showed that clofibrate ameliorates glucose tolerance in this patient population without changing the number of insulin receptors and that this increased insulin sensitivity occurs via an unknown post-receptor mechanism [373]. Strikingly, chronic fenofibrate treatment completely prevents the spontaneous sequential hypertrophy and atrophy of pancreatic islets from obese diabetes-prone Otsuka Long Evans Tokushima Fatty (OLETF) rats, decreases body weight and visceral fat, and improves insulin action in skeletal muscle [374]. 5.2. Insulin Signaling and PPARβ/δ PPARβ/δ cross-reacts with insulin signaling at several points. At first, PPARβ/δ senses elevated glucose levels. Glucose overload leads to cPLA2 activation and the subsequent hydrolysis of arachidonic and linoleic acid and their peroxidation, producing endogenous ligands of PPARβ/δ [384]. In the mouse pancreas, PPARβ/δ represses insulin secretion and the β-cell mass [385]. In adipocytes, it prevents IL-6–dependent STAT3 activation by repressing ERK1/2 and STAT3–Hsp90 association. This effect is thought to prevent cytokine-induced insulin resistance in these cells [386]. Similarly, PPARβ/δ represses IL-6-induced STAT3 activation and suppressor of cytokine signaling-3 (SOCS-3) upregulation in human liver cells and thereby halts the development of insulin resistance [387]. In skeletal muscle cells, PPARβ/δ attenuates ER stress-associated inflammation and prevents insulin resistance in an AMPK-dependent manner [387,388]. Moreover, PPARβ/δ ameliorates hyperglycemia by increasing glucose flux through the pentose phosphate pathway, which enhances FA synthesis. Coupling PPARβ/δ-dependent increased hepatic carbohydrate catabolism and the promotion of β-oxidation in muscle allows PPARβ/δ to regulate metabolic homeostasis and enhance insulin action by complementary effects in distinct tissues [389]. In a primate model of metabolic syndrome, GW501516, an agonist of PPARβ/δ, dose-dependently lowers plasma insulin levels without side effects on glycemic control [390]. GW501516 treatment also markedly improves diabetes by decreasing blood glucose and insulin levels in ob/ob mice [391]. In addition, the treatment of healthy people who are moderately overweight with GW501516 results in a significant reduction in fasting plasma insulin [392], and the dual PPARα/δ agonist GFT505 (elafibranor) improves hepatic and peripheral insulin sensitivity in men with abdominal obesity [393]. 5.1. Insulin Signaling and PPARα Along the same line of observations, fenofibrate treatment significantly reduces hyperinsulinemia and hyperglycemia in C57BL/6 mice with insulin resistance triggered by a high-fat diet and in a model of genetic insulin resistance (obese Zucker rats) [375]. Similarly, the treatment of db/db diabetic mice with PPARα agonists significantly reduces plasma insulin and insulin resistance, improves hyperglycemia, albuminuria, and kidney glomerular lesions, and causes a 50% reduction in FA oxidation, with a concomitant increase in glycolysis and glucose oxidation [376,377]. PPARα-deficient ob/ob mice with obesity-related insulin resistance develop pancreatic β- cell dysfunction characterized by reduced mean islet surface area and decreased insulin secretion in response to high glucose [366]. Similarly, PPARα KO mice develop marked age-dependent hyperglycemia [366], and after 24-h fasting, severe hypoglycemia accompanied by elevated plasma insulin concentrations [54,378]. However, PPARα KO mice are protected from high-fat diet-induced insulin resistance, which is most likely because of the development of increased adiposity [379]. Of note, PPARα gene variation in humans can affect the age of onset and progression of T2D in patients with impaired glucose tolerance [51,52]. In the liver, the insulin-stimulated activation of Akt induces the phosphorylation of NCoR1 on serine 1460, which selectively favors its interaction with PPARα. Phosphorylated NCoR1 inhibits the activity of PPARα, attenuating oxidative metabolism, whereas it derepresses liver X receptor α (LXRα), resulting in increased lipogenesis [380]. Glucose levels also affect PPARα activity. The exposure of islets or INS(832/13) β-cells for several days to supraphysiological glucose concentrations, which are detrimental to insulin secretion, leads to a 60–80% reduction in PPARα mRNA expression, DNA-binding activity, and target gene expression, which results in diminished FA oxidation and increased TG accumulation that are potentially associated with pancreatic lipotoxicity [381]. Moreover, insulin-activated MAPK and glucose-activated PKC stimulate PPARα transcriptional activity in HepG2 cells [382]. Strikingly, glucose itself can modulate PPARα activity because PPARα binds glucose and glucose metabolites with high affinity, prompting changes in its secondary structure [383]. Overall, based on the effects of PPARα on glucose homeostasis and its important regulatory role in the transition from feeding to fasting, PPARα might be involved in protecting against hypoglycemia during CR. 15 of 75 15 of 75 Cells 2020, 9, 1708 5.3. Insulin Signaling and PPARγ The increased adiponectin levels improve insulin sensitivity and free FA oxidation and reduce glucose production in the liver [413,414]. The signaling of PPARγ involves the previously mentioned executor of insulin signaling, FOXO. FOXO1 acts as a transcriptional repressor of Pparγ by binding to its promoter and may reduce PPARγ transcriptional activity through a transrepression mechanism involving direct protein–protein interaction between FOXO1 and PPARγ. This interaction seems to be a crucial part of the pathway responsible for insulin sensitivity in adipocytes [415–417]. Moreover, insulin signaling in the liver directly affects PPARγ, as Akt2 stimulates the expression and activity of PPARγ in hepatocytes, resulting in elevated aerobic glycolysis and lipogenesis [260]. p γ g p g p g p An important contributor to the insulin-sensitizing effect of PPARγ ligands is the suppression of local and systemic cytokine production. TZD as treatment for patients with obesity and without diabetes reduces circulating levels of inflammatory cytokines and other pro-inflammatory markers, which are accompanied by improved insulin sensitivity [409]. Moreover, hepatic PPARγ reduces the expression of SOCS-3, which has been suggested to play a crucial role in linking inflammation and hepatic insulin resistance [399]. SOCS-3 promotes the ubiquitination and degradation of IRS-2 and thus modulates insulin signaling [410,411]. In vitro studies have confirmed that PPARγ agonists may also exert their antidiabetic activities by counteracting the negative effects of TNFα [412]. In addition, PPARγ elevates blood levels of adipocytokines, such as adiponectin, which are present at low concentrations in the plasma of patients with T2D. The increased adiponectin levels improve insulin sensitivity and free FA oxidation and reduce glucose production in the liver [413,414]. The signaling of PPARγ involves the previously mentioned executor of insulin signaling, FOXO. FOXO1 acts as a transcriptional repressor of Pparγ by binding to its promoter and may reduce PPARγ transcriptional activity through a transrepression mechanism involving direct protein–protein interaction between FOXO1 and PPARγ. This interaction seems to be a crucial part of the pathway responsible for insulin sensitivity in adipocytes [415–417]. Moreover, insulin signaling in the liver directly affects PPARγ, as Akt2 stimulates the expression and activity of PPARγ in hepatocytes, resulting in elevated aerobic glycolysis and lipogenesis [260]. As a result of this effect on regulatory pathways, TZDs improve insulin sensitivity, glucose tolerance, and the lipidemic profile in T2D as well as in obesity without diabetes [418]. 5.3. Insulin Signaling and PPARγ PPARγ is an established regulator of insulin sensitivity, making it an excellent drug target (Figure 5). TZDs form a class of PPARγ agonists that reverse insulin resistance in liver and peripheral tissues, reducing plasma glucose through specific PPARγ activation. Troglitazone was the first TZD approved for this use, but it was withdrawn from the market following reports of serious hepatotoxicity in some patients. TZDs not only improve insulin sensitivity but also preserve pancreatic β-cell function, thus reducing T2D incidence, as demonstrated in clinical trials of T2D prevention in high-risk people [394,395]. Figure 5. Pathways in which PPARγ activity leads to increased insulin sensitivity. PPARγ affects insulin sensitivity by managing glucose uptake and disposal, enhancing insulin signaling, and maintaining functioning WAT and pancreas. Figure 5. Pathways in which PPARγ activity leads to increased insulin sensitivity. PPARγ affects insulin sensitivity by managing glucose uptake and disposal, enhancing insulin signaling, and maintaining functioning WAT and pancreas. Cells 2020, 9, 1708 16 of 75 PPARγ exerts its insulin-sensitizing properties in several ways. First, it generates functional WAT, which is required for proper glucose homeostasis because lipodystrophy is associated with severe insulin resistance [396]. An early consequence of PPARγ activation that precedes decreased blood TG and glucose is the stimulation of TG production and a reduction in circulating free FA because of FA retention in fat rather than muscle and pancreas. Consequently, increased fat mass triggered by PPARγ activation results in improved glycemic control [397]. Accordingly, the level of insulin sensitization following PPARγ activation is correlated with the reduction in lipid accumulation in skeletal muscle [398]. Furthermore, in mice fed a high-cholesterol/fructose diet, the selective PPARγ agonist pioglitazone improves insulin sensitivity by affecting its signaling pathway, as measured by induction of IRS-2 expression and increased phosphorylation of Akt and GSK-3β [399]. In fact, PPARγ induces the expression of several proteins in the insulin-signaling pathway, including IRS-1 [400], IRS-2 [401], the p85 subunit of PI3K [402], and Cbl-associated protein (CAP) [403,404]. In 3T3-L1 adipocytes and diabetic rodents, PPARγ directly binds the promoter of the Cap gene. Increased CAP expression results in increased insulin-stimulated c-Cbl phosphorylation [403] and consequently in increased glucose uptake [405]. The activation of PPARγ in muscle cells and adipocytes increases the expression and translocation of GLUT1, GLUT2, and GLUT4 to the cell membrane, thus increasing glucose uptake and consequently reducing glucose plasma levels [406– 408]. 5.3. Insulin Signaling and PPARγ In parallel, PPARγ regulates the expression of genes responsible for glucose disposal [400–404]. PPARγ exerts its insulin-sensitizing properties in several ways. First, it generates functional WAT, which is required for proper glucose homeostasis because lipodystrophy is associated with severe insulin resistance [396]. An early consequence of PPARγ activation that precedes decreased blood TG and glucose is the stimulation of TG production and a reduction in circulating free FA because of FA retention in fat rather than muscle and pancreas. Consequently, increased fat mass triggered by PPARγ activation results in improved glycemic control [397]. Accordingly, the level of insulin sensitization following PPARγ activation is correlated with the reduction in lipid accumulation in skeletal muscle [398]. Furthermore, in mice fed a high-cholesterol/fructose diet, the selective PPARγ agonist pioglitazone improves insulin sensitivity by affecting its signaling pathway, as measured by induction of IRS-2 expression and increased phosphorylation of Akt and GSK-3β [399]. In fact, PPARγ induces the expression of several proteins in the insulin-signaling pathway, including IRS-1 [400], IRS-2 [401], the p85 subunit of PI3K [402], and Cbl-associated protein (CAP) [403,404]. In 3T3-L1 adipocytes and diabetic rodents, PPARγ directly binds the promoter of the Cap gene. Increased CAP expression results in increased insulin-stimulated c-Cbl phosphorylation [403] and consequently in increased glucose uptake [405]. The activation of PPARγ in muscle cells and adipocytes increases the expression and translocation of GLUT1, GLUT2, and GLUT4 to the cell membrane, thus increasing glucose uptake and consequently reducing glucose plasma levels [406– 408]. In parallel, PPARγ regulates the expression of genes responsible for glucose disposal [400–404]. An important contributor to the insulin-sensitizing effect of PPARγ ligands is the suppression of local and systemic cytokine production. TZD as treatment for patients with obesity and without diabetes reduces circulating levels of inflammatory cytokines and other pro-inflammatory markers, which are accompanied by improved insulin sensitivity [409]. Moreover, hepatic PPARγ reduces the expression of SOCS-3, which has been suggested to play a crucial role in linking inflammation and hepatic insulin resistance [399]. SOCS-3 promotes the ubiquitination and degradation of IRS-2 and thus modulates insulin signaling [410,411]. In vitro studies have confirmed that PPARγ agonists may also exert their antidiabetic activities by counteracting the negative effects of TNFα [412]. In addition, PPARγ elevates blood levels of adipocytokines, such as adiponectin, which are present at low concentrations in the plasma of patients with T2D. 5.3. Insulin Signaling and PPARγ Dominant- negative mutations in human PPARγ can lead to severe metabolic syndrome, insulin resistance, and diabetes at an unusually young age [419,420], and several point mutations in the PPARγ gene are associated with severe insulin resistance (with or without T2D) and familial partial lipodystrophy phenotypes [421–425]. Both partial and generalized lipodystrophies have consistently been associated with insulin resistance in animals and humans [426]. Therefore, it is likely that the dramatic reduction in limb and gluteal fat found in subjects with PPARγ mutations contributes to their insulin resistance. In addition, the residual adipose tissue in these individuals is dysfunctional, likely resulting in unregulated FA fluxes and impairing insulin action in skeletal muscle and liver [420]. Of interest, lipodystrophic, WAT-specific PPARγ KO mice show an increased expression of PPARγ in the liver, which promotes insulin sensitivity [427,428]. In this context, it is important to note that insulin sensitivity declines with age in humans and is accompanied by a lower expression of PPARγ in preadipocytes [429]. Hence, FA metabolism becomes altered with aging in preadipocytes, which correlates with increased susceptibility to lipotoxicity and impaired FA- induced adipogenesis. In line with these observations, PPARγ, PPARα, and RXR levels are all 17 of 75 Cells 2020, 9, 1708 increased in the liver of GHR-KO long-lived animals [131]. Thus, the enhanced insulin sensitivity in GHR-KO mice may be the result of the increased hepatic activity of PPAR family members. y p y y In addition to TDZs, several other PPARγ agonists influence insulin and glucose management. FMOC-L-Leucine (F-L-Leu) is a partial agonist that selectively activates some PPARγ pathways. F-L- Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant mice and in diabetic db/db mice, yet it has a lower adipogenic activity [430]. Of interest, INT131 besylate, which is a potent non- TZD-selective PPARγ modulator, induces a dose-dependent reduction in fasting plasma glucose without evoking fluid retention or weight gain, which are both unwanted side effects often triggered by TZDs [431]. In addition, food-derived active compounds may contribute to the management of glucose levels. The plant polyphenols quercetin and kaempferol serve as weak partial agonists of PPARγ and increase insulin sensitivity and glucose uptake via PPARγ agonism [432,433]. Another compound, 13-oxo-9(Z),11(E),15(Z)-octadecatrienoic acid (13-oxo-OTA), a linolenic acid derivative in the extracts of tomato (Solanum lycopersicum), Mandarin orange (Citrus reticulata), and bitter gourd (Momordica charantia), modulates gene expression and the production of adiponectin through PPARγ in adipocytes [434]. 5.3. Insulin Signaling and PPARγ The reduction of PPARγ activity by antagonists improves the metabolic profile in mice [435,436], and haplodeficient Pparγ+/− mice exhibit increased insulin sensitivity compared with their wild-type littermates [437,438]. These animals are characterized by reduced fat deposits and lower levels of TG accumulation and lipogenesis in WAT, skeletal muscle, and liver [439]. Similarly, genetic variants Pro(12)Ala (heterozygotes) and Ala(12)Ala (homozygotes) of PPARγ, which result in decreased receptor activity, are associated with leanness and improved insulin sensitivity [440–442]. A complex U-shaped curve has been proposed to characterize the relationship between PPARγ activity and insulin sensitivity [99]. Altogether, overwhelming evidence points to an important role for all three PPARs in insulin signaling and glucose level management, and to several compounds with similar potential, including some that block the endogenous ligand-induced activation of PPARγ for the treatment of the metabolic syndrome and T2D [436,443,444]. 6. Sirtuins As already mentioned, a CR-related decrease in energy levels leads to the activation of several signaling cascades. Decreased glucose intake reduces the flow of carbon through the glycolytic pathway and the regeneration of ATP from ADP, which eventually alters the NAD+:NADH ratio. This shift activates SIRTs, which serve as both energy sensors and transcriptional effectors by acting as NAD+-dependent HDACs. In addition to CR and fasting, exercise activates SIRTs [445,446], which are remarkably conserved and can even be found in archaebacteria [447]. Originally categorized as class III HDACs, SIRTs are involved in the proper functioning of nucleic acids including DNA repair, homologous recombination, and DNA deacetylation, and they promote transcriptional gene silencing [448,449]. The seven subtypes of SIRTs (SIRT1–7) in mice and humans vary in their cellular distribution and function. SIRT1–SIRT3, SIRT5, and SIRT6 catalyze deacetylation, whereas SIRT4 and SIRT6 have ADP-ribosylation capacity. In addition to histones, SIRT substrates include several transcriptional regulators, such as the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), p53, FOXO, and PGC-1α, but also enzymes, including acetyl coenzyme A synthetase 2 (AceCS2), long- chain acyl-coenzyme A dehydrogenase (LCAD), HMGCS2, superoxide dismutase 2, and structural proteins, such as α-tubulin [450–454]. Therefore, SIRTs influence a wide range of cellular processes including circadian clocks, cell cycle, mitochondrial biogenesis, and energy homeostasis, and on the whole-body level regulate aging, apoptosis, inflammation, and stress resistance [455,456]. SIRT1 is the most thoroughly investigated mammalian SIRT and is closely involved in metabolism. Studies in S. cerevisiae have shown that an extra copy of the Sir2 gene, a yeast homolog of mammalian Sirt1, increases lifespan in a dose-dependent manner [457,458], and the deletion of this gene shortens lifespan [457]. In yeast and Drosophila, a lack of Sir2 and dSir2, respectively, prevents CR-associated life extension [459–461]. SIR2, a yeast analog of Sirt1, assists in DNA repair and regulates genes that change expression with age [462]. Cells 2020, 9, 1708 18 of 75 The most important metabolic regulator affected by SIRT1 is PGC-1α, which is activated by SIRT1-mediated deacetylation [463,464]. Deacetylated PGC-1α increases hepatic gluconeogenic activity [463], whereas in muscle and BAT, PGC-1α enhances mitochondrial activity. The activity of PGC-1α translates into increased exercise capacity and thermogenesis, leading to protection against the onset of obesity and associated metabolic dysfunction [465]. 6. Sirtuins The deacetylation of PGC-1α by SIRT1 depends on cellular NAD+ levels, so the status of cellular energy affects PGC-1α activity, which adapts cellular energy production through mitochondrial biogenesis and function. Furthermore, among the SIRT1 substrates are factors that control cell proliferation and apoptosis, including the tumor suppressor protein p53 [466]. The overexpression of SIRT1 hinders p53 transcriptional activity and p53-dependent apoptosis triggered by DNA damage and oxidative stress, whereas the overexpression of dominant-negative SIRT1 can enhance cellular stress responses [466,467]. SIRTs also regulate the activity of the FOXO family of transcription factors [468,469], which affects cell differentiation, transformation, and metabolism as well as plays an important role in CR and longevity regulation [470–472]. SIRT1-mediated deacetylation of FOXO1 affects its shuttling between the nucleus and cytoplasm, influencing the expression of FOXO1 target genes and promoting gluconeogenesis and glucose release from hepatocytes [473]. The deacetylation of FOXO3a by SIRT1 increases its translocation from the cytoplasm to the nucleus [474] and its DNA- binding activity. In the nucleus, SIRT1 and FOXO3a form a complex that induces cell-cycle arrest and resistance to oxidative stress, also inhibiting the ability of FOXO3a to induce apoptosis [473]. SIRT1 directly suppresses the expression of uncoupling protein 2 (UCP2), leading to an improved coupling of mitochondrial respiration and ATP synthesis, which induces insulin secretion in β-cells [475]. Confirming the role of SIRT1 in the pancreas, SIRT1−/− mice are characterized by impaired insulin secretion in response to glucose compared with wild-type littermates [475,476]. Conversely, β-cell- specific SIRT1-overexpressing mice exhibit improved glucose tolerance and an enhanced glucose- stimulated insulin secretion [476]. In contrast, SIRT4 has an inhibitory effect on amino acid-stimulated insulin secretion. It represses the activity of glutamate dehydrogenase, reducing α-ketoglutarate production and ATP generation, which are known to activate insulin secretion in pancreatic β-cells [477]. To avoid amino acid-stimulated insulin secretion during CR, when amino acid turnover increases, CR decreases SIRT4 activity, which is opposite to the induction of SIRT1 activity during CR [477]. Considering that NAD+ controls the activities of both SIRT4 and SIRT1, their opposing effects on insulin secretion are surprising, and the full implications remain to be understood. The role of other SIRT family members has been less investigated; thus, their function is less well known. SIRT2 is localized mainly in the cytoplasm, where it deacetylates tubulin filaments, HOXA10, and FOXO [478–481]. 6. Sirtuins It takes part in multiple processes including cell cycle regulation [482], lifespan extension [457,483], and glucose and lipid metabolism [451,484]. SIRT3 plays an important role in mitochondria maintenance by acting as a deacetylase for a number of mitochondrial matrix proteins [485,486]. During a prolonged fast, SIRT3 activates FA breakdown by the deacetylation of LCAD [453] and stimulates the production of ketone bodies by activating HMGCS2 [452]. Of note, SIRT3 is genetically linked to lifespan in the elderly [487]. SIRT4 has ADP-ribosylation activity and in addition to blocking amino acid-induced insulin secretion [477], it regulates FA oxidation in hepatocytes and myocytes [488]. Both SIRT4 and SIRT5 show mitochondrial localization [477,489]. SIRT6 resides in the nucleus and is involved in genomic DNA stability and promotes the repair of DNA double-strand breaks [490]. SIRT6-deficient mice present a shortened lifespan and a degenerative aging-like phenotype [491]. In contrast, transgenic male mice overexpressing SIRT6 display lower serum levels of IGF-1, higher levels of IGF-1-binding protein, and modified phosphorylation patterns of different components of the IGF-1 signaling pathway, possibly contributing to about a 15% increase in lifespan when compared to wild-type animals [492]. SIRT1 and SIRT6 are both connected with CR-triggered extension of ovarian lifespan, which is mediated by the inhibition of the transition from primordial to developing follicles and by a delay in the growth phase of follicles to preserve the supply of germ cells [493]. SIRT7 is associated with nucleoli and is implicated in the activation of transcription by RNA polymerase I [494] as well as the 19 of 75 Cells 2020, 9, 1708 repair of double-strand breaks by non-homologous end-joining [495]. SIRT7 knockout mice display features of premature aging [495]. SIRT1, SIRT6, and SIRT7 facilitate DNA repair, and this repair slows the aging process. During CR, except for SIRT4, the expression and activity of SIRTs are increased in many tissues, including adipose and brain [496–498], heart [499,500], and liver [501]. SIRT1 mediates a broad array of physiological effects of CR. The overexpression of SIRT in worms and flies increases their lifespan [460,461], and accordingly, mutants of SIRT do not show lifespan extension by CR [459,502]. Moreover, transgenic mice overexpressing SIRT1 show phenotypes similar to those of CR mice [503]. The previously mentioned role of yeast Sir2 in lifespan is particularly critical in the context of CR. 6. Sirtuins Resveratrol, a polyphenolic compound present in, for example, red grapes and wine, stimulates SIRT1 expression, resulting in extended lifespan and health span in treated animals [504]. SIRT1 activation by resveratrol mimics CR and delays aging in a wide range of organisms, from S. cerevisiae [505] to C. elegans to Drosophila [506] and mice [507]. Resveratrol is considered one of the mimetics not only of CR but also of exercise [504,508]. In mice, resveratrol inhibits gene expression profiles associated with muscle aging and age-related cardiac dysfunction [509]. The compound protects mice against diet-induced obesity and the associated insulin resistance through enhanced mitochondrial function mediated by PGC-1α [465]. 6.3. SIRT1 and PPARγ The interaction between PPARγ and SIRT1 is twofold (Figure 6). PPARγ inhibits SIRT1 expression by binding to the Sirt1 promoter, and PPARγ also directly interacts with and inhibits SIRT1 activity, forming a negative feedback loop [522]. Pioglitazone prevents NF-κB activation through a reduction in p65 acetylation via the AMPK-SIRT1/p300 pathway [523], whereas SIRT1 represses PPARγ actively via docking with two of its cofactors, NcoR and SMRT [524]. Conversely, the treatment of 3T3-L1 adipocytes with resveratrol represses the expression of PPARγ target genes as well as of PPARγ itself. Furthermore, this treatment increases targeting of the PPARγ protein to the ubiquitin–proteasome system for degradation [525]. Hence, SIRT1 acts as a corepressor of PPARγ- mediated transcription. From a functional point of view, the repression of PPARγ by SIRT1 counters adipogenesis, and the upregulation of SIRT1 triggers lipolysis and the release of fat from differentiated adipocytes [22,524]. Following food withdrawal, SIRT1 promotes fat mobilization by repressing PPARγ, which reduces the expression of genes mediating fat storage [524]. In line with these observations, SIRT1+/− mice show a compromised mobilization of FAs from adipose tissue during fasting [524]. 6.2. SIRT and PPARβ/δ PPARβ/δ markedly increases the transcription [516] and protein levels of SIRT1 [517], whereas PPARα and PPARγ do not stimulate SIRT1 expression [516]. Moreover, PPARα and PPARβ/δ promote osteogenic differentiation in an SIRT1-dependent manner [518,519], and PPARγ prevents it [520]. Based on the PPARβ/δ–SIRT1 interaction, a reasonable inference is that during starvation, increased levels of lipolysis-derived free FAs activate PPARβ/δ. This activation leads to enhanced SIRT1 expression, promoting the deacetylation of factors involved in mitochondrial beta-oxidation and cell survival [516]. The regulation of SIRT1 and PPARβ/δ activity operates bidirectionally. First, in human HaCaT keratinocytes, GW501516 modulates inflammation by acting via AMPK and SIRT1 to reduce TNFα-induced IL-8 mRNA levels and NF-κB DNA-binding activity [517]. Second, the upregulation of SIRT1 by PPARβ/δ attenuates premature senescence in angiotensin (Ang) II-treated human coronary artery endothelial cells. Resveratrol can mimic the action of PPARβ/δ on Ang II- induced premature senescence and reactive oxygen species (ROS) generation [521]. 6.1. SIRT and PPARα During fasting, SIRT4 levels decrease in the liver and SIRT4-null mice display an increased expression of hepatic PPARα target genes associated with FA catabolism [510], indicating that PPARα is a negative downstream target of SIRT4. In contrast, the hepatocyte-specific deletion of SIRT1 impairs PPARα signaling and decreases FA β-oxidation, whereas the overexpression of SIRT1 induces the expression of PPARα targets (Figure 6). In fact, SIRT1 interacts with PPARα and is required to activate PGC-1α by deacetylation. Of note, SIRT1-deacetylated PGC-1α can function as a coactivator in PPARα complexes controlling the expression of several metabolic genes. Therefore, SIRT1 activates PPARα to promote FA oxidation in the liver [511]. Similarly, in the heart, PPARα and SIRT1 modulate FA metabolism [512]. Both PPARα and SIRT1 are upregulated by pressure overload in the heart. The haploinsufficiency of either PPARα or SIRT1 reduces pressure overload-induced cardiac hypertrophy and failure, whereas the simultaneous induction of PPARα and SIRT1 aggravates cardiac dysfunction. PPARα and SIRT1 jointly suppress genes involved in mitochondrial functions that are controlled by the estrogen-related receptors (ERRs). PPARα binds and recruits SIRT1 to the ERR response element. In doing so, it represses ERR target genes in an RXR-independent manner. Suppression of the ERR transcriptional pathway by PPARα/SIRT1 also is a physiological response to fasting [513–515]. Figure 6. The interaction between sirtuin 1 (SIRT1) and PPARs. Caloric restriction (CR)-triggered energy shortage leads to the activation of SIRT1 and its interaction with PPARs. Each of these interactions results in a distinct outcome. Figure 6. The interaction between sirtuin 1 (SIRT1) and PPARs. Caloric restriction (CR)-triggered energy shortage leads to the activation of SIRT1 and its interaction with PPARs. Each of these interactions results in a distinct outcome. 20 of 75 20 of 75 Cells 2020, 9, 1708 7.1. Oxidative Stress Reduction WY-14643 and fenofibrate protect mice from acetaminophen-induced hepatotoxicity by upregulating UCP-2, which is a PPARα target gene that reduces the generation of mitochondrial ROS [540]. In a gentamicin- induced model of ROS production, different types of PPARα and PPARγ agonists (fenofibrate, pioglitazone, tesaglitazar) provide protection from toxicity. These ligands prevent oxidative stress by increasing the expression of genes controlling ROS production and detoxification (SOD1, glutathione peroxidase 1 (GPx1), CAT, UCP-2), which will restore the ratio of reduced to oxidized glutathione and prevent apoptosis [541]. PPARγ directly modulates the expression of several antioxidant and pro-oxidant enzymes as well as oxidative stress-related proteins. It transcriptionally regulates mouse, rat, and human catalase, which is a major antioxidant enzyme converting H2O2 to O2 and H2O [542,543]. Similarly, it directly regulates the expression of manganese superoxide dismutase (MnSOD), which performs the dismutation of O2- to O2 and H2O. Conversely, heart-specific PPARγ knockout mice show downregulated levels of MnSOD in cardiac muscle with a consequent increase in O2- levels, suggesting that PPARγ protects cardiomyocytes from oxidative damage [544]. In human skeletal muscle cells, the TZD-mediated activation of PPARγ induces GPx3 and protects against oxidative stress [545] because GPx reduces H2O2 to H2O and O2 and scavenges for oxidized lipids. PPARγ also represses the expression of inducible NO synthase (iNOS) and stimulates eNOS [546–550]. These enzymes produce NO from arginine, which forms highly reactive peroxynitrite when it reacts with O2-. In mice with an endothelial-specific knockout of PPARγ, aortic segments release less NO than those from controls, and this reduced expression correlates with an increase in oxidative stress parameters [548]. Cyclooxygenase-2 (COX-2) is an inducible form of cyclooxygenase that contributes to the metabolism of arachidonic acid-forming prostaglandin H2 [551,552], which requires the presence of free radicals and may produce O2-, contributing to oxidative stress. PPARγ regulates COX-2 expression, but both induction [553,554] and reduction [555,556] in PPARγ expression have been reported, leaving the issue for further investigation. In rats, the activation of PPARγ by oral intake of rosiglitazone upregulates UCP-2 [557], which protects against oxidative stress by preventing O2- accumulation in the mitochondria and facilitating the export of mitochondrial ROS to the cytosol [558]. Moreover, a major target gene of PPARγ, CD36, may act as a scavenger receptor that mediates the recognition and internalization of oxidized lipids [559–561]. 7.1. Oxidative Stress Reduction ROS are generated as a by-product of cellular respiration, contributing to the accumulation of oxidative damage and the formation of a range of oxidation products of different macromolecules including lipids, proteins, and nucleic acids [526]. A small amount of ROS is normally beneficial because it plays an important role in cellular processes such as cell cycle progression, the regulation of signaling pathways in response to intra- and extracellular stimuli, and inflammation [527]. However, high uncontrolled levels of ROS are detrimental. During oxidative stress, the sustained production of ROS and reactive nitrogen species leads to a perturbed equilibrium between pro-oxidants and antioxidants. Consequently, macromolecules, organelles, and cells are altered, and if much damage accumulates, necrotic or apoptotic cell death occurs. The “free radical theory” of aging [528] proposes that the generation of oxidative stress is a major factor contributing to the onset of the aging process and age-related diseases. Therefore, the mammalian lifespan is reduced in relation to the mitochondrial production of oxidizing free radicals [527]. CR likely exerts its diverse benefits through reducing ROS levels and suppressing age-related oxidative stress while supporting the antioxidant defense system [529–531]. CR diminishes the impact of ROS through three processes: reduction of oxygen free-radical generation by slowing metabolism, the acceleration of ROS neutralization, and stimulation of the repair of ROS-damaged molecules [532–536]. Cells 2020, 9, 1708 21 of 75 The oxidative stress-related role of PPARs is first suggested by their name: they were first identified as receptors stimulating peroxisome proliferation. Peroxisomes have oxidative functions that involve use of molecular oxygen and that yield hydrogen peroxide (H2O2). The name of these organelles comes from their hydrogen peroxide-generating and scavenging activities. In addition to the conversion of ROS, peroxisomes play a key role in metabolism, catabolizing very long-chain FAs, branched-chain FAs, bile acid intermediates (in the liver), D-amino acids, and polyamines. The induction of oxidative stress is associated with the downregulation of PPARs, which also occurs during aging [140,537,538]. The reduced expression of PPARα in aging [137,539] has been attributed to increased oxidative stress, and CR has been suggested to prevent this decrease through antioxidative action [140]. PPARα-deficient mice present increased oxidative stress at an earlier age than aged-matched wild-type controls [137]. In fact, the administration PPARα agonists to aged mice restores the cellular redox balance, documented by reduced tissue lipid peroxidation, reduced spontaneous inflammatory cytokine production, and the elimination of constitutively active NF-κB [137]. 7.2. Mitochondrial Function One of the several theories tightly connected with the effects of ROS is the “mitochondrial theory of aging”, which proposes that mitochondria are the critical component in the aging process. In fact, mitochondrial DNA damage and dysfunction increase with aging and are associated with a vast number of pathologies. Defective mitochondria determine the turnover not only of the organelles themselves but also whole cells, resulting in the acceleration of aging [527,582,583]. Aging has been linked to a reduced capacity for oxidative phosphorylation in the muscle and heart, most likely because of a decline in mitochondrial content and/or function [584–586]. Accordingly, young individuals have higher respiratory function compared to the elderly [587–589]. Disturbed mitochondrial electron transfer increases the likelihood of electron leakage and ROS production. Consequently, components of the electron transport chain and mitochondrial DNA become damaged, leading to further increases in intracellular ROS levels and a decline in mitochondrial function. Since mitochondrial DNA is spatially close to the source of ROS production, it is thought to be particularly vulnerable to ROS-mediated lesions [528,590]. An interesting feature of CR, one associated with ROS and changes in metabolism, is mitochondria biogenesis, which is relatively high in various tissues such as in the brain, heart, liver, and particularly the BAT of mice [498,591]. It is associated with activation of the master regulator of mitochondrial biogenesis, PGC-1α [428,592,593]. PGC-1α is expressed at a high level in BAT, heart, skeletal muscle, brain, and kidney, whereas its expression is low in the liver and very low in WAT [594]. Various physiological stimuli highly induce PGC-1α in different organs. It is increased in BAT by cold exposure and in skeletal muscle by exercise and decreased ATP level, whereas in the liver, it is mostly affected by CR [595]. When ectopically expressed in fat or muscle cells, PGC-1α strongly increases mitochondrial biogenesis and oxidative metabolism, which correlates with an increase in mitochondrial DNA and the expression of multiple mitochondrial genes [595,596]. To prevent a mitochondrial biogenesis-associated increase in ROS levels, PGC-1α also induces expression of the antioxidant genes GPx1 and MnSOD [597]. One hypothesis regarding the beneficial outcomes of CR proposes is that CR preserves mitochondrial function by maintaining protein and DNA integrity through decreasing mitochondrial oxidant emission and increasing endogenous antioxidant activity [598,599]. Its impact on mitochondria biogenesis remains a matter of discussion [600,601]. In addition to affecting mitochondria biogenesis, PGC-1α also influences metabolism. 7.1. Oxidative Stress Reduction Finally, PPARγ also has been shown to protect cardiomyocytes and glial cells from oxidative stress-induced apoptosis by increasing Bcl-2 [562,563]. In addition to direct transcriptional regulation, PPARγ can modulate the inflammatory and oxidative status by acting on transcription factors such as NF-κB [547,550,564,565]. NF-κB action is usually pro-inflammatory and pro-oxidant, inducing the expression of genes encoding the inflammatory cytokines sIL-1β, IL-6, and TNFα, as well as the pro-inflammatory enzymes COX-2 and iNOS, but it may also regulate the expression of superoxide dismutases and other anti-inflammatory genes [552,566–570]. PPARγ reduces NF-κB activities in various ways: (1) by transrepressing NF-κB Cells 2020, 9, 1708 22 of 75 activation through forming a repressor complex in the promoter of NF-κB-target genes; (2) by directly binding with NF-κB [547,550,564]; or (3) by catalase-mediated H2O2 reduction, which activates NF- κB [542,543,571]. Conversely, NF-κB negatively regulates PPARγ transcriptional activity via a mechanism that requires the presence of HDAC3 [572,573]. Of note, PPARγ interacts with a major regulator of the antioxidative response, the nuclear factor erythroid 2-related factor 2 (NRF2). NRF2 is a redox-sensitive transcription regulator that plays a vital role in cryoprotection against oxidative and electrophilic stress as well as in inflammation suppression [574]. NRF2 targets multiple genes, including NADPH-generating enzymes [575], glutathione S-transferases [576], CD36 [560,577], and HO-1 [578,579] and it stimulates the production of defense proteins during oxidative stress. NRF2 also induces PPARγ expression by binding the upstream promoter region of the nuclear receptor [580,581]. Conversely, PPREs have been identified on the NRF2 gene promoter [576,581], confirming a positive feedback loop between PPARγ and NRF2. Therefore, the ability of PPARs to extinguish oxidative stress overlaps with CR effects. 7.2. Mitochondrial Function It mediates a fasting-induced increase in FA metabolism and the downregulation of pyruvate dehydrogenase, which is part of the mitochondrial pyruvate dehydrogenase complex that catalyzes the reaction representing pyruvate entry into the tricarboxylic acid cycle. In PGC-1α knockout mice, pyruvate dehydrogenase fails to adapt to CR, and the ability of the mice to endure prolonged starvation is decreased [602]. PGC-1α knockout mice also show a reduced content of mitochondrial electron transport chain proteins in skeletal muscle [603,604]. The activity of PGC-1α is directly regulated by the energy sensors SIRT1 and AMPK [276,463]. Functionally, the transcriptional activity of PGC-1α relies on its interactions with transcriptional factors for controlling FA metabolism. Of 23 of 75 Cells 2020, 9, 1708 note, all three PPAR isotypes are subject to transcriptional coactivation by PGC-1α and are major executors of PGC-1α-induced regulation [72,594,605,606]. Evidence has accumulated for an important role of PPARs in maintaining healthy mitochondria. Agonists of PPARα and PPARγ modulate mitochondrial fusion and fission in neurons, leading to a better response to oxidative stress and neuron protection [607]. The abnormal expression of PPARα is linked to an altered mitochondrial structure and metabolic function, with an increase in number of cristae, and myocardial damage and fibrosis in PPARα knockout mice [608]. Through its key role in FA β-oxidation, PPARα is inevitably associated with mitochondrial function [35,609]. The activation of PPARα rescues mitochondrial depletion and failure to oxidize FA in the liver-specific class 3 PI3K- deficient mice. In this model, PPARα stimulates mitochondrial biogenesis and lipid oxidation by the inhibition of HDAC3 [610]. In addition, fenofibrate ameliorates insulin resistance accompanied by an improved mitochondrial oxidative capacity in pediatric burn patients [611]. Fenofibrate and gemfibrozil also reduce mitochondrial membrane potential depolarization, resulting in apoptosis inhibition in lymphoblast cells in Batten disease [612]. Pretreatment of rats with gemfibrozil prior to global cerebral I/R results in neuroprotection by modulating mitochondrial biogenesis and apoptosis [613]. WY-14,643 and fenofibrate protect mice from acetaminophen-induced hepatotoxicity by upregulating UCP-2, which is a PPARα target gene that reduces the generation of mitochondrial ROS [540]. However, fibrates may also trigger mitochondrial dysfunction because they inhibit the activity of mitochondrial respiratory chain complex I in rat skeletal muscles [614]. Moreover, gemfibrozil and WY-14,643 alter mitochondrial energy production by promoting mitochondrial permeability transition, as documented by membrane depolarization and calcium-induced swelling, which inhibits the oxidative phosphorylation and ATP synthesis in the rat liver [615]. 7.2. Mitochondrial Function Finally, chronic treatment with WY-14,643 impairs myocardial contractile function while decreasing mitochondrial respiratory function and increasing mitochondrial uncoupling in rats [616]. PPARβ/δ has been reported to be essential for the exercise-induced increase in the number of muscle mitochondria [617]. In high-fat–fed C57BL/6 mice, the administration of GW501516, a PPARβ/δ agonist, increases the metabolic rate, reduces fatty liver, decreases lipid accumulation, and increases mitochondrial biogenesis in the muscle [391]. In C2C12 muscle cells, GW501516 induces the mRNA expression of UCP-1, UCP-2, and UCP-3 [618], which are responsible for uncoupling mitochondrial respiration [619]. Accordingly, muscle-specific PPARβ/δ KO mice show a reduction in the expression of genes connected with energy uncoupling, mitochondrial electron transport chain, FA uptake, and catabolism. They develop obesity and diabetes with aging and present a mild defect in glucose metabolism when challenged with a high-fat diet [72,78]. The overexpression of either wild-type PPARβ/δ or constitutively active VP16-PPARβ/δ in muscle results in fiber-type switching and augmented capacity for mitochondrial pyruvate oxidation, which is accompanied by an induction of mitochondria numbers [80,310,620]. This phenotype resembles that of the muscle- specific overexpression of PGC-1α [621] and accords with PGC-1α acting as a co-activator of PPARβ/δ to control mitochondrial biogenesis and muscle fiber-type plasticity [72,78,80]. Furthermore, the expression in BAT of a constitutively active PPARβ/δ results in the increased expression of genes involved in FA oxidation and lipolysis and in energy uncoupling in the mitochondria. However, some studies have shown that the impact of PPARβ/δ on energy metabolism in muscles does not necessarily involve de novo mitochondrial biogenesis [622,623]. PPARγ and TZDs also have roles in mitochondrial structure and function. Transgenic mice overexpressing PPARγ2 have a significantly increased expression of mitochondrial UCP-1, elevated levels of PGC-1α, and reduced mitochondrial ATP concentrations in their subcutaneous fat [624]. The overexpression of cardiac PPARγ results in the production of mitochondria with a distorted architecture of the inner matrix and disrupted cristae [625]. Rosiglitazone treatment of ob/ob mice leads to mitochondrial remodeling, enhanced oxygen consumption, and increased energy expenditure in WAT [626]. Moreover, in adipose tissues of patients with diabetes, agonists of PPARγ increase the relative amounts of mitochondria and mitochondrial DNA copy number and stimulate the expression of factors involved in mitochondrial biogenesis, respiratory complexes I–IV, and FA 24 of 75 Cells 2020, 9, 1708 oxidation [627,628]. 7.3. Reduction of Inflammation The “inflammation hypothesis of aging” posits a molecular mechanism of aging based on inflammation. Inflammation is a complex defense reaction to insult and both physiological and nonphysiological stress, which is induced by agents such as chemicals, drugs, or microbial entities. Inflammation responses are activated by well-coordinated, sequential events controlled by humoral and cellular reactions. Elevated tissue levels of TNFα, IL-1, and IL-6, among other pro-inflammatory mediators, have been observed in experimental animal models of inflammation. With aging, inflammatory responses may be overactive or even cause damage, resulting in pathological conditions [14]. During aging, a shift occurs in the ratio of naive to memory T cells, with associated changes in the cytokine profile in favor of inflammatory cytokines such as TNFα, IL-1, IL-6, INFγ, and transforming growth factor β [637–640]. There is also a progressively higher dysregulation of immune cells and pro-inflammatory responses. Macrophages from old mice produce more prostaglandin E2 than those from young mice because of higher COX-2 activity [641]. One major causative factor in tissue inflammation is the uncontrolled overproduction ROS/reactive nitrogen species. The transcriptional regulator NF-κB is an inflammatory reaction factor of major importance that is extremely sensitive to oxidants [552,566–570]. Enhanced IL-6 production by activated NF-κB has been implicated in many pathophysiological dysfunctions of aging ranging, from Alzheimer’s disease to atherosclerosis [642]. CR exhibits a broad and effective anti-inflammatory effect. It blunts age- triggered increases in COX-2 levels and activity through the modulation of NF-κB and IκB, in which COX-2-derived ROS generation decreases. In addition, the production of iNOS, IL-β, IL-6, TNFα, and prostanoids such as thromboxane A2 (TXA2), prostacyclin 2, and prostaglandin E2 is suppressed [14,531]. The prevention of the age-related decline triggered by CR correlates with dampening the reduction of PPAR expression and activity seen during aging. Therefore, under CR conditions, higher PPAR expression may play a role in the suppression of the age-induced increase in inflammation [140]. PPARs are implicated in inflammation at the transcriptional level by interfering with pro- inflammatory mediators such as NF-κB, STAT-1, and activating protein-1, leading to the downregulation of the gene targets of these factors [643–646]. In this way, PPARα and PPARγ inhibit the expression of inflammatory genes, such as COX-2, iNOS, cytokines, metalloproteases, and acute- phase proteins [549,644]. Inflammatory eicosanoids serve as ligands for PPARs, and the levels of these signaling molecules, including prostaglandins and leukotrienes, increase with age [647]. 7.2. Mitochondrial Function A similar response has been described in differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with rosiglitazone, which showed increased mitochondrial biogenesis, oxygen consumption, and mitochondrial citrate synthase activity [629]. Finally, rosiglitazone also protects T lymphocytes from apoptosis by preventing the loss of mitochondrial membrane potential [630]. The mitochondria-related impact of TZDs has been identified as a basis for their neuroprotective effect [607,631–634]. Of interest, TZDs can also exert PPARγ-independent effects on mitochondrial respiration, leading to changes in glycolytic metabolism and fuel substrate specificity [635,636]. Taken together, the evidence strongly suggests that these three PPARs contribute to the maintenance of mitochondria in a tissue-specific manner. 7.3. Reduction of Inflammation Each of the three PPAR isotypes exhibits a set of individual anti-inflammatory properties [58]. The anti-inflammatory activity of PPARα is in a great part the result of its interaction with NF-κB. The deletion of PPARα results in a premature and enhanced age-dependent increase in oxidative stress and NF-κB activity [137]. Similarly, aged PPARα KO mice display higher oxidative stress at a younger age and an exacerbated inflammatory response to LPS stimulation [137,648]. In contrast, the administration of PPARα agonists to aged wild-type mice restores the cellular redox balance, as attested by the elimination of constitutively active NF-κB and a loss in spontaneous inflammatory cytokine production [137,649]. The interaction of NF-κB and PPARα is intriguing, because high doses of the PPARα ligands activate NF-κB, whereas low or therapeutic doses of the ligands cause decreased NF-κB activation accompanied by reduced IL-6 production and lipid peroxidation [137]. 25 of 75 Cells 2020, 9, 1708 During CR, PPARα is required, at least partially, to mediate the downregulation of acute-phase genes (C4bp, C9, Mbl1, Orm1, Saa4) that are responsive to inflammatory cytokines [141]. During CR, PPARα is required, at least partially, to mediate the downregulation of acute-phase genes (C4bp, C9, Mbl1, Orm1, Saa4) that are responsive to inflammatory cytokines [141]. PPARβ/δ also shows anti-inflammatory properties and can suppress, in a ligand-independent manner, inflammatory bowel disease by the dampening of inflammatory signaling [650]. In cultured cardiomyocytes, the PPARβ/δ agonist GW0742 inhibits LPS-induced TNFα secretion, whereas the absence of PPARβ/δ exaggerates LPS-induced TNFα production [651]. The intracerebroventricular administration of high-affinity PPARβ/δ agonists significantly decreases the infarct volume at 24 h of reperfusion after cerebral ischemia in rats, again underscoring the anti-inflammatory and neuroprotective properties of PPARβ/δ [652]. Lastly, the activation of PPARβ/δ by GW0742 protects skeletal muscle against metabolic disorders caused by chronic exposure to a high concentration of sugars by affecting the insulin and inflammatory cascades, including reversal of the diet-induced activation of NF-κB and the expression of both iNOS and intercellular adhesion molecule 1 [653]. on of NF-κB and the expression of both iNOS and intercellular adhesion molecule 1 [653]. p [ ] PPARγ is undeniably one of the most important and best documented anti-inflammatory factors. PPARγ agonists mitigate inflammatory bowel disease symptoms, reduce inflammation, and are effective in multiple models of ulcerative colitis as well as in Crohn’s disease [654–667]. 7.3. Reduction of Inflammation Functionally, the binding of PPARγ to a DNA-bound repressor complex in macrophages blocks the expression of inflammatory genes by preventing the 19S proteasome-mediated degradation of the repressor complex [668]. Accordingly, the ligands of PPARγ inhibit macrophage activation, stimulate macrophage differentiation into non-inflammatory type M2, and suppress the production of inflammatory cytokines in macrophages and dendritic cells, resulting in increased susceptibility to infection in PPARγ-deletion mouse models [549,644,669–672]. Of interest, the Pro(12)Ala substitution in PPARγ (rs1801282 C>G), which results in a modest decrease in its transcriptional activity and adipogenic potential, mediates anti-inflammatory benefits. The Pro(12)Ala substitution is associated with a 10-year delay in the onset of multiple sclerosis [673] and with a decreased risk for T2D [442]. Males carrying the 12Ala allele and having coronary artery disease show less widespread atherosclerosis and are protected against 10-year vascular morbidity and mortality [674]. Furthermore, another PPARγ polymorphism (rs 1801282 C>G, rs3856806 C>T) is associated with colorectal cancer risk [675,676]. Mice deficient in colonic PPARγ display more acute infectious colitis [663] and are resistant to conjugated linoleic acid therapy for colitis [677]. The molecular mechanism behind the anti-inflammatory activities of PPARγ includes inhibition of the expression of inflammatory genes encoding cytokines, metalloproteases, and acute-phase proteins, and the regulation of multiple signaling pathways, such as those related to p53 [678], Bcl2 [89], c-Myc, [679], Cox-2 [91,680–682], iNOS [683], and Apc/β-catenin [684,685]. Most importantly, PPARγ inhibits NF- κB and NF-κB-driven transcription [89,682]. PPARγ may reduce NF-κB activities in various ways (see the section on PPARs and oxidative stress). Therefore, it is likely that PPARs mediate, at least in part, the anti-inflammatory properties of CR. 7.4. Metabolic Adaptation The shortage of energy during CR leads to a sequence of metabolic changes. Following the depletion of dietary glucose, glycogen is mobilized as an energy supply, and upon prolonged CR, hepatic metabolism shifts to gluconeogenesis to prevent hypoglycemia. Some enzymes connected with hepatic glycolysis, gluconeogenesis, and glycogen metabolism are under the control of PPARα. During fasting, PPARα stimulates glucose import, glycolysis, and glycogenolysis [686–689]. Accordingly, the expression of several genes involved in gluconeogenesis and glycogen metabolism is reduced in PPARα KO mice [368], and these animals show impaired gluconeogenesis regulation and marked hypoglycemia during fasting [54,55]. Upon prolonged energy restriction, carbohydrate depletion triggers a shift to fat recruitment and ketone body production. This switch between energy sources relies on PPARs. Exercise-elicited glycogen depletion activates PPARβ/δ in rat muscle [690]. We speculate that a similar regulation takes place in fasting-related carbohydrate shortage, which would contribute to PPARβ/δ-driven FA oxidation in muscles. Similarly, the upregulation of the expression of PPARα by CR has been suggested to act as a direct stimulus to enhance FA β-oxidation in the heart [139]. Cells 2020, 9, 1708 26 of 75 PPARs also regulate the expression of many genes involved in insulin signaling, glucose uptake, lipid metabolism, and ketogenesis, which are affected by CR. Particularly, the metabolism of lipids and ketone bodies in the liver employs PPARα to regulate the expression of most of the rate-limiting enzymes of β-oxidation including ACOX1 (acyl-CoA Oxidase 1), EHHADH (enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase), carnitine palmitoyltransferases I and II, MCAD (medium- chain acyl-CoA dehydrogenase), LCAD (long chain acyl-CoA dehydrogenase), VLCAD (very long chain acyl-CoA dehydrogenase), and fibroblast growth factor 21, and of ketogenesis, such as HMG- CoA synthase [141,691–700]. y [ , ] During fasting, PPARα promotes cellular FA uptake and β-oxidation and mediates the adaptation to FA catabolism, lipogenesis, and ketone body synthesis in response to energy depletion [53–55]. Consequently, fasting-induced hepatic responses, including elevated FA oxidation and ketogenesis, are all impaired in PPARα-null mice, resulting in hypoketogenesis and liver steatosis [53–55]. Similarly, in aged mammals, including humans, the capacity for FA oxidation and hepatic ketogenesis decreases, resulting in reduced energy metabolism as well as increased dyslipidemia [223–225]. In healthy men, the L162V substitution of PPARα is associated with higher fasting total cholesterol, low-density lipoprotein cholesterol, and apoB, but not with postprandial parameters [50]. 7.4. Metabolic Adaptation Both PPARα and PPARβ/δ are essential regulators of FA oxidation, and their roles in this process overlap. Of importance, the two PPARs show a distinct primary area of activity, with PPARα activating FA oxidation mainly in the liver and BAT, whereas PPARβ/δ controls lipid metabolism in the pancreas, heart, and skeletal muscle. PPARα does not seem to be involved in the metabolic adaptation of the liver to every-other-day fasting [701]. Reduced energy intake accompanied by increased mobilization of the fat reservoir results in weight loss. The release of energy from WAT involves the inhibition of expression of the lipid-storing PPARγ. Collectively, all three PPARs act as metabolic sensors and play essential roles in lipid and FA metabolism. However, PPARγ is more responsible for fat storage and PPARα and PPARβ/δ are more responsible for energy expenditure. Likely for that reason, a high-fat diet increases the expression of PPARγ in the liver, whereas intermittent fasting decreases it [702]. Genetic variation in the Pparγ gene and its target gene Acsl5 determine the capacity for weight loss under CR [703], and six Pparγ single nucleotide polymorphisms are significantly associated with weight reduction in response to CR [704]. Most of the data concerning Pparγ polymorphisms focus on the Pro(12)Ala substitution. Based on a report of a population of children in Mexico, Pro(12)Pro homozygosity is the more represented, followed by Pro(12)Ala heterozygosity, and more rarely Ala(12)Ala homozygosity (73.9%:24.5%:1.6%) [705]. The (12)Ala PPARγ protein shows a decreased binding affinity for PPRE and consequently is a weaker stimulator of target gene expression [441,706]. The presence of (12)Ala PPARγ and resistance to CR-induced weight loss were associated in a comparison of women with obesity losing the most weight to those losing the least after 6 weeks of a 900 kcal/day CR [703]. Moreover, PPARγ polymorphism is associated with changes in body mass index (BMI) in response to the total fat intake [707,708], FA composition in the diet [709], and plasma TG response to ω3 FA [710]. This polymorphism also influences weight regain following CR, with women homozygous for Ala(12)Ala gaining more weight compared to women with Pro(12)Pro homozygosity [711], likely indicating lesser metabolic flexibility for Ala(12)Ala individuals. Long-term CR leads to energy-saving adaptations that can result in a lower resting metabolic rate and decreased body temperature [712–714], which is possibly because of reduced thyroid hormone levels. 7.5. Physical Exercise Exercise, similar to CR, yields multiple beneficial effects. Research outcomes point toward the effectiveness of regular moderate exercise in preventing and delaying several metabolic disorders, chronic diseases, and premature death. Increased physical activity reduces mortality risk from many age-related diseases, including cardiovascular disease, stroke, T2D, certain cancers, hypertension, obesity, depression, and osteoporosis [719–723]. However, in rodents, exercise improves the mean lifespan without increasing maximum longevity [724,725]. Similarly, high physical activity fails to extend maximum lifespan in humans [726]. Compared to exercise, long-term CR in humans improves several biomarkers related to aging [727,728]. Accordingly, exercise has been deemed as unable to fully mimic the beneficial hormonal and/or metabolic changes associated with CR [729]. Therefore, despite a mutual influence with CR on similar molecular pathways and providing multiple advantages, physical activity has been recognized as yielding inferior benefits compared to CR. Physical activity results in the release of stored energy and elevated levels of FAs, increasing the availability of ligands for PPARs [730]. In rat liver, exercise increases PPARα expression and transcription, and PPARα may mediate the impact of exercise on plasma glucose, TG, and cholesterol [731]. Similarly, PPARα may be involved in the protective effects of exercise against myocardial infarction and for cardiac function by changing the expression of metabolic and inflammatory response regulators and by reducing myocardial apoptosis [732]. However, among PPARs, PPARβ/δ is particularly well-known for its impact on physical performance. First, PPARβ/δ controls muscle development and the adaptive response to exercise, and its overexpression results in a switch to type I muscle fiber [620]. Second, exercise can increase PPARβ/δ expression in skeletal muscle, and this activation is essential for increasing the number of exercise-induced muscle mitochondria [617]. As previously mentioned (see the AMPK and PPARβ/δ section), AMPK and PPARβ/δ are exercise mimetics [322], and their stimulation significantly increases running parameters and promotes muscle remodeling [321,322]. During exercise, the depletion of carbohydrates in skeletal muscle limits endurance. PPARβ/δ represses glycolytic genes in muscle to slow glucose catabolism, reducing the use of carbohydrates at the period near exhaustion. In parallel, PPARβ/δ induces a shift to FAs as the main energy source and thus extends the possible exercise time [623,733]. Consequently, transgenic mice overexpressing muscle-specific PPARβ/δ show enhanced exercise performance, but PPARα-overexpressing animals do not [80,310,620]. Exercise is associated with increased PPARγ DNA-binding activity and expression of its target genes in leukocytes [734]. 7.4. Metabolic Adaptation Bezafibrate, a panagonist for all three PPARs, has been reported to induce WAT beiging and thus shows potential for regulating body temperature [715]. Similarly, the activation of PPARα in WAT and BAT results in increased UCP-1 expression and consequently elevated energy dissipation and higher body temperature [716]. Crosstalk between thyroid hormone receptors and PPARs appears to be important for regulating thermogenesis and metabolism [717,718]. In summary, the involvement of PPARs in the metabolic feeding-to-fasting adaptation places these receptors at the center of the proper body response to CR. 27 of 75 27 of 75 Cells 2020, 9, 1708 7.5. Physical Exercise Similarly, in skeletal muscle and subcutaneous WAT, PPARγ and PGC-1α mRNA expression increases in response to physical training, and these expression changes are proposed to mediate the effect of exercise on insulin sensitivity [735]. Furthermore, the beneficial outcome of low-intensity exercise on plasma lipid levels is exerted via PPARγ [734], and PPARγ1 promotes exercise-induced lipoprotein lipase expression [736]. In addition, the Pro(12)Ala substitution in PPARγ polymorphism is associated with reduced glucose and insulin levels as well as body weight loss in response to exercise [737–740]. Therefore, as in the case of CR, PPARs play an active role in upstream molecular signaling and beneficial outcomes of exercise. 7.7. Longevity and Aging According to the “rate-of-living” theory, lifespan differs between species and it correlates with energy metabolic rate and, in general, with body size [757,758]. Thereupon, the level of CR needed to prolong life by a certain percentage varies from species to species. In fact, CR increases lifespan to different extents with a stronger impact on short-living animals, such as C. elegans (up to 150%) [759], Drosophila [760], or rodents (up to 50%) [761] compared to long-lived species including lemurs [762] and rhesus monkeys [6,763]. Therefore, it is not possible to extrapolate the results obtained for one species to others. Importantly, the level of restriction (10–50%) applied in various studies strongly impacts the outcome [11,12]. Although it is proven that CR causes beneficial metabolic modifications in women and men, the exact amount of calorie intake that is necessary to reach maximum longevity and sustain good health is not known, but it is likely different from person to person. It is also obvious that excessive CR leads to malnutrition with adverse health effects. Both genetic and environmental factors control the progression of aging. Aging is associated with immunosenescence, increased oxidative stress, decreased hormonal secretion, changes in metabolic rate, mitochondrial function, insulin resistance, and dysregulated lipid metabolism [764– 766]. The preservation of insulin sensitivity by reducing levels of blood glucose and insulin without compromising glucose fuel may prevent age-related metabolic phenotypes [353]. Glucose metabolism maintenance is a key feature of the anti-aging actions of CR [3]. In fact, genes connected with the insulin/IGF-1 signaling pathway have been proposed as longevity candidate markers [356,357,767]. Paradoxically, impaired insulin signaling through the insulin receptor or its substrates increases rather than decreases lifespan in a number of mouse models [351,356,357]. With regard to insulin sensitivity, the undeniable role of PPARα and PPARβ/δ has already been described above. Worth highlighting here in the context of PPARγ is the role of WAT in proper glucose homeostasis, as attested by the association of lipodystrophy with severe insulin resistance [396]. Aging is accompanied by increased body fat, particularly in the visceral areas as well as in liver and skeletal muscle, which initiates the development of age-associated hypertension, atherosclerosis, inflammatory diseases, dyslipidemia, and T2D [764–766]. In fact, WAT, which has also been directly associated with lifespan, is a central physiological component of energy metabolism [351,396], and its development and function rely on PPARγ [106,768,769]. 7.6. Hunger Food withdrawal or limitation inevitably results in a hunger sensation. The physiology of hunger involves a complex network of sensors, hormones, and neuronal signaling. Hunger signaling relies on PPARs, particularly PPARα. For example, allele “A” in PPARA rs4253747 (a single nucleotide polymorphism in an intron region) in young men of Han Chinese ancestry is significantly associated with an increased risk for appetite loss at high altitude. In contrast, the “AC” haplotype of PPARA rs7292407-rs6520015 in the same cohort had a protective role for high altitude appetite loss [741]. If PPARα is implicated in appetite control, some of its natural ligands should affect hunger sensation. Oleoylethanolamide (OEA), an endogenous ligand of PPARα, is one such compound. This endocannabinoid is produced by enterocytes in response to fat consumption [742], and bile acids modulate its biosynthesis, which requires sympathetic innervation [743] [744]. The administration of OEA has an anorectic effect by acting peripherally, reducing meal size or prolonging eating latency, 28 of 75 Cells 2020, 9, 1708 leading to body weight loss [742,745–747]. PPARα activation in the proximal small intestine mediates this effect [742,748,749]. The intraperitoneal administration of OEA acutely decreases energy expenditure, as well as ambulatory and spontaneous locomotor activity [750]. Via PPARα, OEA stimulates lipolysis and decreases the neutral lipid content in hepatocytes, as well as serum cholesterol and TG levels, and thereby regulates lipid metabolism [751]. OEA engages afferent sensory fibers of the vagal nerve in the intestine, leading to an increased expression of proto-oncogene c-fos in the nucleus solitary tract and the paraventricular nucleus of the brainstem and hypothalamus, respectively [751], which promotes oxytocin secretion and satiety [752]. Since enterocytes in the small intestine are the first cells to respond to dietary fat intake by increasing OEA production, OEA has been suggested to serve as a gut-derived satiety factor [742]. The other PPARs may also indirectly affect appetite. As a master regulator of adipogenesis, PPARγ plays a crucial role in regulating food intake because WAT secretes a number of endocrine and paracrine satiety mediators, including leptin, adiponectin, and resistin [753]. Inflammation, which is well established as being under PPAR influence, reduces appetite [754]. Particularly, NF-kB, which interacts with all PPARs, has been implicated in appetite suppression [755,756]. 7.7. Longevity and Aging In addition to the direct regulation of adipogenesis from fat-produced PPARγ, intestinal PPARγ regulates body adiposity by sympathetic nervous system signaling in mice on CR [101]. Therefore, through the coordination of glucose homeostasis and adipogenesis, PPARγ might affect longevity [770]. A low expression of PPARγ reduces the lifespan in both lipodystrophic PPARγ1/2-hypomorphic and PPARγ2-deficient mice [771]. We suggest that the reduced fat mass observed during CR is not what results in longevity, but rather that the key factor is proper adipose tissue functionality, including insulin-sensitizing effects. Correspondingly, the human genetic variant genotypes 29 of 75 Cells 2020, 9, 1708 Pro(12)Ala and Ala(12)Ala of PPARγ are associated with leanness, improved insulin sensitivity, and increased lifespan in both humans and mice [440–442]. Moreover, gene network analysis has identified PPARγ as one of the “longevity genes” in mouse WAT [771]. However, reports are contradictory concerning the expression of PPARγ in WAT in response to CR. One group found that both 8 weeks of 50% CR and intermittent fasting downregulated PPARγ mRNA and protein expression in the adipose tissue of obese rats [132]. A similar effect was observed in the subcutaneous adipose tissue of obese humans following 10-week CR [772]. In contrast, in intermittently fasting rats, PPARγ2 mRNA levels were approximately two-fold higher than in control or CR animals [773], resulting in a not well-understood effect of restrictive diets on PPARγ2 expression in WAT. Pro(12)Ala and Ala(12)Ala of PPARγ are associated with leanness, improved insulin sensitivity, and increased lifespan in both humans and mice [440–442]. Moreover, gene network analysis has identified PPARγ as one of the “longevity genes” in mouse WAT [771]. However, reports are contradictory concerning the expression of PPARγ in WAT in response to CR. One group found that both 8 weeks of 50% CR and intermittent fasting downregulated PPARγ mRNA and protein expression in the adipose tissue of obese rats [132]. A similar effect was observed in the subcutaneous adipose tissue of obese humans following 10-week CR [772]. In contrast, in intermittently fasting rats, PPARγ2 mRNA levels were approximately two-fold higher than in control or CR animals [773], resulting in a not well-understood effect of restrictive diets on PPARγ2 expression in WAT. Most of the prominent features of aging are related to PPAR activity, mTOR activity, oxidative stress, inflammation, and metabolism. Moreover, changes in PPAR expression and activity often occur in aging and are reversed by CR [140,224,539,774]. 7.7. Longevity and Aging PPAR activity also may be affected indirectly through age-dependent decreases in RXR, the heterodimerization partner of PPARs [775–777]. The impact of PPARs can be particularly well observed in mutant models of longevity, such as the dwarf mice. Snell dwarf, Ames dwarf, and “Little” mice display low levels of GH or a defect in GH signaling because of a mutation (generating the GHR-KO strain). All of these dwarf mice are characterized by having a markedly longer lifespan than their wild-type counterparts and share a number of beneficial phenotypic characteristics with rodents on CR diets. Similar to CR animals, dwarf mice are protected from spontaneous and chemically induced cancer, age-dependent declines in immune function, collagen cross-linking, decreased levels of insulin and IGF-1, and increased insulin sensitivity [428]. The increased expression of PPARα and constitutive activation of some of its target genes have been detected in the liver of the dwarf mice [131,778]. The increased expression of genes involved in β- and ω-oxidation of FAs (Acox1, Cyp4a10, Cyp4a14) in the liver of these mice suggests increased FA oxidation, which could be beneficial for insulin sensitivity. PPARα levels are decreased in the muscle of GHR-KO animals, and PPARβ/δ protein levels are downregulated in the liver and skeletal muscle, which mimics the expression profile in wild-type CR mice [136]. The protein levels of PPARγ are elevated in the liver but downregulated in the skeletal muscle of the GHR-KO animals [136]. Furthermore, the overexpression of fibroblast growth factor 21, previously mentioned as a PPARα target gene, extends the lifespan in mice without affecting AMPK or mTOR but blunting GH/IGF-1 signaling in the liver [779]. In contrast to GHR-KO mice, animals overexpressing the bovine GH gene have a markedly shorter lifespan in comparison to their wild-type counterparts. The hepatic expression of PPARα is decreased in these mice, as is the expression of genes involved in FA activation, peroxisomal and mitochondrial β-oxidation, and the production of ketone bodies. Consequently, bovine GH mice exhibit a reduced ability to produce ketone bodies in the fed and fasted states [780]. The antagonistic relationship between PPARs and GH is demonstrated by the fact that the surgical removal of the pituitary gland (hypophysectomization) of rats enhances the expression of PPAR-inducible proteins, which can be reversed by GH infusion [781]. Moreover, STAT5b, a GH-inducible transcription factor, inhibits the ability of PPARα to activate PPARα-dependent reporter gene transcription [782,783], and PPARα downregulates STAT5b [784]. 7.7. Longevity and Aging Consequently, PPARs may control lifespan at the level of glucose and lipid metabolism and hormonal regulation. 7.8. Microbiota Composition Microbiota composition changes upon CR have been repeatedly observed [138,785–788]. CR increases the abundance of bacteria that positively correlate with lifespan, mainly Firmicutes including Lactobacillus, Allobaculum, Papillibacter, or Lachnospiraceae. In parallel, CR reduces the occurrence of bacteria that negatively correlate with lifespan, such as Clostridiales, Riminococcaceae, Alistipes, or Rikenella [787–791]. The exact effect of microbiota on the outcome of CR is not fully known, but the microbiota mediates some of the beneficial outcomes of CR, including reduced body weight and decreased blood leptin and insulin levels [791]. We could speculate that there is an effect on metabolism, body fat storage, and the endocrine system of microbiota-driven changes in the production of signaling molecules and ligands for nuclear receptors, including PPARs [101]. Indeed, 30 of 75 Cells 2020, 9, 1708 the interaction of PPARs with the microbiota has been well documented. The expression of PPARα and its target genes coding for rate-limiting enzymes of ketogenesis depends on stimulation by commensal gut microbiota [691,698,792]. Using germ-free mice, we have shown that the microbiota not only promotes harvesting energy from the food but is also generating signals, which regulate the hepatic clock genes and their effector genes such as the PPARs, and several PPARα target genes [793]. Of note, PPARα also mediates signals received from the microbiota via TLRs and contributes to the circadian expression of genes in the intestine and intestinal corticosterone production [794]. Thus, PPARα forwards information from the gastrointestinal flora, which affects host physiology. Furthermore, PPARα has been identified as an important factor in the inflammatory response of the intestine to commensal microbiota [795]. It regulates the expression of IL-22, the antimicrobial peptides Reg3β and Reg3γ, and calprotectin [795]. In the context of restrictive diets, the microbiota mediates the stimulatory effect of intermittent fasting on beige fat development [796]. Similarly, the deletion of PPARα triggers the upregulation of UCP1 expression in WAT [796]. PPARβ/δ, which is constitutively expressed in the intestine at a high level [45] and takes part in the differentiation of intestinal cells, is indirectly involved in the secretion of antimicrobial peptides [74,650,797]. Therefore, it influences gut microbiota composition. The expression and activity of PPARγ are induced in the gut by multiple nutrients [114], bacterial metabolites, and bacterial by-products [115–118], and the presence of specific bacterial strains [117,119,120] stimulates PPARγ expression and activity. 7.8. Microbiota Composition However, CR has been shown to limit the production of butyrate [787], which is one of the short chain fatty acids (SCFAs) that is known to activate PPARγ [116,117]. Moreover, the microbiota affects the liver circadian rhythm by modulating the activity of PPARγ expressed in the liver [798]. Of note, PPARγ is responsible for the selective killing of bacteria associated with inflammatory bowel disease by stimulating the expression of β- defensins and the maintenance of innate antimicrobial immunity in the colon [799]. Thus, there are reciprocal interactions between PPARs and gut microbiota in which PPARs can be activated by bacteria and regulate the intestinal microbiota composition [800]. The additional impact of CR on the expression PPARs points to a potential role for PPARs in the response of microbiota to CR. References 1. Osborne, T.B.; Mendel, L.B.; Ferry, E.L. The Effect of Retardation of Growth Upon the Breeding Period and Duration of Life of Rats. Science 1917, 45, 294–295, doi:10.1126/science.45.1160.294. 1. Osborne, T.B.; Mendel, L.B.; Ferry, E.L. The Effect of Retardation of Growth Upon the Breeding Period and Duration of Life of Rats. Science 1917, 45, 294–295, doi:10.1126/science.45.1160.294. 2. McCay, C.M.; Crowell, M.F.; Maynard, L.A. 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PPARs take part in managing the initial shortage of energy, modulating the main signaling pathways, adjusting metabolism to prolonged low energy intake, and mediating direct and long-term outcomes. Each of these roles could contribute to improvements related to common diseases such as diabetes and cancer, and even neurodegenerative diseases. Therefore, PPAR agonist treatments together with CR has great potential for synergistic effects to be explored in future experimental and clinical studies. Author contributions: K.D. wrote the manuscript and prepared the figures, A.G. assisted with research and figure preparation, H.G. edited the manuscript and supported the writing with expert advice, J.K. supported the writing with expert advice, and W.W. counseled on content, corrected the manuscript, and supported the writing with expert advice. All authors have read and agreed to the published version of the manuscript. Funding: Open access funding was provided by University of Vienna. Funding: Open access funding was provided by University of Vienna. Funding: Open access funding was provided by University of Vienna. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 8. Conclusions After remarkable achievements in medical research that have translated into a notable increase in life expectancy, the current focus is more on increasing disease-free years. With the potential to alleviate numerous health conditions while extending the lifespan, CR remains a relevant candidate in health-related research. Therefore, the current recommendation on energy intake should be revised, particularly for individuals with a high risk of developing metabolic, inflammatory, or neurodegenerative diseases. The beneficial impact of several restrictive approaches including multiple models of intermittent fasting [801–804] and fasting-mimicking diet [805–807] has been proven. These diets imply temporal restriction without long-term energy deprivation or prolonged hunger making them less restrictive than CR, easier to apply in everyday life, more flexible for various lifestyles, and therefore more plausible for a wide population. The majority of available studies concerning intermittent fasting are observational studies focused on weight loss, cardiovascular risk, and inflammation. However, knowledge of the molecular mechanism behind the observed effects is still limited [808,809]. More studies comparing CR and other specific restrictive diets, in terms of molecular pathways and health outcomes, are needed to identify which restrictive approach is more efficient. Eventually, the aim is to encourage the use of such diets as a means to prevent diseases. Currently, the large variety of intermittent fasting and CR protocols increases the complexity of this task. Interestingly, the involvement of PPARs in the impact of the intermittent fasting and fasting-mimicking diet has not yet been verified. Despite the lack of a fully revealed network of pathways underlying CR, the current review gives a detailed overview of how CR exerts its effects on the whole organism and which of the many outcomes are mediated by PPARs. During CR, the energy and nutrient sensor pathways involving 31 of 75 Cells 2020, 9, 1708 mTOR, AMPK, insulin signaling, and SIRT are tightly interconnected, resulting in reduced oxidative stress and inflammation, increased autophagy, improved mitochondrial function, and the regulation of metabolism, hunger, and microbiota composition. As discussed, multiple connections have been found between PPARs and regulatory responses to CR and fasting. 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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/W2895194007	http://www.scielo.br/pdf/edreal/v43n4/en_2175-6236-edreal-2175-623675587.pdf	English	null	Biopolítica, Educação e Resistência na Contemporaneidade	Educação & Realidade	2,018	cc-by	8,635	Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. http://dx.doi.org/10.1590/2175-623675587 1537 Biopolitics, Education and Resistance in the Contemporary World Leonardo Balbino MascarenhasI Leonardo Balbino MascarenhasI IUniversidade Federal de Alfenas (UNIFAL), Varginha/MG – Brazil ABSTRACT – Biopolitics, Education and Resistance in the Contemporary World. The present work has two main objectives: first, to examine the ways in which the technologies of power in contemporary capitalism pro- duce forms of subjection and lowering, in life in general and in education in particular. Next, it seeks to analyze the conditions for the production of resistance strategies to biopower. In this way, the aim is to interrogate the present time, using analytical tools that allow to stress the practices insti- tuted in the education field, as well as to put in motion the processes of subjectivation created and experienced there. In this sense, it is important not only to diagnose a certain condition (of the school, of education, of the contemporary world), but above all to cross it with problematizations that may open new possibilities for the construction of existence. K d Ed ti Bi liti R i t RESUMO – Biopolítica, Educação e Resistência na Contemporaneidade. O presente trabalho possui dois objetivos principais: em primeiro lugar, examinar os modos pelos quais as tecnologias de poder no capitalismo contemporâneo produzem formas de assujeitamento e rebaixamento, na vida em geral e na educação em particular. Em seguida, busca-se analisar as condições para a produção de estratégias de resistência ao biopoder. Pretende-se, desta maneira, interrogar o tempo presente, valendo-se de in- strumentos de análise que permitam tensionar as práticas instituídas no campo da educação, assim como colocar em movimento os processos de subjetivação ali criados e vivenciados. Nesse sentido, importa não apenas diagnosticar uma certa condição (da escola, da educação, do mundo con- temporâneo), mas sobretudo atravessá-la com problematizações que pos- sam abrir novas possibilidades de construção da existência. Palavras-chave: Educação Biopolítica Resistência p ç Palavras-chave: Educação. Biopolítica. Resistência. Biopolitics, Education and Resistance in the Contemporary World Introduction Education, as a domain of knowledge, has always been crossed by multiple practices and discourses, which put in motion and dispute different worldviews and political projects. As a meeting point of knowl- edge that came from sociology, psychology, philosophy, pedagogy, economy, among others, it is an arena in which countless power games are produced, with the action of different forces, producing multiple ef- fects. The school – this emblematic institution that materializes and is often viewed, in a reductionist way, as the place of education par excel- lence – is perhaps the device in which tensions and relations of power have greater visibility. The school is a modern institution, which means that, previously, it was common to educate and learn in different ways. It was only from the end of the 18th century on, with the emergence of industrial soci- eties, that the model of school we know and practice today, on a large scale, was constituted. We created an institution in the image and like- ness of factories and prisons: it strives to organize and distribute human bodies in time and space; to apply the same syllabus to all, based on dis- cipline and domestication; to define the expected output parameters, check the quality of the products at the end of the process, release the appropriate ones and retain the misfits, applying the punishments and corrections that best suit them; also, it repeats the process infinitely, in order to guarantee standardization and scale (Foucault, 1995). When the role of educating was relied to a disciplinary institution, with the shape of an industrial factory, what happened was not exactly a change of conception as to the role of education, but rather a continuity: the disciplines came to certify that the ideals of equality, freedom and fraternity, flags of the new social order, submitted themselves to social and class control, guaranteeing the maintenance of social differences and privileges, and the reproduction of the prevailing order and moral- ity. However, along with this update of power technologies, at the turn of modern societies, many forms of resistance and projects dedicated to criticizing the school institution have also been developed, seeking to make it effectively contribute to social transformation and reduction of inequalities. The Name of Fear Image 1 – Pieces of the video for dissemination of The Name of Fear, an exhibition by the artist Rivane Neuenschwander Image 1 – Pieces of the video for dissemination of The Name of Fear, an exhibition by the artist Rivane Neuenschwander Image 1 – Pieces of the video for dissemination of The Name of Fear, an exhibition by the artist Rivane Neuenschwander Source: Artist Talks (2015). Image by the author. Source: Artist Talks (2015). Image by the author. In 2015, the Brazilian plastic artist Rivane Neuenschwander pre- sented in London the results of her project titled The Name of Fear (Im- age 1). The work involved children from four British schools who, during a series of art workshops, reported their fears and designed capes that could protect them against these fears. The drawings were transformed by the artist, in partnership with other professionals, into real capes to be worn. The fears listed by the children were absolutely unique: fear of the end of the world; of the first day of class; of nightmares; of sadness; of cookie crumbs; of fire; of God; of clowns; of death; of airplanes1. Double movement produced by the project: on one hand, to think about what bothers and terrifies; on the other, to produce some form of protection against these annoyances and fears. The use of this invented protection, that is the result of a work of care, reflection and creation, is not an end in itself; it carries an ability to make cease something that presses, constrains, subjects and lowers the power, thus creating conditions of possibility for other forces to de- velop – forces that provide other forms of life, lighter, more joyful and safer of themselves. It would not hurt if we could all learn to create our own protection capes. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1538 Mascarenhas Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1539 Introduction A large number of education professionals and think- ers2 have dedicated, especially since the 20th century, to denounce this silent permissiveness of the school, as well as managed to transform it, where it is possible to see its effects: in the daily life of the classrooms and school corridors, in the midst of attendance control diaries, physi- cal and curricular grids, assessments and tests of all kinds, beadles and school managers, control and serialization of the times, legitimation of certain knowledge to the detriment of others, imposition of rules of conduct and forms of control of clothing and language, among others. Currently, although they are still minority when analyzed in a broad educational spectrum, we can find a wide diversity of alternative experiences of education, such as democratic school movements3; new forms of rural education and indigenous education; slow learning prac- ucação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1539 Biopolitics, Education and Resistance in the Contemporary World tices4, home education, and informal education, among others. Each of these experiences has its peculiarities and singularities, but they share at least one fundamental aspect: they all criticize the modern model of education, whose aims, mechanisms of functioning and results dis- tracts its actors from the possibility of constructing dignified, autono- mous and inventive ways of life. We could then shift education from this place of control and sub- jectivation, to think of it not as training, transmission and assimilation of contents, but as a constant process of transformation of what one is, of production of singularities, in multiple territories. An education that takes place in the movements of life and in the multiplicities that it im- plies. To think of an education that creates for itself new values, that allows for a lighter, more cheerful and powerful life. However, the mutations of advanced capitalism, together with the intensification of the globalization processes and the neoliberal agenda, threaten even the most progressive experiences in education, as they make everything potentially registrable and attachable to its production machines. The capitalist machine acts, on the one hand, extracting from everything its maximum usefulness and productivity, and on the other, diminishing its potency and lowering life (Deleuze; Guattari, 1983). As a result, forms of resistance are often weakened. The risks are multiplying everywhere. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1540 Introduction They are perceptible, for instance, in the increasing institutionalization and bureaucratization of the edu- cational practices that, among other effects, transform educators into school managers; in legal frameworks that affect the alternative edu- cational practices, limiting them or even making them impossible; in the constant threat of reduction and lack of resources with which the educational experiences coexists; in technological transformations and in the labor world, which, together with the public administration, lead to casualization of the teacher’s labor, pressure the schools and demand new content agendas and new ways of making; in the massification of consumption and in the American way of life, which devalue the singu- larities and knowledge of each culture and emphasize only one way of life to the detriment of all others; etc. In light of this, this text is an invitation to analyze some of the mutations of contemporary capitalism, its current forms of control and the processes of subjectivation derived from it, as well as the strate- gies that can be invested against them. A double movement, therefore. However, it is not intended here to exhaust the theme, or to approach it in a totalizing or definitive way. Neither was it sought to make a reper- tory of resistance practices in order to facilitate their control or to shut down their potential in manuals and other forms of reproduction. The purpose of this text is to try to interrogate the present time, seeking to achieve this through analysis instruments that allow stressing the prac- tices instituted in the field of education, as well as putting in motion the subjectivation processes created and experienced there. In this sense, it is important not only to diagnose a certain condition (of the school, of education, of the contemporary world), but above all to cross it with problematizations that may open new possibilities for the construction Mascarenhas of existence. And we try to do this in dialogue and inspired by the works of Foucault, Deleuze and Pelbart, among others. Finally, it is a matter of finding – and even inventing – in the inter- stices of contemporary forms of biopower, their carelessness, small in- terruptions, gaps, in short, their fleeting openings, where the capitalist machine decides to doze, ways of believing again in the world, breaths, blows that allow us to increase our power to act and affirm life – even in procedural forms. Introduction Because, as Pelbart said (2013, p. 13), “[…] there is a biopolitical strangulation that calls for gaps, however small they may be, to reactivate our political, theoretical, affective, bodily, territorial, existential imagination”. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1541 Biopolitics and Education Biopower operates in the sense of “[…] distributing the living in the domain of value and utility” (Foucault, 1976, p. 144); it selects knowledge, organizes and ap- plies techniques and instruments; it brings together linguistic, medical, judicial, domestic, artistic, religious and architectural discourses and institutions, among others, therefore producing the naturalization of some codes of conduct and ways of thought and eliminating others that are considered to be misfits, nonconformities, residuals, unproductive, useless, abnormal. Biopolitics refers to the transformation of disciplinary societies, to an improvement of practices of subjection. It is what can correspond to what Deleuze called societies of control: if disciplinary societies were marked by confinement and disciplines, societies of control are rather modular, they operate through amorphous, subtle and diffuse practic- es, but are felt directly on the human body. Confinements are molds, different moldings, while con- trols are a modulation, like a self-transmuting molding continually changing from one moment to the next, or like a sieve whose mesh varies from one point to another. [...]. Individuals become ‘dividuals’ and masses become samples, data, markets, or ‘banks’ (Deleuze, 1995, p. 178- 180, author’s emphasis). Disciplinary societies were characterized by the emphasis on ownership of the means of production; by the centrality of closed in- stitutions (factories, hospitals, prisons, asylums, schools, the family it- self), whose logic consisted in concentrating, organizing and distribut- ing human bodies through space; also, they were characterized by the use of energy machines and an economy based on the massive produc- tion of goods. Foucault accurately placed the student’s body beside the bodies of the madman, the prisoner, the sick and the worker. The disciplinary practices are the same: concentrating the bodies and organizing them in time and space; correct the postures and define the precise times and movements that can make the bodies. Training, modeling and cor- rection: it is a whole machinery, of knowledge and power, that turns against the body: A ‘political anatomy’, which was also a ‘mechanics of power’, was being born; it defined how one may have a hold over others’ bodies, not only so that they may do what one wishes, but so that they may operate as one wishes, with the techniques, the speed and the efficiency that one determines. Thus, discipline produces subjected and practised bodies, ‘docile’ bodies. Biopolitics and Education What mutations of capitalism and subjectivation processes are we talking about? What risks, possibilities and threats do they present to us? Nowadays, the terminologies are multiplying: post-Fordist society/ capitalism, information and knowledge society, advanced capitalism, cultural capitalism, cognitive capitalism, post-modern capitalism... It is not the case to define and distinguish each one here: it matters to us to investigate how some specific ways of thinking and living nowadays are becoming massified, crystallized, and at the same time are prevent- ing other life forms from happening. In other words, it is a question of analyzing how worlds that are ready to be inhabited in certain forms, or forms ready to inhabit certain worlds, are being fabricated, at the ex- pense of others. Foucault used the term biopolitics to designate the set of practices of life control and management. This set is developed by the articula- tion of two distinct forms of power-knowledge: on the one hand, the dis- ciplines, born in the 17th century, dedicated to the training of the body, to the organization and extraction of its forces and utilities; on the other hand, the control and regulation of populations, which began to emerge in the second half of the 18th century and which focus on attention to births and mortality, health length and longevity. Thus, a new form of power is being born, based on an anatomy-politics of the body: it is the biopower, mark and greater expression of biopolitics: […] one would have to speak of bio-power to designate what brought life and its mechanisms into the realm of ex- plicit calculations and made knowledge-power an agent of transformation of human life. […] There is no need either to lay further stress on the proliferation of politi- cal technologies that ensued, investing the body, health, modes of subsistence and habitation, living conditions, the whole space of existence (Foucault, 1978, p. 143-144). Power that seeks to manage life, power to foster life or let die, un- like the old sovereign power, which was based on take life or let live. This biopower also differs from the disciplinary power, since it aims not only at the control of individual behaviors, but also at the normalization of Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1541 Biopolitics, Education and Resistance in the Contemporary World the conduct of the human species in a broad way. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1542 Biopolitics and Education Discipline increases the forces of the body (in economic terms of utility) and diminishes these same forces (in political terms of obedi- ence) (Foucault, 1995, p. 138). At school, disciplinary practices are still quite visible today: in classroom architecture and ergonomics, whose prescriptions define Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1542 Mascarenhas specific modes of occupying school, ways of sitting, talking, listening, and using the bodies; in the hypervaluation of abstract knowledge to the detriment of the body and life, which results in the fetishization of the concept and discourse, and in the devaluation of the knowledge that can mobilize the body differently in school, especially dance, the- ater, physical education and music, always relegated to less important roles in formal education. Also, in the setting of training cycles, division and serialization of contents, in the crystallization of learning times, regular assessments and continuous checks, which focus on learning and behavior, determining what, how and when to learn, all of this call- ing for an obedience that wants to domesticate the student’s body and soul (Image 2). In turn, the societies of control operate according to a distinct logic, of continuous modulation and connection, of floating exchanges, with an abstract and diffuse control, no longer through confinement and discipline. The factory was replaced by the company, by the pos- sibility of distance work or without an inspector, but accompanied by the regime of productivity goals. In the economy, financialization and capital markets have become so global and important that production itself becomes expendable. In education, the school can be replaced by distance education, the training cycles never end, and there is always a need for continuing education imposed by the market. In health we witness the growth of preventive medicine, the culture of permanent health care, and the multiplication of the health insurance market, whose fundamental principle is pay but do not use. The energy ma- chines of disciplinary societies have given way to computational tech- nologies, to biotechnology and to artificial intelligence. Even money be- comes gradually obsolete, more abstract, the currency being replaced by electronic financial transactions (Deleuze, 1995). Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1543 Biopolitics and Education In the Brazilian case in particular, but following a global trend, it is evident several manifestations of these biopolitical practices in edu- cation: the growing intertwining of educational policies with market rules and principles; the growth of the culture of continued formation, not as a will to learn, driven by curiosity and the pleasure one can draw from knowing something new, but while searching for titles, fear of un- employment, not having the so-called employability, or simply fear of leaving the university behind and ending a cycle; the increase of aca- demic demands, molded by the market, which reduce the free time ex- perience, fundamental for the intellectual work and for a healthy life; the financial precariousness of schools and universities, the privati- zation waves of the public education, and the emphasis on technical and pragmatic courses that correspond to the market demands; the productivity goals in the postgraduate programs as a mechanism for control and distribution of resources, and the regulation they have ini- tiated (such as disputes between the postgraduate programs for scarce resources; stimulation of the overvaluation of the academic résumé, of the production of papers and irrelevant academic events; progressive transformation of teachers into managers; facilitation in building abu- sive relationships between advisors and advisees, etc.). Biopolitics, Education and Resistance in the Contemporary World Biopolitics, Education and Resistance in the Contemporary World It should be noted that we are currently living an overlap between these two forms of social organization: it is a time of passage from one to the other, from disciplines to controls, but also, and above all, a time of coexistence, in which these two models meet, combine and cross us, constituting subjectivation processes, that is, habitable surfaces, ways in which we are led to live. In all fields of social life, it is possible to per- ceive the exercise of a biopower, trying to conform us to be something, to live in specific ways, stiff and little open to differences. Beyond the educational field, it is neither an exaggeration nor a novelty to affirm that we are currently experiencing a widespread tor- por, a narcotization and demotion of the power to act, an extreme lack of political imagination and a heavy disbelief in the world, results of a broad and gradual process of automatization of subjectivities. Biopolitics and Education On all sides we are led to establish and reproduce relationships that are little open to new connections and are not very flexible. We live in the age of compulsive consumption, associated with the search for ready worlds; from the cult to an idealized image of the body, with its most varied practices (from fitness to the commodification of good health practic- es, plastic surgeries, pharmacopornography5 and the medicalization of life). It is the time of information overflows, with its most varied deploy- ments and devices (social media and mobile apps shaping behaviors and limiting coexistence with the difference, inserting everybody into virtual bubbles controlled by algorithms and artificial intelligence, and also stimulating an overexposure to images and the fugacity, useless- ness and volatility of information). Also, is the time of a deepening of the transformations in the relations of production and labor (with the increasing casualization and transformation of labor in an abstract cat- egory, the demand of super productivity accompanied by the insecu- rity and constant threat of loss or reduction of employment, etc.); the growing sense that we live in an ever-accelerating time – marked by the excess of tasks to be accomplished, by the self-imputed pressure of having to be successful in everything – reinforced by the ideologies of success spread in the media; by the thought far from the present and al- ways in what is yet to come, with anxiety being considered the evil of the century; also, by the culture of best sellers and TV series, which inces- santly repeat their narrative and dramatic formulas that only appease the thought and entertain; by the increasing instrumentalisation of the practices of meditation, yoga and other oriental models of existence, sold as a remedy for the accelerated urban life. Jorge Larrosa6 didactically suggested that we “never saw so many things happening, but yet the experience is increasingly rare” (Bondía, 2002, p. 21). The experience pointed out by Larrosa is that one capable of producing intensities, variations in the power to act on the world, affec- tions, some degree of loss of self, decentration: “It is experience what- ever ‘crosses us’, or touches us, or happens to us, and by passing us it forms and transforms us. Only the subject of experience is, therefore, open to his own transformation” (Bondía, 2002, pp. 25-26). Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1544 Mascarenhas However, this vital dimension of human life which is experience, which is to differ from itself, which is indeed the very foundation of all education, is now being reduced, becoming precarious, even eliminat- ed, as a result of different biopolitical devices. In this sense, Jorge Lar- rosa analyzes the ways in which three distinct elements are arranged in the social field: excessive information, overwork and the sensation of an accelerated time (Bondía, 2002). On the one hand, this rarity of the event, and the growing sense of emptying of the meanings of existence that accompanies it, is produced as we submit ourselves to the excesses of signs, images and other visual and sound stimuli, so present in everyday life now. We are all exposed to an absurd amount of information, often confusing quantity with quali- ty, and we are summoned at all times to give our opinion on everything, to express ourselves... Often without sufficient elaboration, without taking the time and reflection enough for something really interesting and relevant to be said, shared, for something to happen. Deleuze had already pointed out – long before the popularization of the internet and social media, we must say – that we are in need of silence: We sometimes go on as though people can’t express them- selves. In fact, they’re always expressing themselves. The sorriest couples are those where the woman can’t be pre- occupied or tired without the man saying ‘What’s wrong? Say something...’, or the man, without the woman say- ing..., and so on. Radio and television have spread this spirit everywhere, and we’re riddled with pointless talk, insane quantities of words and images. Stupidity’s never blind or mute. So, it’s not a problem of getting people to express themselves but of providing little gaps of solitude and silence in which they might eventually find some- thing to say. Repressive forces don’t stop people express- ing themselves but rather force them to express them- selves. What a relief to have nothing to say, the right to say nothing, because only then is there a chance of framing the rare, and ever rarer, thing that might be worth saying (Deleuze, 1995, p. 129). Along with the excess of information, we also suffer from over- work, resulting from a broad process of transformation of the economy and the forms of production. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1545 Biopolitics and Education Experience can be understood, then, as a condition for the affirmation of existence, for the singularization and production of differences and freedom. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1544 Mascarenhas It is that the current capitalism, with the deepening of neoliberal technologies at the global level, whose effects multiply misery and increase the income concentration, no longer hides its agenda. The casualization of jobs is now openly advocated (un- der the false argument that this will solve employment crises and boost economies, a discourse that serves to conceal the maintenance of social privileges and inequalities); it is done a wide misleading propaganda of meritocracy and entrepreneurship (sold as solutions for the market deregulation and flexibilization of labor laws, and which act by blaming those who are excluded by the system); more and more, the submission to the laws of fiscal adjustment and the cut of social expenses are pro- moted, as a way of maintaining the capital gains and the speculation in ucação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1545 Biopolitics, Education and Resistance in the Contemporary World stratospheric levels; it is promoted the financialization of life on an en- larged scale, producing what Lazzarato called “[…] indebted man” (Laz- zarato, 2014; 2012). Added to all this are the technological changes and the constant need to master new devices and ways of working, and we have as corresponding effects the increase of anxiety about the future, fear of unemployment, the naturalization of the idea that more training and information are necessary, and subjection to heavier work routines. All this also contributes to the reduction of experiences. Finally, we can mention a certain temporality, the way we per- ceive and relate to time, as another factor that prevents us from hav- ing powerful experiences. It is because we feel the world is accelerating, there is always a feeling that there is no time to lose, or that we must search for the lost time. […] experience is increasingly rarer due to lack of time. Everything that happens goes extremely fast, and in- creasingly faster. And with that it is reduced to an ephem- eral and instantaneous stimulus, immediately replaced by another stimulus or by another excitement equally ephemeral and instantaneous. The event happens to us in the form of a shock, of stimulation, of raw sensation, in the shape of the instantaneous, punctual, and fragment- ed experience. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1546 Mascarenhas The speed with which events are given to us, and the obsession for the novelty and the new, which characterizes the modern world, hinders the meaningful connection between events. They also prevent memory, since each event is immediately replaced by another that equally excites us for a moment, but leaves no trace (Bondía, 2002, p. 23, author’s translation). In addition, our temporal experience is being colonized by the principles of the market: it is considered necessary to instrumentalize the time, to make it always useful (do not waste time, make things that will be useful in the future, that can improve your curriculum or im- prove your social position); even the leisure and recreation moments need to be planned, so that the maximum productivity can be extracted from them (plan the holiday trip or the weekend exhaustively, in order to enjoy time, look for activities that have the best cost-benefit ratio, etc.). In that sense, free time, the one that is not scanned and controlled by planning and functionality, is often viewed as boredom, waste, or even end up lived in a blameworthy way, as an I should be doing some- thing else. This relationship with time is also the result of a social technolo- gy, product of a social construction that hinders the emergence of other temporalities. Modernity decreed the hegemony of a mechanical time, linear and successive, time of quantities. Yet, one could conceive a time of qualities, whose reason is neither numerable nor successive: a time of Aion, time of duration, of intensities, of affirmation of life and event production7: Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1546 Mascarenhas This dead time, which in a sense is a non-time, also bap- tized the ‘in-time’ is Aion. At this level, the event is no longer just the difference of things or states of things; it affects the human subjectivity, it inserts difference in the subject itself. If we call an event what changes the order of meaning (which made sense until the present moment became indifferent, and even opaque to us, and what we are now sensible to didn’t make sense before), it must be concluded that the event has no place in chronological time, since it affects the very conditions of a chronology. Mascarenhas On the contrary, it marks a cut, so that time is interrupted to resume on another plane (hence the expression ‘be- tween-time’) (Zourabichvili, 2004, p. 11-12). is, they produce incessantly singular modes of interpretation and rela- tion with the world: is, they produce incessantly singular modes of interpretation and rela- tion with the world: The schizo has his own system of co-ordinates for situat- ing himself at his disposal, because, first of all, he has at his disposal his very own recording code, which does not coincide with the social code, or coincides with it only in order to parody it (Deleuze; Guattari, 1983, p. 15). It would be necessary, then, to live a little like a madman, a child and a poet. Not bad. Still, it is not simply having the experience of mad- ness, or writing poems in the free time or playing on the weekends, al- though one can do a little of all of this. It is, more profoundly, to think with the thought of the child, the madman or the poet; of producing himself starting from that, of this new way of thinking, and not like this, representing another life. It is about inventing a new form of life, a new set of values, that refer to an affirmation of life in its now, and in its daily events, in its details, endowing them with lightness, joy and intensity, having the experience of sadness, but also overcoming it, giving way to other affections. Put oneself in movement. It is all a critique of the mod- ern subject conception that is at issue here: to kill what is in us of heavy, of easily disappointing, of insecure, of need for the approval of others; do not let ourselves to be stopped in sadness, to overcome jealousy and envy; to kill in us what is potentially frustrating, resentful, idealized, distant from the present. But where to start? How to do it? How can one produce him/her- self in this way? How to educate ourselves in this way? Now, an inven- tion is like a pregnancy, it is something that has not been born yet. That is, invention is precisely what does not yet exist, and therefore is not determinable, its paths and forms are unknown. No invention can be prescribed, shown in formulas, placed in manuals... Because this pro- cedure would transform it into mere reproduction. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1548 Stop Production, Empty the Body We can then make the Nietzschean critique of the value of val- ues, and ask: who needs these values over the human body? What types of existence imply a body thus lived? And, therefore, how can one es- tablish new values over the body? How to produce forms of education, currently, that stimulate and enable other relationships with one’s own body and new compositions with other bodies? How to restore the expe- rience? What forms of care can boost power in the body? To bring forth new bodies, which are not subjected and mortified entities, but terri- tories of the multiple, expressing singularities and intensive relations with the world, where experience is again possible and stimulated: that is a program of education and resistance to biopolitics. The body without organs (BwO), Artaud’s famous expression, much developed by Deleuze and Guattari, refers to a need to engender a paralyzation, to go through anti-production: “From a certain point of view, it would be much better if nothing worked, if nothing functioned [...] The full body without organs is the unproductive, the sterile, the unengendered, the unconsumable” (Deleuze; Guattari, 1983, p. 7-8). In this sense, Pelbart adds: The whole thematization of the body-without-organs is a variation around this biopolitical theme par excellence, the life undoing itself of what imprisons it, of the organ- ism, of the organs, of the inscription of the diverse powers on the body, or even of its reduction to bare life, dead-life, mummy-life, shell-life (Pelbart, 2013, p. 32-33). The body without organs, this interruption, this cut, does not be- come a definitive state or place: it is a process, a form of passage. In other words, it is about blocking certain forces so that new and unique forms can be produced. To carry out continuous processes of de-subjectivation and re-subjectivation. Perhaps it has been the madmen, the poets, and the children who best knew how to create bodies without organs for themselves, each in his own way, the madman finding in the experience of the crisis its most radical expression. It is that these three figures share a schizo logic, that Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1547 Biopolitics, Education and Resistance in the Contemporary World is, they produce incessantly singular modes of interpretation and rela- tion with the world: However, we know where the invention does not take place, where it lacks the conditions of possibility: right there where biopower is felt in an extremely intense way, to the point of paralyzing, suffocating, exhausting; is where biopolitics drains our will to power, turns us into mere survivors. Mascarenhas Image 2 – Body as Political Territory Source: Campbell (2015, p. 69). There is, in the body without organs, a strange potency: the power of non-doing. As Bartleby, well-known Melville’s character: the pref- erence for not doing, the claim to occupy a suspension place, even of impotence. To construct for oneself an impossibility, of saying some- thing, of doing something, of being something, of allowing oneself to be crossed by a certain line of biopolitical force, by a certain subjectivation process. Image 2 – Body as Political Territory Source: Campbell (2015, p. 69). Image 2 – Body as Political Territory Source: Campbell (2015, p. 69). Image 2 – Body as Political Territory Source: Campbell (2015, p. 69). There is, in the body without organs, a strange potency: the power of non-doing. As Bartleby, well-known Melville’s character: the pref- erence for not doing, the claim to occupy a suspension place, even of impotence. To construct for oneself an impossibility, of saying some- thing, of doing something, of being something, of allowing oneself to be crossed by a certain line of biopolitical force, by a certain subjectivation process. The exhaustion of the possible is the exhaustion of a cer- tain possible, that one ‘given beforehand’, the repertoire of possible that is offered to us in the form of multiple choice every day [...]. The end of the possible corresponds precisely to the necessary creation of possible. It is no longer a question of the possible as mere possibility, ideal, fortuitous, gratuitous, interchangeable, but the possible created necessarily, even if from an impossibility (Pelbart, 2013, p. 297, author’s emphasis). We have to see creation as tracing a path between impos- sibilities […] Creation takes place in choked passages. […] A creator who isn’t grabbed around the throat by a set of impossibilities is no creator. A creator’s someone who cre- ate their own impossibilities, and thereby creates possi- bilities (Deleuze, 1995, p. 133). Thus, a creative education is one that creates its own impossibili- ties, especially the impossibility of letting itself be traversed by the hard Educação & Realidade, Porto Alegre, v. 43, n. is, they produce incessantly singular modes of interpretation and rela- tion with the world: 4, p. 1537-1554, Oct./Dec. 2018. 1549 Biopolitics, Education and Resistance in the Contemporary World lines of biopower. The non-doing of Bartebly can be read as not doing in a certain way, which opens alternatives, inserts the possibility of in- venting other ways of doing. It is not simply failing to do, but to do dif- ferently. In a way that we can follow what appears, what is invented, if we dare to affirm: if it is to produce something with no quality, being massacred by time, I prefer not to do it; if it is to reinforce places of im- prisonment (of sexuality, self-esteem, generate more anxiety, etc.) and a state of emptying of power, I prefer not to do so; if it is to think and act in the world in order to restrict or diminish the possibilities, mine and/or of others, I prefer not to do it; if it is to reproduce and strengthen resent- ful, negative and meaningless life forms, I would rather not do so. On the other hand, to produce oneself in other ways implies not only this negative task, of refusal to do something, but also a positive task, of creation. And every creation process involves effort, displace- ment, and also a certain amount of unrest; involves sustaining a ten- sion, letting oneself be crossed by a problem, until one finds the lines of escape that transform that tension and that problem into other things, reconfiguring a certain existential territory, modifying its structures and putting new situations, new problems. The invention is not a spontaneous activity, even if we in- clude under this term the invention of daily life, and not only the great technological, artistic, or scientific inven- tions. Why is there an effort? Because we have deviated from the already open path, and therefore much easier, that previous habits have left behind […]. In invention, things do not go by itself. There is feeling of difficulty, an- noyance, obstacles. The process may involve a large num- ber of ups and downs. Still thinking with Bergson, there is effort when there is deceleration and delay. There must be work, that is to find a solution, because the idea can be dismantled if one does not seek to concretize it in a solu- tion. The search is active and often hard, involving ten- sion and demanding effort. is, they produce incessantly singular modes of interpretation and rela- tion with the world: On the other hand, creation is not just an effort because it includes the encounter. The encounter is the reflux of the search, for in it we are recep- tive […]. The encounter always has an unexpected edge, an element of unpredictability and surprise. We cannot be completely active in an encounter, but we must let our- selves be affected by what we encounter (Kastrup, 2007, p. 66-67, author’s translation). Final Remarks The inexhaustible capacity of the capitalist machine to renew it- self, to operate as a surface of registration, conformation and control of subversive practices, inserts all the time the risk of dissipation of resis- tances. It takes a constant work of mapping the biopower lines, and of its effects on bodies and on the possibilities of existence. In this sense, I believe that the construction of powerful educational practices, today, demands to rethink the ways we deal with the body, time, information Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1550 Mascarenhas and work. The ways we can favor and allow, through our choices and renunciations, the occurrence of intense and potent experiences. The ways in which we resist to what is presented to us as the only (or better) way of doing and knowing, how we learn and bravely assume places that are also a denial of the established logics. Certainly, there are still many others biopolitical forms that we need to identify and examine, as well as strategies to invent, in this continuous movement that is the production of existence. And in the case of education, the additional tasks of paying attention to the risk of stiffening the practices, and to the forms of subjection that they entail. Regardless if it is the teaching workforce, the technical and manage- ment staff or the students themselves of whom we are talking about, one needs to try not to end up reinforcing the negative, not to overbur- den other people with demands, deadlines and ways of doing that are not related to learn and create power, but to serve as a transmission en- gine that responds obediently to the bureaucratic machine, fulfilling the role of a good employee or good student. Learn how to say no to the gears of biopower, to purge of the guilt, of the will and the pressure of following successful models. Learn to reconstruct the relation with the lived time, to slow down the time, to ponder its utilities, its rationality and its linearity; replace its duration and intensity. Cultivate the use- less. Slowly learning, from experience. Seek the singularity, give pas- sage to the affections, seek answers proper to the problems that must, also, be placed within own forms. Biopolitics, Education and Resistance in the Contemporary World Biopolitics, Education and Resistance in the Contemporary World Notes 6 The following quotes on Larrosa’s work was freely translated by me from the Portuguese article published in 2002. It has become a very popular work in Brazil but, unfortunately, until the publication of the present article, I could not find a stable English version of this paper. 7 The following quote on Zourabichvili’s work was translated by me from the Portuguese version. There is an English version of this text available, published by Edinburgh University Press (Zourabichvili, 2012). 7 The following quote on Zourabichvili’s work was translated by me from the Portuguese version. There is an English version of this text available, published by Edinburgh University Press (Zourabichvili, 2012). Final Remarks In short, it can be said that a creative education involves a double movement: on the one hand, to create its own protective capes, its bod- ies without organs, vacuoles of time and space, its I prefer not to do it, that make it possible to suspend momentarily the biopolitical lines and create conditions for new experiences to take place and new meanings to be cultivated. On the other hand, it also involves making a passage, an exit from comfort zones, putting oneself at risk, overcoming oneself, letting one go through other ways of experiencing oneself and of relat- ing to others. Finally, it should be clarified that educating oneself in this way does not depend on revolutionizing, reforming or saving the education system as a whole (although rethinking and transforming it remain necessary and urgent guidelines). It is not a matter of claiming an ac- tion in the macro-political sphere, but instead beginning to act through the micro – knowing that there are lines of continuity, composition and tension between macro and micro, and that modifying one certainly changes the other. Translated by Leonardo Balbino Mascarenhas and proofread by Ananyr Porto Fajardo. Received on August 9, 2017 Approved on December 28, 2017 Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1551 Notes 1 Another version of this work, developed by the artist in Rio de Janeiro and exhibited at the Rio Art Museum in 2017, revealed new curious fears: of pigeon poop; of being alone; of the brother; of squinting eyes; of knives and drunk people; among others. 1 Another version of this work, developed by the artist in Rio de Janeiro and exhibited at the Rio Art Museum in 2017, revealed new curious fears: of pigeon poop; of being alone; of the brother; of squinting eyes; of knives and drunk people; among others. 2 Without trying to exhaust the subject, we can mention some names whose contributions strongly mark the studies in education, and that are considered “mandatory readings” in the field, such as: the works of Bourdieu and Passeron (1977), Paulo Freire (2005; 1996), John Holt (1972) and Ivan Illich (1971). The influence of Foucault’s thoughts on education, presented in a didactic and summarized form by Veiga-Neto (2007) and Gallo (2008), and in more details in the collection organized by Ball (1997). However, it is necessary to emphasize that there is a great diversity in the approaches, theories, critics and proposals of these authors. 3 This is not a univocal terminology; Singer (2010, p. 15) points out other names commonly used to designate these experiences: “romantic […] student-centered pedagogy […] free, progressive, alternative, democratic schools”. 4 Slow learning (or slow education, or slow school) is a movement inspired by the slow-food movement and proposes to rethink the ways in which the school is structured – from the rhythms of learning to the standardization of curricular structures and assessments, the role of parents in the educational process and, above all, the intertwined relationship between school (and learning) and capitalist ways of life (Holt, 2002). 5 This is what Beatriz Preciado suggests, when analyzing how current capitalism exerts a control of subjectivities from the most accurate management of the libido, and the drainage and “resolution” of mental states such as euphoria, excitement and relaxation, by the use of pharmacoponographic products, such as Prozac, testosterone, Viagra, insulin, alcohol, tobacco, cocaine… (Preciado, 2008). 6 The following quotes on Larrosa’s work was freely translated by me from the Portuguese article published in 2002. It has become a very popular work in Brazil but, unfortunately, until the publication of the present article, I could not find a stable English version of this paper. References ARTIST Talks: Rivane Neuenschwander at Whitechapel Gallery. Whitechapel Gallery Chanel, London, Jul. 2015. Available at: <https://www.youtube.com/ watch?v=7PIsEjtv_dk>. Accessed: 14 September 2016. ARTIST Talks: Rivane Neuenschwander at Whitechapel Gallery. Whitechapel Gallery Chanel, London, Jul. 2015. Available at: <https://www.youtube.com/ watch?v=7PIsEjtv_dk>. Accessed: 14 September 2016. BALL, Stephen J. Foucault y la Educación: disciplinas y saber. 3. ed. Madrid: Morata, 1997. BONDÍA, Jorge Larrosa. Notas sobre a Experiência e o Saber de Experiência. Revista Brasileira de Educação, Rio de Janeiro, n. 19, p. 20-28, jan./abr. 2002. Available at: <http://www.scielo.br/pdf/rbedu/n19/n19a02.pdf>. Accessed: 17 August 2015. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1552 Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1552 Mascarenhas BOURDIEU, Pierre; PASSERON, Jean-Claude. Reproduction in Education, Soci- ety and Culture. Beverly Hills: Sage, 1977. BOURDIEU, Pierre; PASSERON, Jean-Claude. A Reprodução: elementos para uma teoria do sistema de ensino. 3. ed. Rio de Janeiro: Francisco Alves Editora, 1992. CAMPBELL, Brígida (Org.). Exercício para a Liberdade. São Paulo: Invisíveis Produções, 2015. Available at: <http://issuu.com/invisiveisproducoes/docs/exerc__ cioparaaliberdadeissuu>. Accessed: 2 July. 2017. DELEUZE, Gilles; GUATTARI, Félix. Anti-Oedipus: capitalism and schizophre- nia. Minneapolis: University of Minnesota Press, 1983. DELEUZE, Gilles. Conversações. São Paulo: Editora 34, 1992. DELEUZE, Gilles. Negotiations, 1972-1990. Nova York: Columbia University Press, 1995. DELEUZE, Gilles; GUATTARI, Félix. O Anti-Édipo: capitalismo e esquizofrenia. São Paulo: Editora 34, 2010. FOUCAULT, Michel. The History of Sexuality Volume One: an introduction. Nova York: Pantheon Books, 1976. FOUCAULT, Michel. Vigiar e Punir: nascimento da prisão. Petrópolis: Vozes, 1987. FOUCAULT, Michel. História da Sexualidade I: a vontade de saber. Rio de Ja- neiro: Edições Graal, 1988. FOUCAULT, Michel. Discipline and Punish: the birth of the prison. 2. ed. Nova York: Vintage Books, 1995. FREIRE, Paulo. Pedagogia do Oprimido. 17. ed. Rio de Janeiro: Paz e Terra, 1987. FREIRE, Paulo. Pedagogia da Autonomia: saberes necessários à prática educa- tiva. 25. ed. São Paulo: Paz e Terra, 1996. FREIRE, Paulo. Pedagogy of the Oppressed. 30. ed. Nova York: Continuum In- ternational Publishing Group, 2005. GALLO, Silvio. Foucault: (re)pensar a educação. In: RAGO, Margareth; VEIGA- NETO, Alfredo (Org.). Figuras de Foucault. 2. ed. Belo Horizonte: Autêntica, 2008. P. 253-260. HOLT, John. How Children Learn. Middlesex: Penguin, 1972. HOLT, Maurice. It’s Time to Start the Slow School Movement. Phi Delta Kap- pan, Los Angeles, v. 84, n. 4, p. 264-271, dez. 2002. Available at: <http://courses. educ.ubc.ca/cite/socials/Articles/Maurice_Holt_Slow_Schools.pdf>. Accessed: 16 December 2017. ILLICH, Ivan. Deschooling Society. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1554 References New York: Harper & Row, 1971. ILLICH, Ivan. Sociedade sem Escolas. 7. ed. Petrópolis: Vozes, 1985. KASTRUP, Virgínia. Flutuações da Atenção no Processo de Criação. In: LECERF, Eric; BORBA, Siomara; KOHAN, Walter. Imagens da Imanência: escritos em memória de H. Bergson. Belo Horizonte: Autêntica, 2007. P. 66-67. LAZZARATO, Maurizio. Signos, Máquinas, Subjetividades – Signes, Machines, Subjectivities (edição bilíngue). São Paulo: Edições Sesc; N-1 Edições, 2014. PELBART, Peter Pál. O Avesso do Niilismo: cartografias do esgotamento – Car- tography of Exhaustion: nihilism inside out (edição bilingue). São Paulo: N-1 Edições, 2013. Educação & Realidade, Porto Alegre, v. 43, n. 4, p. 1537-1554, Oct./Dec. 2018. 1553 Biopolitics, Education and Resistance in the Contemporary World Biopolitics, Education and Resistance in the Contemporary World PRECIADO, Beatriz. Testo Yonqui. Madrid: Espasa, 2008. PRECIADO, Beatriz. Testo Yonqui. Madrid: Espasa, 2008. SINGER, Helena. República de Crianças: sobre experiências escolares de re- sistência. Campinas: Mercado de Letras, 2010. VEIGA-NETO, Alfredo. Foucault e a Educação. 2. ed. Belo Horizonte: Autêntica, 2007. VEIGA-NETO, Alfredo. Foucault e a Educação. 2. ed. Belo Horizonte: Autêntica, 2007. ZOURABICHVILI, François. O Vocabulário de Deleuze. Campinas: Centro In- terdisciplinar de Estudo em Novas Tecnologias e Informação (IFCH/Unicamp), 2004. Available at: <http://escolanomade.org/wp-content/downloads/deleuze- vocabulario-francois-zourabichvili.pdf>. Accessed: 25 July 2017. ZOURABICHVILI, François. Deleuze, A Philosophy of the Event: together with the vocabulary of Deleuze. Translated by Kieran Aarons. Edinburgh: Edin- burgh University Press. Edited by Gregg Lambert and Daniel Smith. Available at: <http://www.jstor.org/stable/10.3366/j.ctt3fgr6f>. Accessed: 13 June 2017. Leonardo Balbino Mascarenhas is a Brazilian educator, and has been in- volved in educational, artistic, social and economic projects since 2005. Currently is teaching at Universidade Federal de Alfenas – UNIFAL, at cam- pus Varginha/MG and developing research and production in the realms of education, literature, theater and contemporary philosophy. p E-mail: leo.mascarenhas@gmail.com p y p p y E-mail: leo.mascarenhas@gmail.com This is an open-access article distributed under the terms of the Creative Commons Attribution License 4.0 International. 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https://openalex.org/W4233830471	https://bmcendocrdisord.biomedcentral.com/track/pdf/10.1186/s12902-020-0514-9	English	null	Establishment of a type 1 diabetes structured education programme suitable for Chinese patients: Type 1 Diabetes Education in Lifestyle and Self Adjustment (TELSA)	Research Square (Research Square)	2,020	cc-by	7,600	Establishment of a type 1 diabetes structured education programme suitable for Chinese patients: type 1 diabetes education in lifestyle and self adjustment (TELSA) Yuting Xie1,2†, Fang Liu1,3†, Fansu Huang4, Chunna Lan5, Jia Guo6, Jing He7, Lezhi Li3, Xia Li1,2* and Zhiguang Zhou1,2 Xie et al. BMC Endocrine Disorders (2020) 20:37 https://doi.org/10.1186/s12902-020-0514-9 Xie et al. BMC Endocrine Disorders (2020) 20:37 https://doi.org/10.1186/s12902-020-0514-9 Open Access Abstract Background: Various guidelines recommend that all adults diagnosed with type 1 diabetes (T1D) should be offered an evidence based, structured education programme (SEP) to optimize self-management care. China has a 13,000 annual increase in newly diagnosed T1D cases, of which 65% are adults. However, there is yet no validated SEP targeted to T1D patients in China. The purpose of this study is to establish a structured T1D self-management education programme—‘Type 1 Diabetes Education in Lifestyle and Self Adjustment’ (TELSA) that is adapted to medical and cultural practices in China. Methods: TELSA programme was developed based on the ADDIE model, following three steps: i) Semi-structured interviews were administered to 10 healthcare professionals (HCPs) and 13 T1D patients. Different topic guides, focusing on 4 dimensions including goals, contents, format of delivery, and quality assurance, were designed for either HCPs or patients. The interviews were recorded and analysed with thematic analysis. ii) Extracted themes were modified according to Delphi consultation. iii) Preliminary courses were conducted as pilot study to evaluate the effects of TELSA and optimization of the curriculum was finalized accordingly. (Continued on next page) * Correspondence: lixia@csu.edu.cn †Yuting Xie and Fang Liu should be considered co-first author and contributed equally to this work. 1Department of Metabolism and Endocrinology, The Second Xiangya Hospital, Central South University, No.139 Middle Renmin Road, Changsha, China 2National Clinical Research Center for Metabolic Diseases, and Key Laboratory of Diabetes Immunology, Ministry of Education, Changsha, China Full list of author information is available at the end of the article Methods Study design The training programme was developed based on the ADDIE instructional design process [12–14]. Three es- sential steps: semi-structured interviews, nationwide Delphi consultation, and preliminary courses, were in- cluded in the development of TELSA. (Continued from previous page) (Continued from previous page) Results: A total of 18 themes in 4 dimensions of the programme design were identified in the final version: i) goals: ‘behaviour modification’ and ‘outcome improvement’; ii) contents: ‘living with T1D’, ‘self-monitoring of blood glucose’, ‘knowing insulin’, ‘insulin dose adjustment’, ‘carbohydrates and carbohydrate counting’, ‘hypoglycaemia’, ‘complications of diabetes’, ‘managing psychological issues’, ‘physical activity’, and ‘question-and-answer’; iii) format: ‘multidisciplinary team combined with peer support’, ‘face-to-face education followed by remote learning’, and ‘2- day programme held on weekends’; and iv) quality assurance: ‘after-class quiz’, ‘patients’ feedback’, and ‘long-term evaluation on effectiveness’. Conclusions: A type 1 diabetes structured education programme in China was set up and shown to be applicable under local medical, social, and cultural environment. Trial registration: NCT03610984. Date of registration: August 2, 2018. Trial registration: NCT03610984. Date of registration: August 2, 2018. Keywords: Type 1 diabetes, Structured education programme, Qualitative interviews, Delphi me have been diagnosed with diabetic retinopathy reached 17.3% in their investigation [11]. The severe status quo on diabetes-related comorbidity is largely due to the lack of diabetes self-management education from routine clinical care [11]. To date, there is no evidence based and validated SEP available to T1D patients in China. The lack of standard T1D educational approaches has resulted in remarkable regional disparities in diabetes control as well as varied clinical practice routines across different healthcare settings. © 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. Page 2 of 10 Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders Background Type 1 diabetes (T1D) patients suffer from continuous loss of pancreatic β-cell function and need life-long ex- ogenous insulin treatment. Adjusting insulin dose to keep glucose levels at target in a flexible daily routine re- mains an enormous challenge in T1D management. Dia- betes self-management education (DSME) is critical for patients to achieve HbA1c targets, minimise hypoglycaemia, and optimise quality of life. Self- management training programmes such as dose adjust- ment for normal eating (DAFNE) and the diabetes teaching and treatment programmes (DTTP) have con- sistently demonstrated positive outcomes in diabetes control as well as quality of life in multiple studies [1– 4]. Therefore, consensus has been reached across various guidelines that all adults diagnosed with T1D should be offered an evidence based, structured education programme (SEP) [5, 6]. An SEP is defined as ‘a planned and graded process that facilitates the knowledge, skills and ability for diabetes self-management and empowers individuals to live healthily, to maintain and improve their quality of life, and assume an active role in their diabetes care team.’ [5] It incorporates the needs, goals, and life experiences of patients [7, 8]. Five quality cri- teria of an SEP include i) patient-centered philosophy, ii) structured written curriculum, iii) trained educators, iv) quality assurance, and v) regular audit [5]. However, al- though structured education for T1D is an absolute ne- cessity, only limited countries have established proper SEPs [7]. Due to lifestyle and social differences, SEPs should be tailored to each country. There is an urgent unmet need for a high-quality, standard T1D curriculum that em- powers Chinese T1D patients with self-management skills and psychological support. Hence this study fo- cused on establishing the ‘Type 1 Diabetes Education in Lifestyle and Self Adjustment’ (TELSA) programme, the first T1D SEP designed to match the medical, social, and cultural environment in China. Delphi procedure The Delphi questionnaires were delivered to participants in person or via emails and responses were collected within two weeks. The questionnaire started with an introduction that briefly explained the purpose of the study, and included general information on participants. The main part of the questionnaire included 17 items developed from qualitative interviews. The five-point Likert scale was used to evaluate the degree of import- ance for each item (5 for ‘extremely important’ to 1 for ‘unimportant’). Participants’ basis of judgment and de- gree of familiarity for each item were also surveyed in this questionnaire. Free-text columns were included for collection of suggestions. Data were collated and re- presented to the research team for further refinement of TELSA. Here we defined a consensus regarding a spe- cific item as at least 80% agreement (with Likert score 4–5) [17]. Part 1: Semi-structured interviews Data collection A recent study showed that China has 13,000 newly diagnosed T1D cases every year in spite of a low inci- dence rate [9], and is now the fourth largest country in the number of T1D cases in the 0–14 age group [10]. Even with expanding categories of insulin and new tech- nologies, the mean HbA1c across all age groups reported in the cost, coverage, and care (3C) study in China remained as high as 8.9% [11]. Although only 35.2% of patients had eye exams, the percentage of patients that A qualitative, descriptive design was adopted. Healthcare professionals (HCPs) certified for T1D clinical care and adult T1D patients (≥18 years of age) were recruited with purposive sampling strategy as interviewees. Patients with diabetes duration ≥3 months and no medical his- tory of mental disorders or cognition impairment were recruited from 2 tertiary hospitals in Beijing and Chang- sha. HCPs who were certified for T1D clinical care and fa- miliar with diabetes education programmes were selected Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders (2020) 20:37 Page 3 of 10 Xie et al. BMC Endocrine Disorders Table 2 Characteristics of healthcare professionals interviewed Characteristics Professionals (n = 10) Gender, n (%) Male 1 (10.0) Female 9 (90.0) Age in years, mean (range) 37.9 (30.0–53.0) Highest level of education, n (%) Doctor 6(60.0) Master 4(40.0) Professional, n (%) Diabetologist 5 (50.0) Diabetes specialist nurse 2 (20.0) Diabetes educator 3 (30.0) Years of working, mean (range) 14.4 (6.0–31.0) from 4 tertiary hospitals in Beijing, Xi’an, Suzhou, and Changsha. All interviewees gave their informed consent. Interviews were conducted by a diabetes education nurse. Interview guidelines for either patients or HCPs (Add- itional file 1) were created by the research team and piloted in two interviews (not included in the analysis) to assess comprehensibility and avoid content that could be misconstrued or offensive. Questions were open-ended and leading questions were avoided. Each interview lasted 30 to 60 min and was audio-recorded with participants’ consent. In the last two interviews in each group, no new information related to the topic was obtained, which sug- gested that saturation was achieved and data collection was ended [15]. The final sample consisted of 13 T1D pa- tients and 10 HCPs (Tables 1 and 2). Data analysis professional network. Purposive sampling was conducted. Ultimately, a multidisciplinary and geographically diverse group of 25 Chinese-based experts were invited in this Delphi panel (Additional file 2). The five stages of thematic analysis were followed [16]. Records were transcribed verbatim within 24 h of each interview by the interviewer and were re-read and veri- fied. NVivo 11.0 (QSR International Pty Ltd) was used for data analysis. Inductive content analysis was per- formed [16]. The transcripts were then coded and themes were identified, followed by discussion and examination with a second researcher. Disagreements were discussed within the research team to aid refinement. Part 2: Delphi consultation Participants Experts were selected as potential participants if they are: i) recognized experts in the field of T1D or diabetes edu- cation, ii) researchers with expertise in diabetes, educa- tion, or psychology, identified through literature review, and iii) researchers recommended through experts’ Table 1 Characteristics of patients interviewed Characteristics Patients (n = 13) Table 1 Characteristics of patients interviewed Characteristics Patients (n = 13) Gender, n (%) Male 6 (46.2) Female 7 (53.8) Age in years, mean (range) 30.9 (19.0–52.0) T1D duration in years, mean (range) 10.1 (0.5–41.0) Highest level of education, n (%) Junior high 2 (15.4) Senior high 1 (7.7) College/University 7(53.8) Postgraduate 3(23.1) Marital status, n (%) Married 5 (38.5) Single 8 (61.5) Goals Theme 1: Outcome improvement All patients and HCPs ranked improving diabetes outcome, including both physiologically and psychologically, the most im- portant goal of diabetes education. To live ‘a normal life’ or have ‘better quality of life’ is also of top concern. In addition to basic concepts, patients were gener- ally more concerned about ‘to build a healthy attitude towards diabetes’ in the first place. 8 out of 13 pa- tients mentioned an ice-breaking start with establish- ing their confidence to live with diabetes for the rest of their lives. ‘A qualified education programme is supposed to im- pact on patients’ metabolic control. It is critical in improving the quality of life for patients and their family.’ [Healthcare professional (H) 1]. ‘I think in the first class, it should be clarified what the benefits are for controlling diabetes. It should be pointed out that we can still have a normal life if diabetes is under control, so as to build our confi- dence.’ ‘I want to reduce the number of hypoglycaemic epi- sode as well as incidences of very high blood sugar, and ultimately, to avoid complications.’ ‘What I hope is to be able to relieve my anxiety, and thus live a normal life.’ [P9]. Theme 2: Behaviour modification HCPs in China will sometimes encounter T1D patients admitted with dia- betic ketosis due to discontinuation of insulin therapy, because of invalidated treatments that can ‘cure’ T1D. Moreover, patients are frequently confused with differ- ent insulin categories and risk purchasing a wrong cart- ridge. Therefore, most HCPs recognized the importance of educating patients to achieve sufficient basic know- ledge to avoid unnecessary cost. Theme 2: Self-monitoring of blood glucose (SMBG) The importance of SMBG has been greatly underesti- mated by most patients [18]. Over 60% patients in our T1D clinic tested blood glucose levels less than 4 times per day (data unpublished). However, only a few patients in the interview mentioned SMBG. ‘I started with testing my blood glucose levels at least 7 times per day in the hope that I could figure out my glucose pattern. But soon I found it was a mis- sion impossible. Then I just gave up and let it be. Now I just test randomly. As long as it is neither too high nor too low, I’m satisfied.’ [P4] ‘The most important goal of education is to reduce fundamental errors. [H2]. comments column. Questions covered degree of satisfac- tion, course comprehensibility, acceptability, and useful- ness. The quiz contained at least 2 questions per session, with an emphasis on carbohydrate (carb) counting and in- sulin dose adjustment. Pre-testing of the quiz was con- ducted among researchers to avoid misunderstandings. Curriculum was reviewed and modified by the research team according to patients’ responses. [ ] ‘Through education, patients will have a correct un- derstanding of diabetes self-management. … With various online resources that might be misleading, patients need easy access to professional educational materials that can teach correct from incorrect.’ [H4]. Part 1: Semi-structured interviews Part 1: Semi-structured interviews Totally 17 themes in the 4 dimensions of curriculum were identified. ‘In the very beginning patients need to clearly under- stand what they are dealing with every day. … They should know, for example, what causes T1D, and what some of the most common misunderstandings of T1D are.’ [H7]. Results Theme 1: Living with T1D HCPs all agreed that the teaching session should start with a basic introduction to T1D. ‘I’ve heard there is a way to check my blood glucose Part 3: Preliminary courses y The first two courses were piloted to evaluate the ac- ceptability and organization of the curriculum. An- nouncements of the courses were released in the WeChat (Tencent Corp) public group where all the registered T1D patients in our T1D clinic had access to. For each course, the first 10 adult patients volun- teered to join were selected as attendees. After the course, all attendees completed feedback question- naires and a quiz designed by the research team (Additional file 3). Patients were also encouraged to leave their free text comments. Questionnaires were pre- sented in a multiple-choice format with free-text Xie et al. BMC Endocrine Disorders (2020) 20:37 Page 4 of 10 Xie et al. BMC Endocrine Disorders (2020) 20:37 correct time.’ [H7]. HCPs also emphasize on self-adjustment of insulin doses in daily routine in order to help patients to main- tain ‘a normal life’. Patients’ negligence on SMBG is exactly why HCPs emphasize this topic. New techniques for glucose testing such as continuous glucose monitoring also need to be introduced. ‘Blood glucose levels may vary every day. Instead of fixed doses prescribed by the doctors, it’s more im- portant for the patients to learn how to adjust insu- lin doses properly according to their own meals and activities.’ [H10]. ‘In addition to the normal range, frequency, and cor- rect procedures of testing blood glucose levels, the importance of SMBG should also be emphasized in different conditions, helping patients to form a habit of testing.’ [H7]. Theme 5: Carbohydrate and carb counting Carbohy- drates are the staple food in Chinese cuisine. Although cooking methods and ingredients are more diversified and harder to predict compared to western food culture, consensus was still reached that, along with insulin, identifying carbohydrates, knowing carb counting and carb-insulin ratio, are fundamental. Theme 3: Managing psychosocial stress Almost all pa- tients were stressed at different levels and occasions— stress in the process of schooling, working, and estab- lishing relationships. Some patients have difficulties in communicating with people around them about diabetes. ‘Through understanding how carbohydrates and other ingredients affect blood glucose levels, patients can learn to inject boluses before meals or snacks. If they want to live a less restricted life while keeping stable blood glucose levels, carb counting is a basic skill.’ [H9]. ‘My previous research found that T1D patients are under great stress but generally don’t know how to relieve it, or to effectively communicate with families and friends to gain support.’ f g pp [H3]. ‘Since I have been diagnosed with diabetes, I have not been in the mood for starting a romantic rela- tionship. I strongly believe no one will accept a young man with diabetes as a boyfriend.’ ‘Since I have been diagnosed with diabetes, I have not been in the mood for starting a romantic rela- tionship. I strongly believe no one will accept a young man with diabetes as a boyfriend.’ ‘I heard from other patients about reading food la- bels, but I still have no idea what to look at. Energy? Carbohydrates? What’s the meaning of reading these numbers anyway? Goals For instance, some patients choose invalidated Chinese traditional medicine in- stead of insulin in the hope of curing T1D; some pa- tients misuse NovoRapid 30 Mix as bolus instead of NovoRapid. These can be avoided through proper education right after diagnosis.’ ‘I’ve heard there is a way to check my blood glucose Page 5 of 10 Page 5 of 10 Page 5 of 10 Xie et al. BMC Endocrine Disorders Xie et al. BMC Endocrine Disorders (2020) 20:37 levels without finger prick. I’d like to know more about that.’ [P10]. correct time.’ [H7]. correct time.’ [H7]. correct time.’ [H7]. ‘There need to be a part where patients can freely and directly ask whatever questions they have during the course.’ ‘Question & Answer must be included… To guaran- tee that each attendee can have at least one actual problem solved.’ [P5]. ‘I don’t know what type of exercise fits me. I assume exercise can lower blood sugar, but my sugar level can’t even drop for 1 mmol/L after running for 5 kilometres.’ Format of delivery Theme 1: Multidisciplinary team combined with peer support SEPs in other countries are mainly conducted by a team of one diabetes education nurse and one diet- ician. Nevertheless, most HCPs interviewed embraced a multidisciplinary team of educators, with psychologists and physical therapists included. ‘Physical activity definitely has profound effects on glucose levels thus should be discussed. For example, patients need to learn how glucose levels might fluc- tuate with different types, duration, and strength of activities; how to make certain adjustments on food intake and insulin dose before, during, and after physical activities.’ ‘A multidisciplinary team is essential. Different con- tents should be elucidated by specific specialists—- diets by dieticians, dose adjustment by diabetologists, and so on.’ [H4]. ‘A multidisciplinary team is essential. Different con- tents should be elucidated by specific specialists—- diets by dieticians, dose adjustment by diabetologists, and so on.’ [H4]. Patients also mentioned the lack of time, and more importantly, perseverance to exercise regularly. ‘I know the benefits of regular exercises, but it’s really easier said than done. I am so exhausted to even move my legs after work every day. I do want to know whether there is a better way to set myself in motion.’ [P7]. From patients’ perspectives, peer support was indis- pensable. Patients are more likely to gain strength from people in the same condition. ‘Experiences from well-managed patients will be quite valuable. ‘Long illness makes the patient a good doctor’. We cannot learn those personal experi- ences from textbooks or professionals. Plus, they can inspire us to never lose hope.’ [P12]. ‘Experiences from well-managed patients will be quite valuable. ‘Long illness makes the patient a good doctor’. We cannot learn those personal experi- ences from textbooks or professionals. Plus, they can inspire us to never lose hope.’ [P12]. Theme 8: Complications of diabetes Screening for complications is recommended by diabetes guide- lines [5, 6]. A comprehensive introduction to dia- betic complications in plain language should be available. Theme 2: Face-to-face education followed by remote learning Face-to-face group learning remains the most effective setting by all participants. However, different from the large learning groups commonly seen among T2D patients in China, ‘small-group education’ was preferred for T1D. Meanwhile, with the rapid devel- opment of mobile health (mHealth) technologies, dia- betes remote learning apps has become a welcomed supplement. ‘There must be a session for (chronic) complications, especially for the screening part.’ [H4]. correct time.’ [H7]. I am so afraid of being in the supermarket now, dare not to buy anything.’ [P9]. ‘All my classmates know that I’m diabetic. Every time I inject insulin before a meal, I think they are pitying me. So I just hide. I don’t like discussing it with others.’ ‘All my classmates know that I’m diabetic. Every time I inject insulin before a meal, I think they are pitying me. So I just hide. I don’t like discussing it with others.’ Theme 6: Hypoglycaemia Hypoglycaemic events occur to every T1D patients, but not everyone knows the right procedure to handle it. All basic dimensions of hypoglycaemia need to be elucidated. Theme 4: Insulin Insulin is essential for blood glucose control in T1D. Both the dose and timing of bolus injec- tions are key factors in controlling post-prandial glucose [19]. However, it is not easy for the patients to under- stand how different types of insulin work. ‘In fact, quite a few patients don’t realize that they are correcting hypoglycaemia in a wrong way. They need to learn how to treat and prevent hypoglycaemia correctly.’ [H3] ‘In fact, quite a few patients don’t realize that they are correcting hypoglycaemia in a wrong way. They need to learn how to treat and prevent hypoglycaemia correctly.’ [H3] ‘I want to learn the effect (PK-PD) of different insu- lin, like the time of onset, peak time, and etc.’ ‘Besides, we need to let them understand what causes low blood glucose levels.’ [H9] ‘Patients need to know the basics of insulin, such as its physiological effects and classification. This is the prerequisite of administering correct insulin at ‘I want to know how to reduce hypoglycaemic events.’ [P2] Page 6 of 10 Page 6 of 10 Page 6 of 10 Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders Theme 7: Physical activity When it comes to the pa- tients’ physical activity, the biggest obstacle is know- ing the trend of glucose fluctuations during and after exercises. Some experienced repeated hypoglycaemia after household chores. Some didn’t know their glu- cose changes because they never checked before or after. Format of delivery ‘…How to prevent and detect early signs of diabetic complications.’ [H5]. ‘If we could know what diabetic complications feel like, probably we would pay more attention to them.’ [P4]. ‘There must be a session for (chronic) complications, especially for the screening part.’ ‘I prefer patients sitting together because we can learn from each other. However, I’m not good at memorizing things, so it would be very helpful if we can watch video recordings back home.’ [P4]. Theme 9: Question-and-answer Participants consid- ered an opportunity for further discussion with HCPs and experienced patients a good summary of the whole course. ‘Small-group teaching followed by remote learning is better. Remote learning should be via video Page 7 of 10 Xie et al. BMC Endocrine Disorders (2020) 20:37 recordings. It is more direct and intuitive.’ [H8]. how well they have learned during class, and what needs to be explained again.’ how well they have learned during class, and what needs to be explained again.’ ‘An extremely important indicator is patients’ re- sponses, such as their experiences, degree of satisfac- tion, and so forth.’ ‘An extremely important indicator is patients’ re- sponses, such as their experiences, degree of satisfac- tion, and so forth.’ Theme 3: 2- to 3-day programme held on weekends or holidays Most interviewees agreed that the length of face-to-face education should not exceed three days. ‘I think the final evaluation needs to be thorough, in- cluding both biomedical and psychological out- comes.’ ‘Weekends are good options. It won’t conflict with any work. Besides, I think it will be too much to di- gest if the course lasts for more than 3 full days.’ [P8]. [H3, H5, H8]. Quality assurance All 17 themes reached consensus and results are shown in Table 3. Therefore, all items were preserved and no more rounds of Delphi were performed. According to experts’ suggestions, the research group modified the curriculum by i) changing the Chinese title for each lesson to make the course more intriguing, ii) splitting ‘insulin’ to ‘knowing insulin’ and ‘insulin dose adjust- ment’, iii) adding knowledge on diabetic ketoacidosis in the session ‘complications of diabetes’, and iv) arranging the course as a 2-day weekend event. Questions about quality assurance were only adminis- tered to HCPs. Three themes were considered useful for developing an assessment system: ‘After-class quiz’, ‘Pa- tient’s feedback’, and ‘Long-term evaluation of effective- ness’. While the first two themes mainly aim at examining immediate knowledge capture, general ac- ceptance by patients and comprehensibility of the programme, the third theme evaluates effectiveness to provide insights for further refinement of the programme. aAgreement ratio is the proportion of experts scoring the item 4–5 points with Likert scale Part 3: Preliminary courses ‘A quiz held right after class is the most straight for- ward way to test the acceptance and outcome of teaching. You could know from attendees’ responses In total, 20 T1D patients (Additional file 4) attended and 3 family members audited the courses. Degree of satis- faction was 100%, and all attendees rated the programme Table 3 Results of Delphi consultation Dimension Item Agreement ratioa (%) Full mark ratio (%) Degree of importance Mean SD CV Goals Behaviour modification 100.00 92.00 4.92 0.28 0.06 Outcome improvement 100.00 96.00 4.88 0.37 0.08 Sessions Living with T1D 96.00 80.00 4.60 0.63 0.13 Self-monitoring of blood glucose 100.00 92.00 4.88 0.44 0.09 Managing psychosocial stress 92.00 68.00 4.28 0.80 0.19 Insulin 100.00 96.00 4.72 0.33 0.07 Carbohydrates and carb counting 100.00 88.00 4.76 0.37 0.08 Hypoglycaemia 100.00 84.00 4.72 0.54 0.12 Physical activity 100.00 80.00 4.68 0.52 0.11 Complications of diabetes 96.00 84.00 4.48 0.56 0.12 Question-and-answer 96.00 72.00 4.52 0.82 0.18 Format Multidisciplinary team combined with peer support 100.00 96.00 4.84 0.20 0.04 Face-to-face education followed by remote learning 96.00 84.00 4.76 0.63 0.13 2- to 3-day programme held on weekends or holidays 92.00 72.00 4.48 0.93 0.24 Quality assurance After-class quiz 100.00 80.00 4.64 0.70 0.15 Patients’ feedback 96.00 84.00 4.88 0.54 0.11 Long-term evaluation on effectiveness 96.00 80.00 4.60 0.85 0.18 aAgreement ratio is the proportion of experts scoring the item 4–5 points with Likert scale Table 3 Results of Delphi consultation Page 8 of 10 Xie et al. BMC Endocrine Disorders (2020) 20:37 Page 8 of 10 Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders centered approach. In this study, we integrated three procedures employing the ADDIE model. as ‘Extremely helpful’. 13 patients expressed their grati- tude for this programme in comments. The average score on the quiz was 70%, and most incorrect answers were in dose calculation. According to patients feedback, ‘managing psychosocial stress ’ was expanded to ‘man- aging psychological issues’, the teaching methods of ‘carbohydrate counting’ and ‘insulin dose adjustment’ were modified to improve comprehensibility, and lunch break was extended to 2 h. Class schedule is shown in Table S5 (Additional file 5). The final version of TELSA programme is shown in Table 4. Semi-structured interview is a commonly used ap- proach for data collection in qualitative researches [21]. Part 3: Preliminary courses Diverse social and cultural environment of patients must be taken into consideration for educational programme to be feasible and effective. Thus, data were collected from patients and HCPs living and working in China, taking into account their own social and cultural situ- ation [22]. Additionally, the qualitative design of Part 1 helped us to understand patients’ perspectives and daily life. The validity of results from interviews was further verified with Delphi method. Delphi consultation is a validated and widely used technique for obtaining a con- sensus [23, 24]. In our study, the participants were not chosen randomly but were representative of their profes- sional specialty [25], and had extensive experience with diabetes or education, as the average years of working experience was about 18 years. In the preliminary courses, all attendees reported a high level of satisfaction with the programme, regardless of diabetes duration or the current level of self-management skills. This suggests that the programme is well-fit for Chinese culture and social environment. However, patient enrollment methods in the preliminary courses favored patients with stronger initiation and willingness to receive diabetes education, thus a more positive feedback to the course. Further studies with more representative, average T1D patients are needed to evaluate the generalizability of this study to a broader T1D patient population. Discussion DSME is an essential component of diabetes manage- ment, especially in T1D [20]. Unfortunately, few T1D patients in China receive systematic DSME [11] due to the lack of standardized, validated SEPs. Therefore, we developed the first programme specific to T1D educa- tion in China. Validity of the development process The overall curriculum was based on the ADDIE (i.e. Analysis, Design, Development, Implementation, and Evaluation) framework, ‘a cyclic process that evolves over time and continuous throughout the instructional planning and implementation process’ [14]. ADDIE is widely used in the development of training programmes and can assist developers in instituting a learner- Table 4 Final version of TELSA Dimension Theme Goals 1. Behaviour modification 2. Outcome improvement Sessions 1. Living with T1D 2. Self-monitoring of blood glucose 3. Managing psychological issues 4. Knowing insulin 5. Carbohydrates and carb counting 6. Insulin dose adjustment 7. Hypoglycaemia 8. Physical activity 9. Complications of diabetes 10. Question-and-answer Format 1. Multidisciplinary team combined with peer support 2. Face-to-face education followed by remote learning 3. 2-day programme held on weekends Quality assurance 1. After-class quiz 2. Patients’ feedback 3. Long-term evaluation on effectiveness Table 4 Final version of TELSA Comparisons with prior SEPs and perspectives Derived from DTTP in Dusseldorf, Germany [26, 27], structured education courses have been adapted and im- plemented internationally. The principle approach is a flexible intensive insulin therapy adjusted to the carbo- hydrate component of the meal and the blood glucose level informed by SMBG [28]. Nonetheless, education needs to be tailored to fit the local audience. In a study by Grant et al. [29], glucose diary and doctor question- and-answer sessions were not considered as core com- ponents by interviewed educators in UK. However, as was shown in various themes such as ‘SMBG’ from our interviews, the perspectives of patients were quite differ- ent from those of educators. Therefore, our study inte- grated patients’ actual opinions with HCPs’ professional knowledge to maintain intactness of the whole programme, and was designed for the current status of diabetes management in China. Another aspect that gives rise to regional divergence is the intensity of course delivery. Most SEPs in the UK are either 5 consecutive days or over several weeks [29, 30]. PRIMAS and DTTP in Germany are held during a 6-week Xie et al. BMC Endocrine Disorders (2020) 20:37 Xie et al. BMC Endocrine Disorders Page 9 of 10 period with 2 sessions per week [3]. These formats are quite unpractical in China due to diverse working envir- onment as well as challenges in commuting. TELSA is de- signed as a 2-day programme with 10 lessons, which has not yet been reported in other SEPs. Funding h k This work was supported by the National Key R&D Program of China (2017YFC1309604), and the Clinical Nursing Research Fund Project of the Second Xiangya Hospital, Central South University (2017-YHL-03). 2017YFC1309604 coverd printing costs, classroom room services, reading materials for course participants, and payments for teachers. 2017-YHL-03 covered consulting fee for the Delphi panel. The funding body did not in- volve in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Ethics approval and consent to participate The study was approved by the Research Ethics Committee of the Second Xiangya Hospital, Central South University. All participants provided written informed consent prior to participation. Acknowledgments We would like to thank all participants for sharing their perspectives in the course of this research. We are grateful for Weiyan Li PhD for her helpful suggestions and revisions on the manuscript. Validity of the development process A DAFNE course de- livered 1 day a week for 5 consecutive weeks (5-week course) was shown to be equivalent with the traditional 1- week course in terms of biomedical and psychosocial out- comes [31]. A 2.5-day educational intervention delivered through 6 weeks had no significant improvement on HbA1c or severe hypoglycaemia, but did show improve- ments in diabetes treatment satisfaction and patient em- powerment [32]. However, intensity of delivery will influence course attendance [33, 34], and there has been no research focusing on the social-economic aspect of SEPs. We believe the shorter the course is, the less bar- riers there will be for attendance and implementation [34]. Whether 2-day is sufficient for an informative educa- tion course needs further evaluations. Additional file 2: Table S3 Characteristics of participants in Delphi consultation. Additional file 3: Quiz and a sample of feedback questionnaire. Additional file 4: Table S4 Characteristics of T1D patients in preliminary courses. Additional file 5: Table S5 Class schedule of TELSA. Abbreviations SEP St t d SEP: Structured Education Programme; SMBG: Self-monitoring of blood glucose; T1D: Type 1 Diabetes; TELSA: Type 1 Diabetes Education in Lifestyle and Self Adjustment Author details 1Department of Metabolism and Endocrinology, The Second Xiangya Hospital, Central South University, No.139 Middle Renmin Road, Changsha, China. 2National Clinical Research Center for Metabolic Diseases, and Key Laboratory of Diabetes Immunology, Ministry of Education, Changsha, China. 3Clinic Nursing Teaching and Research Section, The Second Xiangya Hospital, Central South University, Changsha, China. 4Department of Clinical Nutrition, The Second Xiangya Hospital, Central South University, Changsha, China. 5Department of Rehabilitation, The Second Xiangya Hospital, Central South University, Changsha, China. 6Xiangya School of Nursing, Central South University, Changsha, China. 7Medical Psychological Center, The Second Xiangya Hospital, Central South University, and Medical Psychological Institute of Central South University, Changsha, China. Availability of data and materials The datasets used and/or analysed during this study are available from the corresponding author on reasonable request. Authors’ contributions ZZ, XL, FL and YX conceived and designed the research. YX and FL drafted the manuscript. YX and LL revised the manuscript. FL conducted interviews and collected data. XL and FL analyzed the data. YX, XL, FL, FH, CL, JG, and JH contributed to the design, implementation and revision of the curriculum. All authors read and approved the final version of the manuscript. Regardless of course intensity, some education ses- sions, such as calculating insulin doses, are generally considered challenging by both educators and course participants [29]. Many participants struggle to sustain behavior changes over time [35]. Results of after-class quiz in our preliminary courses also indicated that it would require patients more efforts to master carbohy- drate counting and insulin dose calculation. Hence long- term improvements in glucose control after attending SEPs is compromised. Additionally, although classic SEPs are all face-to-face group education, this format also becomes a barrier for attendance [36]. Therefore, remote learning was highly advocated in our study to provide sustained education. It also has been suggested that providing practical follow-up support will likely ex- tend the benefits of SEPs [35, 37, 38]. In addition to traditional SEPs, new formats and technologies are worth exploring in the near future. Consent for publication Not applicable. We developed TELSA—the first SEP targeted at Chinese adult T1D patients, using semi-structured interviews and Delphi method. The course was piloted and shown to be applicable in the local medical, social, and cultural environment. We will further conduct randomized con- trolled trials to evaluate the effectiveness of the programme. 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https://openalex.org/W4391311253	https://link.springer.com/content/pdf/10.1007/s40299-023-00801-6.pdf	English	null	The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey in Western Australia and Queensland	The Asia-Pacific education researcher	2,024	cc-by	11,342	The Asia-Pacific Education Researcher (2024) 33:1287–1301 https://doi.org/10.1007/s40299-023-00801-6 REGULAR ARTICLE REGULAR ARTICLE The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey in Western Australia and Queensland Olivia Johnston1 · Rebecca Spooner‑Lane2 · Wei Zhang3 · Suzanne Macqueen4 · Nerida Spina2 Accepted: 6 December 2023 / Published online: 29 January 2024 © The Author(s) 2024 Abstract Grouping students into separate classes accord- ing to their ‘ability’ is an inequitable practice that does not, overall, improve academic outcomes. Research has continued to show that class ability grouping widens the educational gap between students from disadvantaged and privileged backgrounds. PISA data analysis suggests that class ability grouping continues to be used in Austral- ian schools, at least in Year 10. However, no research has characterized the existing class ability grouping practices being used in secondary schools from Years 7 to 9. The findings reported in this paper add quantitative evidence to the literature, showing that students are experiencing differ- ent class ability grouping practices according to their year group, subjects, and locations. An across-state survey about class ability grouping was conducted with respondents from 143 schools providing data about the schools’ class ability grouping practices. The findings reveal variations in how class grouping practices are used by schools in Australia that have been not captured in other research: Class abil- ity grouping was activated differently in different schools, increased as students’ year levels increased, was most preva- lent in Maths and English, and was used more extensively in Western Australia than in Queensland. The findings use descriptive statistics to show that students in Australia are experiencing different grouping practices, with discussion of how some practices are more inequitable than others. This raises questions about how the varied forms of class ability grouping that are being employed are contributing to educa- tional inequalities in Australia and how future research can address this problem. Keywords Ability grouping · Tracking · Social equity · Secondary education Keywords Ability grouping · Tracking · Social equity · Secondary education * Olivia Johnston o.johnston@ecu.edu.au 1 Edith Cowan University, Perth, Australia 2 Queensland University of Technology, Brisbane, Australia 3 The University of Western Australia, Perth, Australia 4 The University of Newcastle, Newcastle, Australia Class Ability Grouping This paper focuses on a type of ability grouping called ‘class ability grouping.’ The phrase ‘class ability grouping’ demarcates the practice from other forms of ability grouping to clearly indicate what this research is, and is not, about. This research is about ability grouping by class, not about ability grouping within the class or selective schooling. It focuses on practices where students are grouped into separate classes within the school according their ‘ability.’ Educators and the general public in Australia often refer to class ability grouping as ‘streaming,’ but this can evoke confusion for international audiences (Chmielewski, 2014). Words and phrases including ‘tracking,’ ‘achievement grouping,’ ‘attainment grouping,’ or ‘course-by-course ability grouping’ are also used to describe variations of class ability grouping across international contexts (Kaya, 2015; Regan & Jesse, 2019; Van Houtte et al., 2013). Previous research has found that more rigid forms of class ability grouping, such as streaming students for all classes in the same groups or inflexible tracking, are more inequitable than more flexible practices, such as subject-by-subject groupings or where students are only grouped for some learning areas (Chmielewski et al., 2013; Francis et al., 2020). Thus, it is important to be precise about the kind of ability grouping being described. Students’ placement into classes by ‘ability’ reflects the students’ pre-existing educational advantages and disadvantages. The term ‘ability’ is often placed in quotations in this paper to acknowledge that that the concept is problematic and can be contested. ‘Ability’ is defined here a social and cultural construct that is used to group students into classes for learning, rather than an indication of any innate or ‘natural’ capacity that students may or may not have (Francis et al., 2017). Research increasingly lends evidence to critical consideration of notions of finite ‘ability,’ which can be associated with deficit thinking and assumptions about students’ prior background and educational advantages/disadvantages (Clarke, 2014; Dweck, 2012; Hart et al., 2014). The history of ‘ability’ in education is not related to any fixed academic potentials – instead, ‘ability’ is a guise through which social and cultural power and privilege are preserved (Clarke, 2014; Hart et al., 2014). This problem is evident in the tools that are often used to assess student ‘ability,’ such as the difficulty in developing equitable and consistent instruments for identifying gifted and talented students (Thraves et al., 2021). Introduction International data collected by the OECD’s Program for International Student Assessment (PISA) indicates that most Australian schools group students into classes by ‘ability’ during Year 10 of secondary school (OECD, 2014, 2018b). This practice is called ‘class ability grouping,’ and it has persisted in Australia despite clear findings from research over many decades that there is no overall academic benefit to students and that in fact many students are disadvantaged by the practice (Ansalone, 2010; Boaler et al., 2000; Hodgen et al., 2022; Ireson & Hallam, 2009; Slavin, 1987). Class ability grouping exacerbates the effect of student background on achievement at school, perpetuating pre-existing disadvantages, and increasing the educational achievement gap (Francis et al., 2020; Razer et al., 2018). To date, there has not been any wide-scale quantitative research characterizing the extent to which class ability grouping is being used in Australian schools. Australian research about class ability grouping has tended to be qualitative, focusing on social equity issues or benefits for high ability learners (Johnston & Taylor, 2023). Some quantitative research has drawn on international PISA data that includes Australia to make comparisons between the effects of ability grouping in different countries (Johnston & Taylor, 2023). What is absent is empirical evidence that illuminates the scope and detail of class grouping practices * Olivia Johnston o.johnston@ecu.edu.au 1 Edith Cowan University, Perth, Australia 2 Queensland University of Technology, Brisbane, Australia 3 The University of Western Australia, Perth, Australia 4 The University of Newcastle, Newcastle, Australia (0121 1288 O. Johnston et al. are used including selective schooling, where whole schools are grouped by ability, and within-class grouping, where teachers form ‘ability’ groups within mixed-ability classes. Thus, the phrase ‘class ability grouping’ distinguishes ability grouping that organizes students into separate classrooms from other forms of ability grouping, which include grouping within the class, or selective schooling. The research presented here characterises class ability grouping during Years 7–9 in two Australian states. being used across the country. Existing research does not provide data on across-state differences or provide detail about specific ability grouping practices. For example, when does ability grouping start and for which subjects? How is it used in Years 7–9, and how many ‘levels’ of class groups are students grouped into? Introduction The answers to these questions could provide details about how class ability grouping is practiced and in doing so highlight potential implications for reducing educational inequalities in Australia through changes to class grouping practices to being used. International and Australian evidence cautions against the use of class ability grouping because it exacerbates social inequalities, segregating students into separate classes according to their backgrounds (Steenbergen-Hu et al., 2016). International comparative research findings show that countries that use class ability grouping have less equity in academic outcomes than countries with more mixed-ability classes, such as Japan and Finland (Luschei & Jeong, 2018; Parker et al., 2021). Researchers have investigated how class ability grouping has detrimental effects on equity outcomes because it increases the influence of students’ socio-economic status (SES) background on academic achievement (Castejón & Zancajo, 2015). Students from lower SES backgrounds achieve less academically than their more privileged peers when they are grouped into classes by ‘ability’ (Castejón & Zancajo, 2015; Chmielewski, 2014). Others have found that class ability grouping increases the association between racial/ethnic background and academic outcomes, particularly for students from traditionally marginalized groups (Razer et al., 2018). The study presented in this paper addresses the need for quantitative research about how class ability grouping is practiced in Australia. A survey about class grouping practices from n = 143 schools in Queensland and Western Australia addresses this gap by characterizing grouping practices in terms of how, where, and when class ability grouping is used in Years 7–9 of secondary school. Class Ability Grouping England is a country characterized in comparative PISA analysis as using similar methods of class ability grouping as Australia, but there has been no examination of how practices in Australia are affecting students (Schmidt et al., 2015). f In secondary schools, ability-based classes may be used for all or only some subjects and are more common for some subjects than others (OECD, 2018b). When schools choose to organize classes by ability, a wide range of approaches can be implemented. Variations have been reported based on which students are grouped for and at what age they are grouped (Johnston et al., 2022). Maths has often been the focus of ability grouping research because ability grouping is common in the subject (Jaremus et al., 2022). Further variations in class streaming practices include whether or not the students are in the same between-class ability groups for all, some, or none of their subjects. Previous research has found that these subtle differences can impact students’ academic outcomes (Chmielewski, 2014), so they are important considerations for class ability grouping practitioners. Thus, further research that characterises the class ability grouping practices being used in countries like Australia is important. There are some perceived benefits of class ability group- ing in Australia, including a reduced load on teachers who hold perspectives that class ability grouping enables them to cater for a reduced range of student ability (Mills et al., 2014). The practice might also seem to be reasonable within Australian culture where beliefs about fixed ‘ability’ and static potential to succeed academically have long prevailed. If one believes that students can be ranked according to such ‘ability,’ then class ability grouping might seem an intuitively reasonable approach. This also might be due to its common use over many decades, it has become a ‘nor- malised’ way of organizing classes and is widely accepted (Perry, 2016; Vialle et al., 2015). Other evidence from Aus- tralia shows that stakeholders in education hold perspectives that class ability grouping is beneficial. Parents, students, and teachers tend to view class ability grouping as benefit- ing high ability students who are placed in the ‘top’ classes (Kronborg & Cornejo-Araya, 2018; Noor, 2018). Teachers can also view ability grouping as making their teaching load more manageable because it is easier to differentiate and that students benefit when they can learn at a pace and level appropriate for them (Francis et al., 2020). Class Ability Grouping To address the language issues in ability grouping research, international research is increasingly adopting the term ‘between-class ability grouping’ to capture all variations of practices that group students into separate classes according to their ‘abilities’ (Papachristou et al., 2021). The phrase is simplified to ‘class ability grouping’ in this paper. The use of this phrase is especially practical in the Australian context where a broad range of practices Notions of ‘ability’ are inextricably linked with students’ backgrounds, including their SES and minority 1 3 3 The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… 1289 uses subject groupings, as opposed to class ability grouping that is less flexible where students are grouped the same way for all classes (Chmielewski, 2014). Previous international comparative research has suggested that the effects of ability grouping depend on the type of grouping being used, with rigid forms where students are fully streamed across all subjects less inequitable than ‘course-by-course’ grouping where students are grouped into different ability levels for different classes (Chmielewski et al., 2013; Razer et al., 2018). This language of rigidity is well established in the literature about ability grouping, which recommends softer approaches where students are grouped with flexibility and more mixed-ability classes are used (Francis et al., 2020). No research has explored the variety of class ability grouping practices being used in Australia in terms of their rigidity or other features. However, there is evidence to suggest a wide variance from small studies using qualitative methods (Johnston et al., 2022). New understandings can be gleaned from new knowledge about how class ability grouping is practiced within a single country, like Australia, on a wider scale. group membership (Hart et al., 2014). When ‘ability’ is used to group students into separate classes, students with educational disadvantages become segregated into lower ability-grouped classrooms (McCardle, 2020; McGillicuddy, 2021). Thus, class ability grouping can widen the educational achievement gap between society’s ‘haves’ and ‘have nots’ (Castejón & Zancajo, 2015; Hodgen et al., 2022). Such widening gaps are detrimental for equity in education, which is ultimately detrimental for a country’s economic performance (OECD, 2018a). A recent study from England found that class ability grouping widened the attainment gap between students grouped in the highest classes for English and Maths (Hodgen et al., 2022). Class Ability Grouping Teachers can see low ability classes as beneficial for students because they can be smaller groups and receive more attention, while higher groups can be more easily accelerated when they are grouped separately for learning (author, 2018). How- ever, empirical evidence from student results in Australia to substantiate these claims is lacking. Meanwhile, such views reflect misconceptions about the philosophy of differentia- tion originally conceived by theorists, such as Tomlinson (2014) and Rose and Meyer (2006). Class ability grouping is inflexible and does not facilitate differentiation- Tomlinson Class Ability Grouping in Australia and Rose both conceived differentiation as a philosophical mindset where all students’ needs are met through flexible grouping in mixed-ability classes, with Tomlinson articu- lating that inflexible ability grouping is the ‘antithesis’ of differentiation (Tomlinson, 2008, p. 4). f The perceived benefits may be one reason that class ability grouping continues to operate widely in secondary schools within countries, like Australia. There is evidence to suggest widespread use of the practice may be compounding inequality in educational outcomes in countries, including Australia, which is an increasingly problematic feature of Australian education (Hetherington, 2018). PISA analyses suggest that most Australian schools are using between- class ability grouping in Year 10 (OECD, 2018b). However, research thus far has only drawn implications of class ability grouping for equity in Australia from international comparative research using PISA data. These findings generalize across class ability grouping practices without exploring differences in how streaming is practiced within a country, such as Australia, including the age that grouping starts and subject variation. In Australia, state educational authorities make a few decisions about whether to group students into public schools by ability. PISA data indicate that class ability grouping is widely practiced in Australian schools (OECD, 2018a, b). Policy in many Australian states limits selective entry requirements for state schools, while Independent and Catholic schools use a range of criteria to determine enrollment. Given the general trend away from public schooling (which dropped from approximately 66% in 2020 to 64% in 2022) (Australian Bureau of Statistics, 2022), research on school structures is a matter of growing importance. In Western Australia, there is only one selective government school (Western Australia Department of Education, 2023b). Students can elect to sit a ‘Gifted and Talented entry’ test in their last year of primary school (Western Australia Department of Education, 2023a). Top performers are offered a place in the selective school, while next ranking performers are offered places in schools with ‘Gifted and Talented programs’ (Western Australia Department of Education, 2023a). These programs are offered at 24 government schools across the state, where they are implemented using class ability grouping. Queensland, for example, has three selective entry “academies”: one in creative industries, one in science, maths, and technology, and another in health sciences. Queensland also has a partially selective (academic) secondary state school. Class Ability Grouping in Australia Class ability grouping is used differently by different schools in Australia, with locally determined practices reported in the qualitative research (Johnston et al., 2022). Many primary and secondary schools use class ability grouping for one or more subjects (Black, 2021; Jaremus et al., 2022; Macqueen, 2013). Students in Australia begin primary school at age four or five and move to secondary at age eleven or twelve. They finish secondary school when they are seventeen or eighteen years old. PISA data report on practices in Year 10, but there are a lack of quantitative data about practices in Years 7–9 when students are eleven to fifteen years old. i International research using PISA data suggests that class ability grouping is more equitable when it is flexible and 1 3 1 1290 O. Johnston et al. Australia suggests that some schools start class ability grouping at the beginning of Year 7, while others delay class ability grouping until the end of Year 10 (Authors, 2022). Some schools use class ability grouping for only one subject, two subjects, or all subjects (Authors, 2022). It is likely that practices range widely in Australia because there is limited policy guidance or regulation for schools. Most schools make local, contextual decisions about if and how to group students into classes. There is limited official advice from educational authorities or policy makers currently guiding these practices. Further variations in practice, and other forms of practices are likely, but no quantitative research has been conducted to quantify the extent to which these practices exist or to further characterize the practices being used. Australia suggests that some schools start class ability grouping at the beginning of Year 7, while others delay class ability grouping until the end of Year 10 (Authors, 2022). Some schools use class ability grouping for only one subject, two subjects, or all subjects (Authors, 2022). It is likely that practices range widely in Australia because there is limited policy guidance or regulation for schools. Most schools make local, contextual decisions about if and how to group students into classes. There is limited official advice from educational authorities or policy makers currently guiding these practices. Further variations in practice, and other forms of practices are likely, but no quantitative research has been conducted to quantify the extent to which these practices exist or to further characterize the practices being used. Class Ability Grouping in Australia In both states, schools that do not offer these government-sanctioned competitive entry programs often create their own ‘gifted and talented’ or other high ‘ability’ class ability groupings within the school. Reasons for selective entry include a desire to attract and retain students with good academic track-records in a high-stakes testing environment and to provide targeted schooling (for example through creative industries or sporting excellence programs) in the context of a competitive educational marketplace (Harris, 2018; authors, under review). j International analyses of PISA data that include Australia characterize Australia as a country that uses a system of ability grouping similar to some other Western nations (Razer et al., 2018). These PISA analyses and other studies show that class ability grouping continues to operate in these countries, including England (Taylor et al., 2020), United States (Loveless, 2013) and New Zealand (Hornby & Witte, 2014). Recent research has begun to explore how practices within the UK can vary widely too (Wilkinson & Penney, 2023). Taylor et al. (2020) conducted a survey on ability grouping practices in English and Maths in England, while Wilkinson and Penney (2023) conducted a survey on Physical Education ability grouping practices in England. Research about ability grouping in England has also explored grouping practices in primary schools. Existing research in Australia has suggested that class ability grouping practices are common in secondary schools but are also present in some primary schools (Cheeseman & Klooger, 2018; Roth, 2017). For example, an Australia- wide study that investigated an Indigenous education reform surveyed teachers and principals from 201 Australian schools and found evidence of ability-based grouping in primary schools (Luke et al., 2013). The report, drawing on further qualitative data, noted that ability grouping was used from the foundation year of schooling onward, for a range of reasons that included managing classroom behavior and providing extension activities. Our review of the Australian literature about between- class ability grouping practices revealed limited evidence of how the practice is being used in schools with autonomy over how students are grouped into classes for learning (Authors, 2023). Existing qualitative research from 1 3 3 1291 The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… subjects, so data pertaining to each of them reflects how students spend most of their time at school. Instrument The Class Ability Grouping Practices Survey was used to determine the extent to which the various class ability grouping practices are being used for students in Year 7–9 core (MESH) subjects. The survey was adapted from a study about grouping practices in the UK and has been validated in that context (Taylor et al., 2020). MESH subjects were chosen as a focus because they are considered the ‘core subject areas’ in Australian education, so all students must take them until at least Year 10. Much research about between-class ability grouping, including PISA analyses, has focused on class ability grouping for Maths only. However, all students in Year 7–9 in Australia take these four MESH Class Ability Grouping in Australia This paper presents analysis of data from a recent survey about class ability grouping practices being used in Queens- land and Western Australia. As PISA data pertain to Year 10 students (15-year-olds), data about how students are grouped in the years leading up to this point (years 7–9) provide the focus for this research. Researching school practices at this point generates useful contextual information about group- ing as students begin secondary schooling and provides a foundation for future research about the effects of ability grouping in Australia. The survey included a question where respondents identi- fied their class grouping practices for each of the MESH sub- jects in Years 7–9. Respondents selected from six types of grouping practices for each of the four subjects, in each year from 7 to 9, representing a variety of practices from com- pletely mixed-ability grouping to a fully streamed grouping practice. These six practices are depicted on a spectrum in Fig. 1 below, ranging from ‘soft’ to ‘hard’ forms of group- ing, which is modeled on the survey by Taylor et al. (2020). Definitions of each practice that were provided to partici- pants are included in Fig. 1 as well: Methods Respondents also answered a question about class grouping practices across subjects, in terms of whether students were in the same class groups for the various subjects. The responses to these questions were used to answer the research question with the descriptive analyses below. The methods for this research were informed by the research literature, which points to class ability grouping as a practice that has the capacity to widen achievement gaps and perpetuate inequalities (e.g., Jaremus et al., 2022). In this way, it can function within education to reproduce and exacerbate existing inequalities (Bourdieu & Passeron, 1977). A pragmatic approach was thus appropriate to best answer the research question, combining approaches from both positivist and interpretivist methodologies (Dewey, 1916). Mixed methods were thus used to answer the research question through an explanatory sequential design that prioritized quantitative data with some qualitative data incorporated to further explain the quantitative findings (Edmonds & Kennedy, 2016). Sampling The sampling procedure was to invite all schools in Western Australia and Queensland with Year 7–9 students to complete the survey. Western Australia and Queensland were chosen for sampling because of convenience, as the authors’ institutions are located in these two states. Ethics approval had been gained from the lead author’s institutional Human Research Ethics Committee, but approval also needed to be gained to conduct the research from all school authorities in the two states. Approval was gained from all state and Catholic authorities except one diocese in Queensland, who did not approve the research on time for schools within that diocese to be invited to complete the survey. All other schools were invited through an email directed to the school principal. A total of 909 schools across the two states were sent an email inviting them to complete the Class Ability Grouping Practices Survey online via Qualtrics in early 2023. The methods used in this research were selected to generate an answer to the research question: To what extent are the various class ability grouping practices being used for MESH (Maths, English, Science, Humanities and Social Sciences [HASS]) in Year 7–9 of secondary schools in Queensland and Western Australia? A survey of class ability grouping practices in secondary schools in Queensland and Western Australia was conducted. Data Analysis A total of 173 schools responded to the survey, representing a 19% response rate. This response rate represents a confidence level of 95% for the population, with a margin of error of 6.5% (Qualtrics, 2023). This confidence level and margin of error assume equal response rates across different parts of the population and that relevant variables like grouping practices or location did not influence the response rate. Of the 173 responses, 143 were included in the analysis after data cleaning, representing a 16% 1 3 1 1292 O. Johnston et al. 1 Range of class grouping practices Fig. 1 Range of class grouping practices Fig. 1 Range of class grouping practices practices being used by the 143 participant schools, affecting 108,019 students who attend these schools. response rate. Cleaning involved screening for outliers and correcting or deleting errors (e.g., scores were not within the range of possible scores) and excluding surveys with large proportions of missing data, leaving 68 Queensland Schools (47.6%) and 75 Western Australian schools (52.4%). The survey was completed primarily by principals (n = 93, 65%) and associate or deputy principals (n = 30, 21.0%). Other respondents included timetablers, curriculum/year leaders, and other leadership roles within the school. The scale in the present study had excellent internal consistency with a Cronbach alpha coefficient reported of = 0.97. Findings Before determining the extent to which the schools in Queensland and Western Australia engaged in class grouping practices, grouping practices were initially collapsed into two categories: completely mixed or class ability grouping before examining the descriptive statistics. The grouping practices are detailed in Table 2. fi Descriptive analysis of the quantitative data occurred using IBM SPSS Version 29. Qualitative data were analyzed by three researchers to improve inter-rater reliability. The data were coded in NVivo to identify themes relating to the research questions. The findings revealed that in Queensland, most classes were completed mixed. This pattern was reflected across English, Maths, HASS, and Science. Ability grouping was highest in Year 9 for Maths (33.8%) and English (11.8%). However, in contrast, Western Australian reported using primarily class ability grouping practices across English, Maths, HASS, and Science. For English, the percentage of schools using ability groupings rose from 73.91% in Year 7 to 85.5% in Year 9. For Maths, ability groupings remained consistent from Year 7 (85.71%) to Year 9 (85.51%). For HASS, class ability grouping rose from 61.76% in Year 7 to 70.59% in Year 9. For Science, class ability groupings rose from 62.86% in Year 7 to 70.15% in Year 9. The frequency tables that follow describe the Australian Curriculum, Assessment, and Reporting Authority (ACARA, 2023) data for the participant schools, illustrating that the participant school profiles generally reflect the averages for Australian schools. Critical readers can evaluate the information presented in Table 1 below and make their own decisions about the extent to which the sample represents a more general population. Findings At the least, the data reported in this paper characterizes the class ability grouping 1 3 3 The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… 1293 q y y p g g y Table 1 Representation of Participant Schools: ICSEA, Location, and Students Note: ICSEA, or Index of Social Community Advantage, is a score used in Australian schooling to indicate the relative level of socio-economic and cultural advantage for the students that attend the school (ACARA, 2016) Representation of participant schools’ Index of Socio-community educational advantage (ICSEA)* ICSEA All Australian schools Participant schools M 1000 993.67 Min 500 611 Max 1300 1182 SD 100 106.10 % of students in participant schools in bottom ICSEA quarter 25 31.78 % of students in participant schools in bottom-middle ICSEA quarter 25 26.68 % of students in participant schools in upper-middle ICSEA quarter 25 22.72 % of students in participant schools in top ICSEA quarter 25 18.89 Representation of participant schools’ locations Location All Australian schools Participant schools % Inner Regional 24.3 17.9 % Major Cities 54.5 47.8 % Outer Regional 15 21.5 % Remote 3.1 7.7 % Very Remote 3.1 5.1 Representation of participant schools’ students No. of Students All Australian schools Participant schools M 423.91 832.74 Min 1 39 Max 5259 2601 SD 434.52 612.22 % Indigenous 10.86 13.97 % language background other than English 24.19 18.76 % Indigenous % language background other than English * Note: ICSEA, or Index of Social Community Advantage, is a score used in Australian schooling to indicate the relative level of socio-economic and cultural advantage for the students that attend the school (ACARA, 2016) * Note: ICSEA, or Index of Social Community Advantage, is a score used in Australian schooling to indicate the relative level of socio-economic and cultural advantage for the students that attend the school (ACARA, 2016) “Split line with ENG [English] and HUM [humanities] in Year 7, 8 but blocked line in Year 9 ENG [English], though semester rotation for 9HUM [Year 9 humani- ties]” [90]. Figure 2 suggests that overall, schools in Queensland and Western Australia reported higher percentages of class abil- ity grouping for the subject areas of Maths and English. Findings We are trialing a stream- Table 2 Class Grouping practices in Years 7–9 for English, Maths, HASS, and Science in Queensland and Western Australia Location Year & Subject n Completely Mixed (f) % Class Ability Grouping (f) % Queensland Yr 7 English 68 67 98.5 1 1.5 Yr 8 English 68 67 98.5 1 1.5 Yr 9 English 68 60 88.2 8 11.8 Western Australia Yr 7 English 69 18 26.09 51 73.91 Yr 8 English 70 13 18.57 57 81.43 Yr 9 English 69 10 14.49 59 85.51 Queensland Yr 7 Maths 68 65 95.6 3 4.4 Yr 8 Maths 68 56 82.4 12 17.6 Yr 9 Maths 68 45 66.2 23 33.8 Western Australia Yr 7 Maths 70 10 14.29 60 85.71 Yr 8 Maths 71 10 14.08 61 85.92 Yr 9 Maths 69 10 14.49 59 85.51 Queensland Yr 7 HASS 68 68 100 – – Yr 8 HASS 68 67 98.5 1 1.5 Yr 9 HASS 68 67 98.5 1 1.5 Western Australia Yr 7 HASS 68 26 38.24 42 61.76 Yr 8 HASS 69 26 37.68 43 62.32 Yr 9 HASS 68 20 29.41 48 70.59 Queensland Yr 7 Science 68 68 100 – – Yr 8 Science 68 67 98.5 1 1.5 Yr 9 Science 68 67 98.5 1 1.5 Western Australia Yr 7 Science 70 26 37.14 44 62.86 Yr 8 Science 70 26 37.14 44 62.86 Yr 9 Science 67 20 29.85 47 70.15 Fig. Findings We are trialing a stream- Table 2 Class Grouping practices in Years 7–9 for English, Maths, HASS, and Science in Queensland and Western Australia Location Year & Subject n Completely Mixed (f) % Class Ability Grouping (f) % Queensland Yr 7 English 68 67 98.5 1 1.5 Yr 8 English 68 67 98.5 1 1.5 Yr 9 English 68 60 88.2 8 11.8 Western Australia Yr 7 English 69 18 26.09 51 73.91 Yr 8 English 70 13 18.57 57 81.43 Yr 9 English 69 10 14.49 59 85.51 Queensland Yr 7 Maths 68 65 95.6 3 4.4 Yr 8 Maths 68 56 82.4 12 17.6 Yr 9 Maths 68 45 66.2 23 33.8 Western Australia Yr 7 Maths 70 10 14.29 60 85.71 Yr 8 Maths 71 10 14.08 61 85.92 Yr 9 Maths 69 10 14.49 59 85.51 Queensland Yr 7 HASS 68 68 100 – – Yr 8 HASS 68 67 98.5 1 1.5 Yr 9 HASS 68 67 98.5 1 1.5 Western Australia Yr 7 HASS 68 26 38.24 42 61.76 Yr 8 HASS 69 26 37.68 43 62.32 Yr 9 HASS 68 20 29.41 48 70.59 Queensland Yr 7 Science 68 68 100 – – Yr 8 Science 68 67 98.5 1 1.5 Yr 9 Science 68 67 98.5 1 1.5 Western Australia Yr 7 Science 70 26 37.14 44 62.86 Yr 8 Science 70 26 37.14 44 62.86 Yr 9 Science 67 20 29.85 47 70.15 Fig. 2 Class ability grouping practices in Years 7–9 for Eng- lish, Maths, HASS, and Science on ability grouping, most schools now operate in envi- ronments where technology offers new opportunities for innovation that sit behind some grouping decisions. For example, one respondent explained that. “The only difference [in how are grouped is] with Maths [63]. We started a new program called Math Pathways which students stay in their class grouping; however, each student is working on a program built around their level and needs. Findings 2 Class ability grouping practices in Years 7–9 for Eng- lish, Maths, HASS, and Science Table 2 Class Grouping practices in Years 7–9 for English, Maths, HASS, and Science in Queensland and Western Australia Location Year & Subject n Completely Mixed (f) % Class Ability Grouping (f) % Queensland Yr 7 English 68 67 98.5 1 1.5 Yr 8 English 68 67 98.5 1 1.5 Yr 9 English 68 60 88.2 8 11.8 Western Australia Yr 7 English 69 18 26.09 51 73.91 Yr 8 English 70 13 18.57 57 81.43 Yr 9 English 69 10 14.49 59 85.51 Queensland Yr 7 Maths 68 65 95.6 3 4.4 Yr 8 Maths 68 56 82.4 12 17.6 Yr 9 Maths 68 45 66.2 23 33.8 Western Australia Yr 7 Maths 70 10 14.29 60 85.71 Yr 8 Maths 71 10 14.08 61 85.92 Yr 9 Maths 69 10 14.49 59 85.51 Queensland Yr 7 HASS 68 68 100 – – Yr 8 HASS 68 67 98.5 1 1.5 Yr 9 HASS 68 67 98.5 1 1.5 Western Australia Yr 7 HASS 68 26 38.24 42 61.76 Yr 8 HASS 69 26 37.68 43 62.32 Yr 9 HASS 68 20 29.41 48 70.59 Queensland Yr 7 Science 68 68 100 – – Yr 8 Science 68 67 98.5 1 1.5 Yr 9 Science 68 67 98.5 1 1.5 Western Australia Yr 7 Science 70 26 37.14 44 62.86 Yr 8 Science 70 26 37.14 44 62.86 Yr 9 Science 67 20 29.85 47 70.15 Fig. 2 Class ability grouping practices in Years 7–9 for Eng- lish, Maths, HASS, and Science on ability grouping, most schools now operate in envi- ronments where technology offers new opportunities for innovation that sit behind some grouping decisions. For example, one respondent explained that. “The only difference [in how are grouped is] with Maths [63]. We started a new program called Math Pathways which students stay in their class grouping; however, each student is working on a program built around their level and needs. We are trialing a stream- Fig. 2 Class ability grouping practices in Years 7–9 for Eng- lish, Maths, HASS, and Science on ability grouping, most schools now operate in envi- ronments where technology offers new opportunities for innovation that sit behind some grouping decisions. For example, one respondent explained that. Findings For these subjects, the most common class grouping practices used in English and Maths for the total sample (n = 143) for Years 7, 8, and 9 were completely mixed-ability, followed by a mixed-ability class with a top and bottom class, and a mixed-ability class with a top class (See Figs. 3 and 4). Our findings suggest that high school students in Years 7 to 9 in Western Australia are more frequently grouped into class ability groups than students in Queensland. This was an unexpected finding as there is nothing in the lit- erature to suggest any reason for this difference between states. Furthermore, there is nothing in our current data to suggest why this might be the case, so further research must be conducted to explain why this is so. However, some data revealed how school leaders make decisions about student groupings under complex circumstances that include teacher shortages and concerns about indi- vidual student achievement. Schools made comments such as “Due to shortages in staffing (we) had to look at Years 7 and 8 to allow teachers to take 2 of the same year group.” In comparison with the contexts of early research We acknowledge the importance of timetabling lines in how students are grouped, as schools work to offer elective subjects to students while minimizing timetable clashes. Subject selection (including electives and participation in excellence programs) during secondary school is a related factor that is linked to both timetable lines and thus class grouping. The following qualitative response data illustrates this point further, “Middle School structure in Years 7 and 8 means some commonality with class lists, but top and tail in Math and English means not exclusively the same” [110]; 1 3 1294 O. Johnston et al. 1294 O. Johnston et al. on ability grouping, most schools now operate in envi- ronments where technology offers new opportunities for innovation that sit behind some grouping decisions. For example, one respondent explained that. “The only difference [in how are grouped is] with Maths [63]. We started a new program called Math Pathways which students stay in their class grouping; however, each student is working on a program built around their level and needs. Findings on ability grouping, most schools now operate in envi- ronments where technology offers new opportunities for innovation that sit behind some grouping decisions. For example, one respondent explained that. “The only difference [in how are grouped is] with Maths [63]. We started a new program called Math Pathways which students stay in their class grouping; however, each student is working on a program built around their level and needs. We are trialing a stream- 1 3 3 1295 The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… ing approach for 1/5 of Maths and English. This is to provide intensive intervention or extension and will be determined by student needs” [64]. Such reasoning for grouping in the qualitative data was provided by schools who used class ability grouping in Western Australia, but there is no reason suggested that this should be different in Western Australian than Queensland. Our analysis of the qualitative data also showed that approach is a trial for this year, as we had previously moved away from ability grouping” [24]; “We are currently exploring the impact of streaming and are very interested in what this research has to say” [8]. These school leaders were eager to make decisions about class ability grouping in their students’ best interest, but funding and resource issues were also considerations for lower ability learners. Fig. 3 Differences in in Class Ability Grouping for Maths across Year Levels and States Fig. 4 Differences in Class Ability Grouping for English across Year Levels and States The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… 1295 Fig. 3 Differences in in Class Ability Grouping for Maths across Year Levels and States Fig. 4 Differences in Class Ability Grouping for English across Year Levels and States ing approach for 1/5 of Maths and English. This is to provide intensive intervention or extension and will be determined by student needs” [64]. approach is a trial for this year, as we had previously moved away from ability grouping” [24]; “We are currently exploring the impact of streaming and are very interested in what this research has to say” [8]. Findings “Maths and English classes are streamed in Years 7 to 10... For all other subjects, students are in mixed- ability learning groups” [8]; “Year 9 Maths has two top streamed classes, one bottom streamed (small) class and two middle mixed classes” [17]; “Students sit an entrance exam focused primarily on Maths and are admitted to the top two classes based on this exam” [25]; “Bottom group mostly students on ICP or working below level. Based on curriculum ability not literacy or numeracy” [24]. “Maths and English classes are streamed in Years 7 to 10... For all other subjects, students are in mixed- ability learning groups” [8]; “Year 9 Maths has two top streamed classes, one bottom streamed (small) class and two middle mixed classes” [17]; “Students sit an entrance exam focused primarily on Maths and are admitted to the top two classes based on this exam” [25]; “Bottom group mostly students on ICP or working below level. Based on curriculum ability not literacy or numeracy” [24]. “Maths and English classes are streamed in Years 7 to 10... For all other subjects, students are in mixed- ability learning groups” [8]; “Year 9 Maths has two top streamed classes, one bottom streamed (small) class and two middle mixed classes” [17]; “Students sit an entrance exam focused primarily on Maths and are admitted to the top two classes based on this exam” [25]; “Bottom group mostly students on ICP or working below level. Based on curriculum ability not literacy or numeracy” [24]. Figure 4 demonstrates that overall, Queensland schools report higher levels of completely mixed classes for English compared to Western Australian schools. Ability grouping for English is highest in Year 9 in Queensland (n = 8, 11.8%). Class ability grouping for English increases from 68% in Year 7, to 76% in Year 8 and 80% in Year 9 in Western Australia. Figure 5 suggests that overall, Queensland schools report higher levels of completely mixed classes for HASS compared to Western Australian schools. In Queensland schools, class ability grouping remains low (i.e., 1.5% for Years 8 and 9). In Western Australian schools, levels of ability grouping for HASS rise from 56% in Year 7, to 57.3% in Year 8 and 64% in Year 9. Findings Such reasoning for grouping in the qualitative data was provided by schools who used class ability grouping in Western Australia, but there is no reason suggested that this should be different in Western Australian than Queensland. Such reasoning for grouping in the qualitative data was provided by schools who used class ability grouping in Western Australia, but there is no reason suggested that this should be different in Western Australian than Queensland. Our analysis of the qualitative data also showed that school leaders made decisions about grouping based on the needs that they perceived their students to have. Some indicative comments included: These school leaders were eager to make decisions about class ability grouping in their students’ best interest, but funding and resource issues were also considerations for lower ability learners. f Our analysis of the qualitative data also showed that school leaders made decisions about grouping based on the needs that they perceived their students to have. Some indicative comments included: Students’ varying experiences of class ability grouping have implications for students’ attainment of educational outcomes, specifically in terms of equity. Equity issues are explored further in the discussion section that follows. Class size is another factor at play, as small schools typically offered only one class per year level, reducing the likelihood “Bottom group mostly students on (independent curriculum program) or working below level. Based on curriculum ability not literacy or numeracy. This 1 1296 O. Johnston et al. Queensland and Western Australia. Class ability grouping is most common in Maths classes in Queensland and Western Australia. The next most common subject grouped by ability levels was English in Queensland and Western Australia. of class grouping. Similarly, specialist schools (such as flexible schooling with residential offerings) made choices based on a range of factors, such as safety. While there were a range of configurations, the qualitative data indicate that Queensland schools often allocated students to “top” and “bottom” classes for English and Mathematics, before placing the remaining students into “middle” classes. The following comments capture typical approaches: Figure 3 reveals that overall, Queensland schools report higher levels of completely mixed classes for Maths compared to Western Australian schools. Ability grouping for Maths is highest in Year 9 in Queensland (n = 23, 33.8%). The percentage of class ability grouping for Maths in Year 7 (80%), Year 8 (81.3%), and Year 9 in Western Australia (78.7%) were relatively consistent. Discussion Our findings show that in Queensland and Western Australian secondary schools, grouping by ability generally increases as students get older, is used more by some schools than others (e.g., larger schools), and is most prominent in Maths and English. The findings also highlight pronounced differences between the two Australian states, where Western Australia uses class ability grouping much more often, for more subjects, and begins class grouping sooner. These findings suggest that some students are experiencing more inequitable grouping practices than others, due to contextual factors, which may complicate and exacerbate existing equity issues associated with ability grouping. This research generated new knowledge that characterizes how streaming practices vary across two states in Australia according to year level, subject, and location. Class ability grouping showed an overall upward trend as students move up year levels, with increases from Year 7 to 9 reflected in Fig. 2. Class ability grouping was most common in Maths classes, with analyses also showing that English was the second highest MESH subject where ability grouping was used. However, Maths indicated a slight dip in Year 9 grouping in the Western Australia schools that was not evident in Queensland. These findings are illustrated in Figs. 3, 4, 5, and 6. Possible reasons and implications for these differences across subjects and states are suggested in the discussion below.f The pronounced differences in the two states that were indicated by the data analyses were an unexpected finding. Figure 2 showed that the majority of schools in Western Australia use class ability grouping for all of the MESH subjects from Year 7–9 inclusive, while the majority of Queensland schools use completely mixed-ability grouping for all of the MESH subjects from Year 7 to 9. This means that most 12–15-year-olds in Western Australia are learning their MESH subjects in ability-grouped classes, while most 12–15-year-olds in Queensland are learning their MESH subjects in completely mixed-ability classes. The possible implications of these findings are discussed in terms of equity in the discussion section that follows, but reasons for this difference were not found within the scope of this research. Inequality in how students are grouped into classes for learning may compound and complicate the implications for equity in education. PISA data from 2012 to 2018 asked principals whether students were grouped into classes by ability for learning in no subjects, at least one subject, or all subjects (OECD, 2014, 2018b). Findings While slight increases for HASS were evident across the year levels, they were much lower than the increases in ability grouping by year level reported in Maths and English that were reflected in Figs. 3 and 4. In Western Australia, qualitative responses indicate that schools seemed more likely to provide focused instruction programs rather than class grouping. Some indicative com- ments include “Some lower ability students in Year 7 receive intensive literacy support in place of their regular English class. From Year 9, entry into the top class for English, Sci- ence, Maths, and HASS is through an application process, involving past results and an external selection test.” [60]; “There is English intervention provided for those with addi- tional needs. This is a ‘push in’ model.” (65]. Descriptive analysis of the survey responses suggested that class ability grouping is most common in Maths classes, but less com- mon in other subjects. Figures 3, 4, 5, and 6 illustrate the rates of class ability grouping in the four MESH subjects in Figure 6 depicts that overall, Queensland schools report higher levels of completely mixed classes for Science compared to Western Australian schools. In Queensland schools, class ability grouping is low (1.5% for Years 8 and 9). In Western Australian schools, levels of ability Fig. 5 Differences in in Class Ability Grouping for HASS across Year Levels and States 1 3 3 The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… 1297 Fig. 6 Differences in Class Ability Grouping for Science across Year Levels and States grouping for Science were 58.7% in Year 7 and 8 and slightly higher in Year 9 (62.7%). Like HASS, although there is a slight increase in class ability grouping for Science across year levels, they were much lower than the increases reported in Maths and English. Discussion Further upcoming publications will address in greater detail why such decisions are made as well as how the decisions are enacted within schools. Some possible reasons for differences may include varying levels of disadvantage, cultural norms, leadership, or past interventions. However, these reasons for differences in respondent schools’ class ability grouping practices can only be speculative until further research is conducted. Such further research could include interviews with school leaders and educators in both states to further explore the reasoning behind their decisions about class ability grouping. Overall, the qualitative data provided by participants indicated that school leadership teams are searching for practices and structures that will best meet the needs of their students and trialing different grouping strategies as a means of increasing the support provided to students. Our analysis of the qualitative data also highlighted that schools are making decisions based on what school leaders consider to be best for their students. It is concerning that students who are commencing Year 7, as young as 11 years old, may have their post-secondary pathways limited by class grouping practices. Teachers’ expectations of students can affect their academic outcomes when they shape how students are treated in classrooms (Papageorge et al., 2020; Wang et al., 2019). Students being grouped into classes associated with defined post-secondary pathways could create self-fulfilling prophesies where students experience little flexibility in their educational trajectory (Boaler, 2005). Once students are allocated to an ability-based class, movement to a ‘higher’ class is both difficult and rare, due to missed curriculum and the need for another student to be moved down, making a space available (Ireson et al., 2002). A student’s previous group level influences subsequent placement regardless of later achievement (Gamoran, 1986). Recent research from Australia has investigated how students placed in lower ability-grouped classes in New South Wales were unable to take university-entry level Maths classes in Years 11 and 12 (Jaremus et al., 2022). These findings confirm international research that has shown that class ability grouping is associated with restricted post-secondary pathways (Francis et al., 2020). Inequality in Australian education is growing with devastating economic repercussions, with conservative estimates that inequality is costing Australia 20.3 billion dollars (Hetherington, 2018). Many aspects of an education system might contribute to inequality, but international research suggests that class ability grouping can be a contributing factor (Castejón & Zancajo, 2015; OECD, 2018a). Discussion The analysis of PISA data above shows that the vast majority of principals in all three testing years selected ‘for at least one subject,’ but there was no identification of subject provided by the data. Furthermore, the PISA data are only for fifteen-year- old students, which is Year 10 in Australia. The research presented in this paper has elaborated on the PISA data in three ways: 1 3 1298 O. Johnston et al. (1) Identifying the specific MESH subjects in which students are grouped into classes by ability; Science and HASS. Interestingly, high-stakes testing in Australia’s NAPLAN focuses on student achievement in these two subjects. Students undertake standardized tests for Science and HASS less frequently and these subjects are not reported about publicly as they are for Maths and English in Australia. Furthermore, schools are held to account by their state government according to NAPLAN scores in Western Australia, with underperforming schools subject to review processes that can involve dismissal of the school Principal and government intervention. Research about class ability grouping in Australia has traditionally focused on the Maths learning area (Forgasz, 2010; Zevenbergen, 2005). The research presented in this paper adds to the literature by comparing class ability grouping in Maths and English with the other MESH subjects of HASS and Science.i g p y y (2) Characterizing the increase in class ability grouping from Year 7 to 9; and (3) Identifying variance in between-class ability grouping practices according to where students live and which school they attend. If Australian educators are committed to following through on policy commitments to equity in education (MYCEETA, 2008), closer scrutiny of class ability grouping practices could be a starting point for change. Our findings confirmed that levels of class ability grouping used in MESH subjects increase as students move from Year 7 to Year 9. This is unsurprising because students’ academic pathways for Years 11 and 12 become more certain (and restricted) as they get older. Schools might consider it preparation for students’ future, according to whether or not the students will be enrolling in university pathway courses in Years 11 and 12. The research findings also begin to reveal the complexity of class grouping practices being used in Australia, emphasizing that vast differences across locations exist. The current research presents what occurs in schools regarding class grouping and begins to consider the reasons why some class grouping decisions are made. 3 Limitations The present study is not without limitations and findings should be considered with some caution. The limitations of the voluntary response method are noted. The present study is not without limitations. We recognize that the study relies on a self-report methodology which is susceptible to subjective bias. We acknowledge the potential bias that may have arisen that those who chose to participate might have had current or prior knowledge (including about the research literature) or a particular interest in ability grouping. Some schools may have chosen to participate because they are currently changing, or have recently changed, their practices. Others may have chosen to participate because they are interested in the topic and have knowledge about it. Although there is little research and policy advice about ability grouping in Australia, the research predominately cautions against the practice. For this reason, the authors believe that the levels of class ability grouping reported in this study are modest representations of the extent to which class ability grouping is being used in these two states. Furthermore, the study used cross-sectional data from two states in Australia. As such, findings should be interpreted with some caution and further research is needed with larger sample sizes across all Australian states and territories. Black, A. (2021). Streaming, social class and schooling in Western Australia: A critical ethnography of post-school reflections (Publi- cation Number 63077) Murdoch University. https://researchreposit ory.murdoch.edu.au/id/eprint/63077/1/Black2021.pdf Boaler, J. (2005). The “psychological prisons” from which they never escaped: The role of ability grouping in reproducing social class inequalities. Forum, 47(2&3), 125–134. https://ed.stanford.edu/ sites/default/files/psychological_prisons_2005.pdf i Boaler, J., Wiliam, D., & Brown, M. (2000). Students’ experiences of ability grouping-disaffection, polarisation and the construction of failure. British Educational Research Journal, 26(5), 631–648. Bourdieu, P., & Passeron, J. C. (1977). Reproduction in education, society and culture. Sage.f Castejón, A., & Zancajo, A. (2015). Educational differentiation poli- cies and the performance of disadvantaged students across OECD countries. European Educational Research Journal, 14(3–4), 222–239. https://doi.org/10.1177/1474904115592489 Cheeseman, J., & Klooger, M. (2018). Mathematics teachers: Deal- ing with difference. Australian Primary Mathematics Classroom, 23(3), 27–29. https://doi.org/10.3316/informit.924803020028042 Chmielewski, A. K. (2014). An international comparison of achieve- ment inequality in within-and between-school tracking systems. American Journal of Education, 120(3), 293–324. https://www. jstor.org/stable/https://doi.org/10.1086/675529?seq=1 Chmielewski, A. K., Dumont, H., & Trautwein, U. (2013). Tracking effects depend on tracking type: An international comparison of students’ mathematics self-concept. American Educational Research Journal, 50(5), 925–957. https://doi.org/10.3102/00028 31213489843 Clarke, M. (2014). References ACARA. (2016). ICSEA Fact Sheet. http://docs.acara.edu.au/resources/ About_icsea_2014.pdf ACARA. (2023). My school. https://myschool.edu.au/f Ansalone, G. (2010). Tracking: Educational Differentiation or Defec- tive Strategy. Educational Research Quarterly, 34(2), 3–17. Australian Bureau of Statistics. (2022). Schools. https://www.abs.gov. au/statistics/people/education/schools/latest-release#:~:text= Media%20releases-,Key%20statistics,13.1%20students%20to% 20one%20teacher Funding Open Access funding enabled and organized by CAUL and its Member Institutions. Discussion 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/. Discussion Future research could inform policymaking about class ability grouping in the Australian context, leading to improvements in class ability grouping practices that create equitable outcomes for our students, without compromising academic outcomes. Such further research could examine the Australian context, investigating how different forms of class ability grouping affect student outcomes, including how this varies across subjects and groups. This might include following students and grouping practices over several years. Such research could examine the characteristics of the students in each class group within the school and explore any differences in their experiences according to how they are grouped. The research could also be expanded to other Australian states. The findings also reveal that class ability grouping is much more widely used in Maths and English than in 1 3 3 1299 The Equity of Class Ability Grouping Practices in Australian Education: Findings from a Survey… Our findings indicated that school leaders’ intentions were to provide the best learning conditions for their students, but further research is needed to see how students are being affected by these choices in Australia. Previous qualitative research by the first author of this study has suggested that students in high ability groups experience benefits of class ability grouping, but that they do not always see it as beneficial or fair (Author et al., 2022). Future research could draw wider comparisons from evidence about how students experience different forms of class ability grouping and how these affect their educational outcomes. We suggest that further research is also needed to determine how education funding and policy could provide school leaders with clear direction and the support needed to tackle the large disparities in student needs and achievement at the school level. As Ball (2012) suggests, most education policy research does not deal with issues of funding. 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https://openalex.org/W3187441743	https://www.researchsquare.com/article/rs-724766/v1.pdf?c=1637369290000	English	null	Association between oligo-residual disease and patterns of failure during EGFR-TKI treatment in EGFR-mutated non-small cell lung cancer: a retrospective study	BMC cancer	2,021	cc-by	7,139	Association Between Oligo-Residual Disease and Patterns of Failure During EGFR-TKI Treatment in EGFR-Mutated Non-Small Cell Lung Cancer: A Retrospective Study Taichi Miyawaki Shizuoka Cancer Center Hirotsugu Kenmotsu (  h.k Shizuoka Cancer Center Hiroaki Kodama Shizuoka Cancer Center Naoya Nishioka Shizuoka Cancer Center Eriko Miyawaki Shizuoka Cancer Center Nobuaki Mamesaya Shizuoka Cancer Center Haruki Kobayashi Shizuoka Cancer Center Shota Omori Shizuoka Cancer Center Ryo Ko Shizuoka Cancer Center Kazushige Wakuda Shizuoka Cancer Center Akira Ono Shizuoka Cancer Center Tateaki Naito Shizuoka Cancer Center Haruyasu Murakami Shizuoka Cancer Center Keita Mori Shizuoka Cancer Center Hideyuki Harada Shizuoka Cancer Center Eriko Miyawaki Shizuoka Cancer Center Nobuaki Mamesaya Shizuoka Cancer Center Ryo Ko Shizuoka Cancer Center Kazushige Wakuda Shizuoka Cancer Center Akira Ono Shizuoka Cancer Center Tateaki Naito Shizuoka Cancer Center Haruyasu Murakami Shizuoka Cancer Center Keita Mori Shizuoka Cancer Center Hideyuki Harada Shizuoka Cancer Center Page 1/20 Masahiro Endo Shizuoka Cancer Center Kazuhisa Takahashi Juntendo University Graduate School of Medicine Toshiaki Takahashi Shizuoka Cancer Center Abstract Background: Local ablative therapy (LAT) may be beneficial for patients with epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) with oligo-residual disease after treatment with EGFR tyrosine kinase inhibitor (EGFR-TKI). However, this has not been fully established. This study aimed to evaluate the predominant progressive disease (PD) pattern limited to residual sites of disease after treatment with EGFR-TKI. Methods: Patients with advanced EGFR-mutated NSCLC treated with EGFR-TKIs as first-line therapy were retrospectively analysed during a 7-year period. Oligo-residual disease was defined as the presence of 1 – 4 lesions (including the primary site) at 3 months from the start of EGFR-TKI treatment. The predictive factors of PD patterns after EGFR-TKI treatment were evaluated. Results: A total of 207 patients were included. Three months after the start of EGFR-TKI treatment, 66 patients (32%) had oligo-residual disease. A total of 191 patients had PD, 60 with oligo-residual disease and 131 with non-oligo-residual disease. Regarding the pattern, 44 patients (73%) with oligo-residual disease and 37 patients (28%) with non-oligo-residual disease had PD limited to the residual sites. Multivariate logistic regression analysis at 3 months from the start of EGFR-TKI treatment revealed that oligo-residual disease (P < 0.001), the lack of residual central nervous system metastases (P = 0.032), and initial treatment with osimertinib (P = 0.028) were independent predictors of PD limited to residual disease sites. Conclusions: This study provided a rationale for LAT to all sites of residual disease in patients with oligo- residual disease during EGFR-TKI treatment. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Cancer on November 19th, 2021. See the published version at https://doi.org/10.1186/s12885-021-08983-2. Page 2/20 1. Background Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have demonstrated clinical activity in the treatment of patients with EGFR-mutated non-small-cell lung cancer (NSCLC). [1] Several clinical trials have indicated the superiority of EGFR-TKIs over conventional chemotherapy in terms of treatment efficacy, progression-free survival (PFS), and objective response rate in patients with EGFR- mutated NSCLC. [1–3] Furthermore, osimertinib, a third-generation EGFR-TKI, was associated with prolonged PFS and overall survival (OS) compared to first-generation EGFR-TKIs, including erlotinib or gefitinib, in a phase III trial. [4,5] However, the development of resistance to EGFR-TKIs remains an obstacle to achieving disease control and long-term survival. [6] Adding local ablative therapy (LAT) to standard systemic therapy could be a treatment option that provides high local control of residual disease in patients with oligometastatic NSCLC. [7–9] The European Society for Medical Oncology defines oligometastatic NSCLC as the presence of 1 – 3 metastases and recommends the addition of LAT in these patients. [10]Previous studies have demonstrated that LAT of all sites of disease provided potential benefits in patients with synchronous oligometastatic NSCLC. [11–13] Page 3/20 Progressive disease (PD) after first-line chemotherapy has been shown to be predominantly limited to the original disease site of disease, providing a rationale for the addition of LAT in patients with oligometastatic NSCLC. [14,15] In patients with EGFR-mutated NSCLC, almost half had PD limited to the original sites of disease after first-line treatment with EGFR-TKIs. [16–18] No significant association was found between synchronous oligometastatic disease and patterns of initial PD after EGFR-TKI treatment. [18] However, several studies have shown that LAT to all sites of residual disease demonstrated favourable PFS and OS in patients with oligo-residual disease. [19–21] The clinical features of oligo-residual disease are unknown. Furthermore, the patterns of initial PD after first-line treatment with EGFR-TKIs remain unclear in patients with oligo-residual disease. Our study aimed to evaluate the clinical impact of oligo-residual disease on patterns of PD after EGFR-TKI treatment and establish a rationale for adding LAT to all sites of oligo-residual disease. 3.1 Baseline patient characteristics A total of 207 patients were included in this study. The median patient age was 68 years (34 – 88 years). The majority of the patients had adenocarcinoma (94.7%), ECOG-PS 0 – 1 (82.6%), and sensitising EGFR mutations (95.1%). Only 6% of patients had synchronous oligometastatic disease, and 87% of patients had 10 or more metastases at the start of EGFR-TKI treatment. (Table 1) 2.3 Statistical analysis PFS was calculated from the residual disease evaluation to the first evidence of disease progression or death from any cause and estimated using the Kaplan-Meier method. The log-rank test was used to compare PFS between patients with oligo-residual disease and those with non-oligo residual disease. The end of the follow-up period was 31 December 2020. All categorical variables were analysed using the chi- square test. Potential predictive factors for oligo-residual disease were assessed using univariate and multivariate analyses with a logistic regression model adjusted for patient characteristics at baseline. Potential predictive factors for PD limited to the residual sites were assessed using univariate and multivariate analyses with a logistic regression model adjusted for patient characteristics at 3 months from the start of EGFR-TKI therapy. For the univariate analyses, the covariates included age (≥ 75 vs. < 75 years), sex, smoking status, ECOG-PS (0 – 1 vs. ≥ 2), histology (adenocarcinoma vs. non- adenocarcinoma), EGFR mutation (exon 19 deletions / L858R point mutations vs. others), residual central nervous system (CNS) metastases (yes vs. no), number of residual metastatic organs (0 – 1 vs. ≥ 2), [25,26] EGFR-TKI type (osimertinib vs. other EGFR-TKIs), and oligo-residual disease. Factors with univariate P-values of < 0.1, were subjected to multivariate analyses. Differences were considered statistically significant at P-values of < 0.05. All analyses were performed using STATA version 14.0 (Stata Corp., Texas, USA). 2.1 Patients The medical records of patients with stage IV EGFR-mutated NSCLC who received EGFR-TKI first-line monotherapy at the Shizuoka Cancer Centre between January 2013 and December 2019 were retrospectively reviewed. This study’s protocol was approved by our institutional ethics review board (approval number: J2020-177-2020-1) and was conducted in accordance with the Declaration of Helsinki. The need for informed consent was waived due to the retrospective nature of the study. The collected data included age, sex, Eastern Cooperative Oncology Group performance status (ECOG- PS), smoking history, EGFR mutation status, metastatic details (sites, number of organs, number of metastases), and type of EGFR-TKIs. EGFR gene mutations were evaluated using polymerase chain reaction amplification using commercially available assays. Age, ECOG-PS, metastatic details, and the number of residual lesions were collected at 3 months ± 6 weeks after the start of EGFR-TKI therapy as previous studies have shown that this is the median time to initial response. [16,23] Synchronous oligometastatic disease was defined as the presence of 1 – 3 metastases (1 – 4 lesions including the primary site) at the time of NSCLC diagnosis. [14,22] Oligo-residual disease was defined as the presence of 1 – 4 residual lesions, including the primary site, at 3 months after the start of EGFR-TKI (Figure 1). [19,22] Residual disease was defined as detectable lesions on imaging evaluation after 3 months of EGFR-TKI therapy. Lesions that had previously been treated with local therapies, such as radiotherapy, were excluded from the definition of "residual disease" unless they had worsened. In all analysed patients the residual lesions were independently evaluated by a thoracic oncologist and radiologist. Among the 308 eligible patients, those with EGFR exon20 insertion orinitialT790M mutation (n = 9), those who did not continue EGFR-TKI therapy for 3 months (n = 61, 42 due to disease progression, and 19 due to side effects), those without an efficacy evaluation at the previously mentioned time frame (n = 17), those transferred to other hospitals during the treatment period (n = 7) and other reasons (n = 7) were all excluded from this study. (Figure 2) Page 4/20 Page 4/20 2.2 Treatment and assessments The baseline disease stage was assessed using systemic and brain imaging. Metastatic lesions were independently evaluated by a thoracic oncologist and radiologist in all patients. Based on a randomised phase II study, any metastatic thoracic lymph nodes (N1 – N3), including those in the supraclavicular fossae, were collectively considered as a single lesion. [22] PD was identified by reviewing follow-up radiological imaging, including CT and MRI, after the initiation of EGFR-TKI. Tumour responses were classified according to RECIST version 1.1. [24] PD patterns were classified as PD limited to residual sites of disease, PD at new sites, and PD at both sites (Figure 1). 3.3 Comparison between the oligo-residual and non-oligo-residual disease The clinical characteristics of patients with oligo-residual disease and those with non-oligo-residual disease are summarised in Table 1. The distribution of ECOG-PS differed significantly between these two groups (P = 0.021). The predominant metastatic organs also differed significantly, as pleura, pulmonary, bone, and brain metastases were more frequently observed in patients with non-oligo-residual disease than in those with oligo-residual disease. The median follow-up time for PFS was 16 months. The median PFS was 11.3 months (95% CI [8.7 – 13.7]) in patients with oligo-residual disease and 9.1 months (95% CI [7.0 – 10.2]) in patients with non- oligo-residual disease. There was no significant difference in PFS between patients with oligo-residual disease and those with non-oligo-residual disease (hazard ratio = 0.81, 95% CI [0.60 – 1.10], P = 0.183). (Figure 3) 3.2 Oligo-residual disease Three months after the start of EGFR-TKI treatment, 32% (n = 66) had oligo-residual disease. All 13 patients (100%) with synchronous oligometastatic disease at baseline had oligo-residual disease. Out of Page 5/20 the 194 patients with non-synchronous oligometastatic disease at baseline, 53 patients (27%) showed oligo-residual disease at 3 months. (Table 1) Baseline clinical factors were investigated to identify the predictive factors of oligo-residual disease. The univariate logistic regression analysis demonstrated that ECOG-PS 0 – 1 (Odds ratio [OR] = 6.41, 95% confidence interval (CI) [1.89 – 21.77], P = 0.003) and the presence of only one metastatic organ (OR = 4.10, 95% CI [2.18 – 7.72], P < 0.001) were predictive factors for oligo-residual disease. The multivariate logistic regression analysis demonstrated that ECOG-PS 0 – 1 (OR = 4.98, 95% CI [1.41 – 17.6], P = 0.013), the presence of only one metastatic organ (OR = 3.59, 95% CI [1.86 – 6.93], P < 0.001) and treatment with osimertinib (OR = 2.33, 95% CI [1.06 – 5.13], P = 0.034) were independent predictive factors of oligo-residual disease. (Table 2) 4. Discussion This is the first study that assesses the pattern of PD in patients with EGFR-mutated NSCLC using the residual disease evaluation at 3 months after the start of EGFR-TKI treatment, including first-, second-, and third-generation EGFR-TKIs. Our study found that, after adjusting for other clinical factors, oligo- residual disease was an independent predictive factor of PD limited to the residual sites in patients with EGFR-mutated NSCLC. Analysing the pattern of PD after EGFR-TKI treatment might be essential to provide the rationale for LAT of all disease sites in patients with EGFR-mutated NSCLC, as well as in NSCLC patients without EGFR mutations. [16,18] Our study also showed that more than 90% of patients had PD after EGFR-TKI. Acquired resistance remains a significant obstacle to achieving a durable response, even if the patients had oligo-residual disease response after EGFR-TKI treatment. Based on these data, adding consolidative LAT to all sites of residual disease at the time of response to EGFR-TKIs could potentially delay the time to progression or even improve survival outcomes in patients with oligo- residual disease. The results of our study show that the majority of patients with oligo-residual disease have PD limited to residual sites after EGFR-TKI treatment. Preclinical studies have demonstrated that synchronous oligometastatic disease does not metastasise to other sites. [27,28] EGFR-TKI treatment might have caused EGFR-mutated polymetastatic disease to become oligometastatic, which was defined as oligo- residual disease. Furthermore, our study showed that only 6% of patients had synchronous oligometastatic disease before treatment, which increased to 32% after treatment. These findings suggest that EGFR-TKI treatment for 3 months increased the number of patients eligible for LAT more than fivefold. Limited to the small number of cases in our study, treatment with osimertinib was found to be an independent predictor of oligo-residual disease and PD limited to residual sites. No previous study has demonstrated the differences in PD patterns between osimertinib and other EGFR-TKIs. The FLAURA study showed that osimertinib was more effective than first-generation EGFR-TKIs, and suggested that the incidence of CNS relapses was lower in patients treated with osimertinib than in those treated with first-generation EGFR-TKIs (19% vs. 43%). [4,5] A recent retrospective study showed that osimertinib might delay the development of CNS metastasis compared to first-generation EGFR-TKIs. 3.4 Pattern of PD A total of 191 patients (92%) had PD, 60 of whom (91%) had oligo-residual disease, and 131 (93%) with non-oligo-residual disease. PD limited to residual sites was observed in 44 patients (73%) with oligo- residual disease and 37 patients (28%) with non-oligo-residual disease. The proportion of PD in the residual sites alone was significantly higher in patients with oligo-residual disease than in those with non- oligo-residual disease (P < 0.001). (Figure 3) The clinical factors at 3 months from the start of EGFR-TKI treatment were investigated to identify the predictive factors of PD limited to residual sites. The univariate logistic regression analysis demonstrated that ECOG PS 0 – 1 (OR = 2.96, 95% CI [1.26 – 6.94], P = 0.012), the lack of CNS metastases (OR = 4.15, 95% CI [1.72 – 10.00], P = 0.002), residual metastatic organ number of 0 – 1 (OR = 2.53, 95% CI [1.39 – 4.61], P = 0.002), oligo-residual disease (OR = 6.98, 95% CI [3.51 – 13.24], P < 0.001), and treatment with osimertinib were associated with PD limited to residual disease sites (OR = 3.13, 95% CI [1.27 – 7.74], P = 0.013). The multivariate logistic regression analysis demonstrated that the lack of CNS metastases (OR = 3.07, 95% CI [1.10 – 8.52], P = 0.032), oligo-residual disease (OR = 5.43, 95% CI [2.57 – 11.46], P < 0.001), Page 6/20 Page 6/20 and treatment with osimertinib (OR = 3.24, 95% CI [1.13 – 9.25], P = 0.028) were independent predictors of PD limited to residual disease sites. (Table 3) and treatment with osimertinib (OR = 3.24, 95% CI [1.13 – 9.25], P = 0.028) were independent predictors of PD limited to residual disease sites. (Table 3) 4. Discussion [29] Intriguingly, the results of our study suggest that more effective EGFR-TKI treatment for CNS metastases may result in oligo-residual disease and PD limited to the residual sites. Additionally, the lack of residual CNS metastases at 3 months after the start of EGFR-TKI treatment was found to be an independent predictive factor of PD limited to residual sites in our study. Previous studies have suggested that patients with brain metastases have a higher risk of developing newer brain metastases and have a poorer prognosis. [30,31] ECOG PS 0 – 1 and having one metastatic organ at baseline were independent predictive factors of oligo-residual disease in patients with EGFR-mutated NSCLC. Previous studies have Page 7/20 Page 7/20 already shown that both were prognostic factors in EGFR-mutated NSCLC patients receiving EGFR- TKIs. [25,32] There were some limitations to our study. Our analysis was limited by its retrospective nature and the inability to account for unknown confounders. A relatively small sample size has the potential to affect its statistical power. This study was conducted in a cohort from a single institution and was not validated independently. Although not all patients had undergone comprehensive imaging at the time of disease progression, the patients were categorised based on all available imaging findings. Furthermore, this study included only patients who were able to continue EGFR-TKI therapy for 3 months, which may have resulted in selection bias. Additionally, since first-line treatment with osimertinib was approved in Japan in August 2018, the median follow-up period for these patients was 15.8 months, shorter than the 37.8 months for patients treated with first- and second-generation EGFR-TKIs. Thus, bias due to differences in follow-up periods cannot be ruled out. A randomised phase II study of osimertinib with or without local consolidation therapy for stage IV NSCLC (ClinicalTrials.gov identifier: NCT03410043) is currently ongoing. The results of a prospective randomised study could confirm our hypothesis. Moreover, from a strictly scientific perspective, counting the number of residual lesions might be a crude surrogate marker of oligo-residual disease. Evaluation of the number of alleles with EGFR mutations in circulating cell-free DNA during EGFR-TKI treatment has been applied in several studies. Recent studies have suggested that minimal residual disease can detect changes in circulating cell-free DNA. [33,34] Thus, this has the potential to become a new method of identifying true oligo-residual disease. [35,36] 5. Conclusions In conclusion, our study revealed that oligo-residual disease was significantly associated with PD limited to residual sites after EGFR-TKI treatment in patients with EGFR-mutated NSCLC. These results provide a rationale for LAT of all disease sites in patients with EGFR-mutated NSCLC and oligo-residual disease. Future studies should focus on the development of treatment strategies, including LAT, in patients with oligo-residual disease during treatment with EGFR-TKI. Central nervous system Confidence interval List Of Abbreviations Central nervous system Progressive disease Progressive disease Declarations Ethics approval and consent to participate: The study was approved by the institutional ethics review board of Shizuoka Cancer Centre (approval number: J2020-177-2020-1) and was conducted in accordance with the Declaration of Helsinki. The need for consent to participate was waived due to the retrospective nature of the study. Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Non-small-cell lung cancer Odds ratio Overall survival Progression-free survival Progressive disease Non-small-cell lung cancer Odds ratio Overall survival Progression-free survival Progressive disease Confidence interval Page 8/20 Confidence interval Eastern Cooperative Oncology Group performance status Epidermal growth factor receptor-tyrosine kinase inhibitor Local ablative therapy Eastern Cooperative Oncology Group performance status Epidermal growth factor receptor-tyrosine kinase inhibitor Local ablative therapy Page 8/20 Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. KT reports C/A and RF AstraZeneca K.K., Pfizer Japan, Inc., Eli Lilly K.K., MSD, and Boehringer Ingelheim as well as grants from Takeda Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., KYORIN Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., GlaxoSmithKline Consumer Healthcare Japan K.K., SHIONOGI & CO., LTD., and Novartis Pharma K.K., which are unrelated to the submitted work. TT reports C/A and RF from AstraZeneca KK, Pfizer Japan, Inc., Eli Lilly Japan K.K., Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., MSD K.K., Boehringer Ingelheim Japan, Inc., and Pfizer Japan, Inc., and Roche Diagnostics K.K., outside the submitted work. TM, HK (Kodama), NN, EM, TN, and, KM declare that they have no competing interests. Funding: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Funding: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contributions: TM and HK wrote the manuscript and researched data. HK reviewed and edited the manuscript. KM is a professional biostatistician and responsible for statistical analysis. All authors reviewed, approved the final version of the manuscript. Authors’ contributions: TM and HK wrote the manuscript and researched data. HK reviewed and edited the manuscript. KM is a professional biostatistician and responsible for statistical analysis. All authors reviewed, approved the final version of the manuscript. Acknowledgements: We would like to thank Editage (https://www.editage.jp) for editing this manuscript. Acknowledgements: We would like to thank Editage (https://www.editage.jp) for editing this manuscript. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. P 9/20 Competing interests: HK (Kenmotsu) reports C/A and RF from Chugai Pharmaceutical Co, Ltd., C/A from Ono Pharmaceutical Co, Ltd., C/A from Boehringer Ingelheim, C/A from Eli Lilly K.K, C/A from Kyowa Hakko Kirin Co., Ltd., C/A from Bristol-Myers Squibb, C/A from MSD, C/A and RF from Novartis Pharma K.K., C/A and RF from Daiichi-Sankyo Co., Ltd., C/A and RF from AstraZeneca K.K., C/A from Pfizer, C/A from Taiho Pharma, outside the submitted work. NM reports C/A from AstraZeneca KK, Pfizer Japan, Inc., C/A from Chugai Pharmaceutical Co., Ltd., C/A and RF from Boehringer Ingelheim, C/A from MSD K.K., personal fees from TAIHO PHARMACEUTICAL CO., LTD., C/A from ONO PHARMACEUTICAL CO., LTD., outside the submitted work. HK (Kobayashi) reports C/A from Eli Lilly K.K, C/A from Taiho Pharmaceutical, personal fees from AstraZeneca, outside the submitted work. SO reports C/A from Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical, AstraZeneca K.K., Boehringer Ingelheim, Taiho Pharmaceutical, and MSD, which are unrelated to the submitted work. RK reports C/A and RF from Boehringer Ingelheim, C/A and RF from AstraZeneca, C/A from Taiho Pharmaceutical, Chugai Pharmaceutical, Ono Pharmaceutical, Pfizer, and Lilly, outside the submitted work. KW reports C/A and RF from Chugai Pharmaceutical Co., Ltd., C/A from Taiho Pharmaceutical, C/A from Boehringer Ingelheim, C/A from Eli Lilly K.K., C/A from Ono Pharmaceutical, C/A from MSD, C/A and RF from AstraZeneca, RF from Novartis, RF from AbbVie, outside the submitted work. AO reports C/A from Taiho Pharmaceutical, C/A from Ono Pharmaceutical, C/A from Chugai Pharmaceutical Co., Ltd., C/A from Novartis Pharma K.K., outside the submitted work. HM reports C/A from AstraZeneca K.K., Ono Pharmaceutical, Bristol- Myers Squibb Japan, Chugai Pharmaceutical Co., Ltd., Pfizer Inc., Novartis Pharma K.K., Boehringer Ingelheim, Taiho Pharmaceutical, Eli Lilly K.K., and MSD, which are unrelated to the submitted work. HH reports C/A and RF from Daiichi Sankyo Pharmaceutical Co. AstraZeneca K.K., Brain Labo Co., and Page 9/20 Page 9/20 Chugai Pharmaceutical Co., and RF from the Japan Agency for Medical Research and Development and the National Cancer Centre Research and Development Fund, which are unrelated to the submitted work. ME reports C/A from Ono Pharmaceutical, C/A from AstraZeneca, C/A from Takeda Pharmaceutical Co., Ltd, C/A from Daiichi-Sankyo Co., Ltd, outside the submitted work. References 1. Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. 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Tables Tables Page 13/20 Table1 Patient Characteristics at baseline and 3 months from EGFR-TKI Characteristics N=207 (%) Baseline 3 months from EGFR-TKI Overall N=207 Oligo-residual n=66 (%) Non-Oligo n=141 (%) P Median age (range) 68 (34-88) 67 (41-87) 68 (34-88) 0.478 Sex Male 77 (37) 22 (33) 55 (39) 0.431 Female 130 (63) 44 (67) 86 (61) ECOG-PS 0 43 (21) 28 (42) 34 (24) 0.021 1 128 (62) 36 (55) 90 (64) 2 26 (12) 2 (3) 15 (11) 3 10 (5) 0 2 (1) Smoking status Ever 109 (53) 34 (52) 64 (45) 0.411 Never 98 (47) 32 (48) 77 (55) Histology Adenocarcinoma 196 (95) 61 (92) 135 (96) 0.321 Non-Adenocarcinoma 11(5) 5 (8) 6 (4) Type of EGFR mutation Del 19 124 (60) 33 (50) 91 (65) 0.391 L858R 73 (35) 30 (45) 43 (30) Other 10 (5) 3 (5) 7 (5) Site of metastatic organs Pleura 122 (59) 0 92 (65) <0.001 Pulmonary 114 (55) 17 (26) 63 (45) 0.009 Bone 95 (46) 12 (18) 62 (44) <0.001 Brain 71 (34) 6 (9) 32 (23) 0 018 Adrenal grand 24 (12) 5 (8) 19 (14) 0.217 Extra-thoracic lymph node 23 (11) 4 (6) 12 (9) 0.538 Others 13 (6) 2 (3) 10 (7) 0.244 Number of lesions 1 0 17 (8) 0 2 0 19 (9) 0 3 3 (1) 20 (10) 0 4 10 (5) 10 (5) 0 5 5 (3) 0 2 (1) 6 5 (3) 0 0 7 1 (0) 0 2 (1) 8 3 (1) 0 1 (0) 9 0 0 0 ≥10 180 (87) 0 136 (66) Number of metastatic organs 0 0 15 (22) 0 1 63 (31) 39 (59) 60 (43) 2 62 (30) 11 (17) 33 (23) 3 46 (22) 1 (2) 20 (14) 4 24 (12) 0 20 (14) 5 5 (2) 0 3 (2) 6 7 (3) 0 5 (4) EGFR-TKI treatment Gefitinib 96 (46) 30 (45) 66 (47) 0.132 Erlotinib 49 (24) 11 (17) 38 (27) Afatinib 24 (12) 8 (22) 16 (11) Osimertinib 38 (18) 17 (26) 21 (15) Eastern Cooperative Oncology Group; PS, performance status; EGFR, epidermal growth facto r; TKI tyrosine kinase inhibitor; CNS central nervous system; DEL 19 exon 19 deletions; L85 ECOG, Eastern Cooperative Oncology Group; PS, performance status; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor; CNS, central nervous system; DEL 19, exon 19 deletions; L858R, Page 15/20 L858R point mutations. Tables Table 2. Predictive factors of Oligo-residual disease using a logistic regression model adjusted for baseline patient characteristics Covariates N=207 Univariate analysis Multivariate analysis OR 95% CI P- value OR 95% CI P- value Age (<75 years vs ≥75 years) 1.74 0.87-3.45 0.114 Sex (male vs female) 1.29 0.69-2.36 0.432 ECOG performance status score (0-1 vs ≥2) 6.41 1.89- 21.77 0.003 4.98 1.41- 17.6 0.013 Smoking status (ever vs never) 1.28 0.71-2.29 0.411 Histology (adeno vs non-adeno) 0.54 0.15-1.84 0.327 EGFR mutation (del19/L858R vs Others) 0.64 0.23-1.77 0.394 No baseline CNS metastases 1.00 0.53-1.85 0.989 Baseline number of metastatic organ (1 vs ≥2) 4.10 2.18-7.72 <0.001 3.59 1.86- 6.93 <0.001 Treatment with Osimertinib 1.98 0.96-4.07 0.063 2.33 1.06- 5.13 0.034 Significant P-values are shown in bold type. OR, odds ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; EGFR; epidermal growth factor receptor; DEL 19, exon 19 deletions; L858R, L858R point mutations; CNS, central nervous system. Significant P-values are shown in bold type. OR, odds ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; EGFR; epidermal growth factor receptor; DEL 19, exon 19 deletions; L858R, L858R point mutations; CNS, central nervous system. Significant P-values are shown in bold type. OR, odds ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; EGFR; epidermal growth factor receptor; CNS, central nervous system m; del 19, exon 19 deletions; L858R, L858R point mutations. Significant P-values are shown in bold type. OR, odds ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; EGFR; epidermal growth factor receptor; CNS, central nervous system m; del 19, exon 19 deletions; L858R, L858R point mutations. g yp Cooperative Oncology Group; EGFR; epidermal growth factor receptor; CNS, central nervous system m; del 19, exon 19 deletions; L858R, L858R point mutations. Figures Page 16/20 Table 3. Predictive factors of PD limited to residual sites of disease using a logistic regression model adjusted for patient characteristics Covariates N=191 Univariate analysis Multivariate analysis OR 95% CI P- value OR 95% CI P- value Age (< 75 vs ≥ 75 years)　 1.14 0.60- 2.17 0.669 Sex (male vs female) 1.05 0.58- 1.90 0.853 ECOG performance status score (0-1 vs ≥2) 2.96 1.26- 6.94 0.012 1.77 0.67- 4.66 0.249 Smoking status (ever vs never) 1.65 0.92- 2.95 0.088 1.41 0.72- 2.74 0.315 Histology (adeno vs non-adeno) 1.11 0.30- 4.07 0.874 EGFR mutation (del19/L858R vs Others) 2.60 0.69- 9.76 0.157 No residual CNS metastases 4.15 1.72- 10.00 0.002 3.07 1.10- 8.52 0.032 Number of residual metastatic organ (0-1 vs ≥2) 2.53 1.39- 4.61 0.002 1.02 0.48- 2.14 0.951 Oligo-residual disease 6.98 3.51- 13.24 <0.001 5.43 2.57- 11.46 <0.001 Treatment with Osimertinib* 3.13 1.27- 7.74 0.013 3.24 1.13- 9.25 0.028 Table 3. Predictive factors of PD limited to residual sites of disease using a logistic regression model adjusted for patient characteristics Page 17/20 Page 17/20 Figure 1 Oligo-residual disease and PD limited to residual sites. EGFR, epidermal growth factor receptor; PD, progressive disease; TKI, tyrosine kinase inhibitor. Figure 1 Oligo-residual disease and PD limited to residual sites. EGFR, epidermal growth factor receptor; PD, progressive disease; TKI, tyrosine kinase inhibitor. Oligo-residual disease and PD limited to residual sites. EGFR, epidermal growth factor receptor; PD, progressive disease; TKI, tyrosine kinase inhibitor. Page 18/20 Page 18/20 Figure 2 Flowchart of included and excluded patients. EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; TKI, tyrosine kinase inhibitor. Page 19/20 Figure 3 A) Kaplan-Meier curves for progression-free survival of oligo-residual and non-oligo-residual disease. The P-value was calculated using the log-rank test. The small vertical lines on the curve indicate patients who were censored. B) Pattern of progressive disease. EGFR, epidermal growth factor receptor; PD, progressive disease; TKI, tyrosine kinase inhibitor. Figure 3 Figure 3 A) Kaplan-Meier curves for progression-free survival of oligo-residual and non-oligo-residual disease. The P-value was calculated using the log-rank test. The small vertical lines on the curve indicate patients who were censored. B) Pattern of progressive disease. EGFR, epidermal growth factor receptor; PD, progressive disease; TKI, tyrosine kinase inhibitor. Page 20/20 Page 20/20
https://openalex.org/W3119974988	https://pathology.medresearch.in/index.php/jopm/article/download/506/824	English	null	Malaria Seroprevalence in the Blood Donors of Malaria Endemic Regions of Northern Andhra Pradesh, India.	Pathology update	2,020	cc-by	2,608	Malaria Seroprevalence in the Blood Donors of Malaria Endemic Regions of Northern Andhra Pradesh, India. Sitalata C.1, Kalyan K.2* DOI: https://doi.org/10.17511/jopm.2020.i08.06 DOI: https://doi.org/10.17511/jopm.2020.i08.06 1 C. Sitalata, Assistant Professor, Department of Pathology, Maharaja Institute of Medical Sciences, Vizianagaram, Andhra Pradesh, India. 2* K. Kalyan, Professor, Department of Pathology, Dr. PSIMS, and RF, Gannavaram, Andhra Pradesh, India. Introduction: Transmission of malaria through blood transfusion continues to be a major threat to safe blood transfusion practice. Transfusion-transmitted malaria occurs at an estimated rate of 0.25 cases per 1 million blood units collected. It is significantly more common in endemic areas. Aim: To study the Seroprevalence of Malaria among the blood donors in the endemic areas of Northern Andhra Pradesh. Materials and methods: The present survey was carried out at the blood bank of Maharaja institute of medical sciences, Vizianagaram. This includes the analysis of seroprevalence of Malaria in the blood donors during the period of 1 year from February 2018 to January 2019. Two ml of the blood sample was collected in the labeled pilot tube at the time of collection of blood from donor tubing of the blood bag. The serum was separated. The samples were tested for Malaria by rapid antigen detection test. Results: Out of the total of 3096 blood donors, replacement donors (86.91%) were more in comparison to voluntary donors (13.08%). The seroprevalence among the replacement blood donors was more compared to voluntary blood donors. Conclusion: Voluntary blood donation, increasing awareness about blood donation in the general population, selection of repeat, non-remunerated, regular voluntary blood donors and diligent donor selection, sensitive screening tests are most important to increase blood safety and prevent transmission of Malaria through blood transfusion. Keywords: Malaria, Seroprevalence, Blood donors, Endemic zone Keywords: Malaria, Seroprevalence, Blood donors, Endemic zone Corresponding Author How to Cite this Article To Browse K. Kalyan, Professor, Department of Pathology, Dr. PSIMS, and RF, Gannavaram, Andhra Pradesh, India. Email: Sitalata C, Kalyan K. Malaria Seroprevalence in the Blood Donors of Malaria Endemic Regions of Northern Andhra Pradesh, India.. Trop J Pathol Microbiol. 2020;6(8):493-497. Available From https://pathology.medresearch.in/index.php/jopm/ar ticle/view/506 Tropical Journal of Pathology and Microbiology 2020 Volume 6 Number 8 November-December E ISSN:245 P-ISSN:245 RNI:MPENG/20 Research Blood Do 2020 Volume 6 Number 8 November-December Publisher www.medresearch.in Publisher www.medresearch.in Publisher Introduction Transfusion of blood and its components is lifesaving as well as it has life-threatening hazards. With every transfusion, there is a chance of transfusion-associated problems. Vector-borne diseases like Malaria, Filariasis, Dengue are endemic in the Northern parts of Andhra Pradesh. Among all, Malaria continues to be a leading public health problem in Northern Andhra Pradesh. It constitutes about 400 million cases and 2.5 million deaths annually [1-5]. Study design: A retrospective cross-sectional study. Sample size: In this study, 3096 samples were collected. Out of these, 2691 samples were from replacement donors and 405 samples were from voluntary donors. These samples are collected from both the sex and from different age groups. The blood donor’s history and lab test records were reviewed at the blood bank. Inclusion criteria: All the donors were required to pass through a panel of questions on previous illnesses and medical conditions for blood donation. They should meet the eligibility criteria which consists of age between 17 to 65 years, body weight > 45 kg, and no history of blood transfusion, hepatitis, jaundice, hypertension, and fever at the time of blood donation. Transmission of malaria through blood transfusion is more common in endemic areas and cases are being reported in non-endemic areas too. Transfusion transmission malaria occurs at an estimated rate of 0.25 cases per 1 million blood units collected. In many establishments, Malaria screening in potential blood donors is based on Interviews. Even though there is a specific test for malaria, prevention of transmission of malaria through blood transfusion is not possible mainly because of the inability of the test to detect the antigen in the window period. Exclusion criteria: Any donor who does not meet the eligibility criteria and also any donor having reaction during blood donation procedure was excluded from the study. This survey aimed to access the Seroprevalence of malaria in blood donors of malaria-endemic regions of Northern Andhra Pradesh. The present study was done on voluntary and replacement donors in Maharaja institute of medical sciences. Sample collection: The samples were obtained from donors using the recommended bio-security measures. 2ml of the blood sample was collected in the labeled pilot tube at the time of collection blood from donor tubing of the blood bag. It is centrifuged at 3500 rpm for 5minutes to obtain clear non- hemolyzed serum. Results Table-1 explained the seropositivity of malaria in different donor categories studied at Maharaja institute of medical sciences, Vizianagaram. Out of the 3096 blood donors, 2691 were replacement donors and 405 were voluntary donors. Introduction The test used to diagnose Malaria was the Rapid Diagnostic test - SD BIO-LINE RAPID TEST (Standard Diagnostic, INC) which is a visual, rapid, and sensitive immunoassay for the qualitative diagnosis of Malaria. Rapid diagnostic test kits are used for the diagnosis of malaria. Routine methods like microscopic visualization of malarial parasites in thick and thin smears are inadequate for wide-field applications. Rapid diagnostic tests detect malarial antigens using immunochromatographic methods. Other diagnostic methods available are microscopy using fluorochromes, polymerase chain reaction tests, and antibody detection by serology. Corresponding Author K. Kalyan, Professor, Department of Pathology, Dr. PSIMS, and RF, Gannavaram, Andhra Pradesh, India. Email: K. Kalyan, Professor, Department of Pathology, Dr. PSIMS, and RF, Gannavaram, Andhra Pradesh, India. Email: Manuscript Received Review Round 1 Review Round 2 Review Round 3 Accepted 2020-11-28 2020-12-08 2020-12-17 2020-12-23 Conflict of Interest Funding Ethical Approval Plagiarism X-checker Note No Nil Yes 8% © 2020 by C. Sitalata, K. Kalyan and Published by Siddharth Health Research and Social Welfare Society. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ unported [CC BY 4.0]. Tropical Journal of Pathology and Microbiology 2020;6(8) 493 Review Round 2 2020-12-17 Ethical Approval Yes Manuscript Received 2020-11-28 Conflict of Interest No Review Round 1 2020-12-08 Funding Nil © 2020 by C. Sitalata, K. Kalyan and Published by Siddharth Health Research and Social Welfare Society. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ unported [CC BY 4.0]. Tropical Journal of Pathology and Microbiology 2020;6(8) Sitalata C. et al: Malaria Seroprevalence in the Blood Donors Also, they prepare different blood components including platelets and plasma. Conclusion The overall seroprevalence of transmission of malaria through blood transfusion was 0.22%. This result does not include transfusion transmission malaria during the window period, which is serological negative. This still poses a threat to blood safety in the environment. Thus, there might be a higher rate of transfusion transmission of malaria in the community. Our survey concluded that the seroprevalence of malaria was more in replacement donors than voluntary donors. The screening of blood bank donors in endemic areas is of higher importance because of the two opposing factors in play: the scarce number of blood donors and the increased risk of disease transmission. So, it is important to increase the no. of voluntary donors who were at low risk. In this study, all the positives blood units were obtained from replacement donors. This can be due to the reason that the replacement donors are less likely to express about previous exposure. Also, there was less number of voluntary donors. Several studies indicate the prevalence rates of transfusion- transmissible infections are found to be higher among replacement donors than voluntary donors. Table-3: Percentage of voluntary and replacement donors in different studies. Chavan SK in their study has reported that over the last 10 years malarial infection has been negligible in the studied population. Prevalence of Malaria was low (0.002 %) with only one case being detected overall (in the year 2013). No cases of Malaria were detected in other years (2004 to 2012) [14]. Though globally malaria constitutes a big health problem in the general population, the prevalence of malaria among blood donors is low in most studies. Authors Voluntary donors Replacement donors Sonawane et al [6] 1.5 98.5 Castillo et al [7] 5.3 94.7 Singh et al [8] 15.5 84.5 Bhattachary et al [9] 94.6 5.4 Bruce-Chwatt et al [10] 64.78 35.22 Present Study 13.08 86.91 Methods and Materials Study Area and Period: The present study was carried out at the blood bank of Maharaja institute of medical sciences, Vizianagaram from February 2019 to January 2020. The blood bank of the department of pathology, Maharaja institute of medical sciences is a licensed blood bank. It has an average collection of 4000 units of blood from healthy donors in and around Vizianagaram. It has been collecting blood from donors by undergoing campaigns and institutes, routinely screens the collector blood, and provide it to recipients in need at hospitals. Table-1: Seroprevalence of Malaria in different donors. Donor Category No. of blood units Screened No. of Seropositive (%) Voluntary 405 0 0 Replacement 2691 7 0.26 Total 3096 7 0.22 and Microbiology 2020;6(8) Table-1: Seroprevalence of Malaria in different donors. Tropical Journal of Pathology and Microbiology 2020;6(8) Tropical Journal of Pathology and Microbiology 2020;6(8) 494 Sitalata C. et al: Malaria Seroprevalence in the Blood Donors Out of the 3096 screened blood samples, seven units of the blood units which constitute 0.22% were positive for malarial parasites. Of the positive units, seven blood units which constitute 0.26% were from replacement donors and zero blood units from voluntary donors. Table-2 gives information about the age-wise distribution of malaria positive donors. The majority of the donor's positive donors belong to the age group of 36-45 years. These imply that there is a need on increasing the number of voluntary donors through creating awareness among the population. By increasing the voluntary donors, it can be ensured the safety of blood transfusion as they were at low risk. In endemic areas, it is better to advise chemoprophylaxis for all the recipients of blood transfusion. Elghouzzi et al [12] studied the result of the new ELISA malaria screening. The observed malaria prevalence was 0.05%. Hildaf Etal [13] have reported 0.01% seropositivity among blood donors. Keshav et al17 have reported 0.04% seropositivity among blood donors. Table-2: Age-wise distribution of Malaria positive donors. Table-2: Age-wise distribution of Malaria positive donors. Table-2: Age-wise distribution of Malaria positive donors. Age Range (years) No of Sero-Positives Percentage 18-25 0 0 26-35 1 14.2 36-45 5 71.4 46 and Above 1 14.2 Total 7 100 Interestingly, PCR analysis of 115 individuals, rejected as potential donors because they had traveled to or lived in malaria-endemic areas within the previous year, showed that none were infected with any of the Plasmodium species [7]. Thus, efficient diagnostic tests for blood donor screening need to be implemented to avoid losing valuable blood donors. Table-3 shows the percentage of replacement and voluntary donors. In a study by Sonawane et al [6] at Ambajogai, out of all the blood units screened, none were positive. Author’s contribution Dr. C. Sitalata: Concept, study design Dr. K. Kalyan: Manuscript preparation Dr. C. Sitalata: Concept, study design Dr. C. Sitalata: Concept, study design Dr. K. Kalyan: Manuscript preparation 10. Singh K, Bhat S, Shastry S. Trend in seroprevalence of hepatitis B virus infection among blood donors of coastal Karanataka. Indian J Infect Dev Ctries. 2009;3(5)376-379. [Crossref] Sitalata C. et al: Malaria Seroprevalence in the Blood Donors 07. Bruce‐Chwatt LJ. Transfusion malaria. Bull World Health Organ. 1974;50(3-4)337-346. [Crossref] Extensive donor selection, sensitive screening procedures, and strict blood donation guidelines can improve blood safety. The emphasis must also be laid on voluntary risk reduction, which will require increased awareness and change in the attitude of people. With the implementation of the above- mentioned strategies, it is possible to reduce the transfusion of malaria through blood transfusion. Extensive donor selection, sensitive screening procedures, and strict blood donation guidelines can improve blood safety. The emphasis must also be laid on voluntary risk reduction, which will require increased awareness and change in the attitude of people. With the implementation of the above- mentioned strategies, it is possible to reduce the transfusion of malaria through blood transfusion. 08. Organization WHO. New perspectives malaria diagnosis. The WHO Organization. 2000;14-15. Available at [Article] [Crossref] 09. Srikrishn A, Sitalaxmi S, Domodhar P. How safe are our safe blood donors?. Indian J Pathol Microbial. 1999;42(4)411-416. [Crossref] What does the study add to the existing knowledge? Based on the results, it was felt that to reduce the risk of transfusion-transmitted malaria non- remunerated repeat voluntary donor services need to be instituted. Tropical Journal of Pathology and Microbiology 2020;6(8) Tropical Journal of Pathology and Microbiology 2020;6(8) 495 Sitalata C. et al: Malaria Seroprevalence in the Blood Donors Reference 01. Fernandes H, D’souza P, D’souza P. Prevalence of transfusion transmitted infections in voluntary and replacement donors. Indian of Heamatol Blood Transfus. 2010;26(3)89-91. 11. Bhattacharya P, Chandra PK, Datta S, Banarjee A. Significant increase in HBV, HCV, HIV and syphilis infection among blood donors in West Bengal, Eastern India, 2004-2005, Exploratory screening reveals high frequency of occult HBV infection. World J Gastroenterol. 2013;13(27): 3730-3733. doi: 10.3748/wjg.v13.i27.3730 [Crossref] 02. Elghouzzi MH, Senegas A, Steinmetz T, Guntz P, Barlet V, Assal A, et al. Multicentric evaluation of the DiaMed enzyme-linked immunosorbent assay malaria antibody test for screening of blood donors for malaria. Vox Sang. 2008;94(1)33-40. doi: 10.1111/j.1423-0410.2007.00998.x [Crossref] 12. Sharma RI, Patel KJ, Patel H, Dharaiya MC. Seroprevalence of transfusion transmissible infections among blood donors in tertiary care centre of Gandhinagar, Gujarat, India. Trop J Pathol Microbiol. 2018;4(4)302-307. Available from: [Article] [Crossref] 03. Castillo CM, Ramírez C. Malaria screening in blood donors in Cali, Colombia. Biomedica. 2005;25;203-210. [Crossref] 13. Chavan SK, Chavan KB. Seroprevalence, trend of transfusion transmittable infections and co- infections rate among blood donors at tertiary care hospital – 10 years study. Int J Res Health Sci. 2014;2(4)1014-1020. [Crossref] 04. Seed CR, Kitchen A, Davis TM. The current status and potential role of laboratory testing to prevent transfusion-transmitted malaria. Transfus Med Rev. 2005;19(3)229-240. doi: 10.1016/j.tmrv.2005.02.004 [Crossref] 14. Irshad M, Peter S. Spectrum of viral hepatitis in thalassemic children receiving multiple blood transfusions. Indian J Gastroenterol. 2002;21(5)183-184. [Crossref] 05. Rodulfo H, de Donato M, Quijada I, Peña A. High prevalence of malaria infection in Amazonas state, Venezuela. Rev Inst Med Trop Sao Paulo. 2007b;49(2)79-85. doi: 10.1590/s0036-46652007000200003 [Crossref] 15. Sulhyan Kalpana R, Anvikar Arti R, Ratnaparkhi Aaditi D. Seroprevalence of transfusion transmissible infections among blood donors at a tertiary care centre in Maharashtra, India. Int J Contemp Med Res. 2017;4(9)1865-1867. [Crossref] 06. Kitchen AD, Chiodini PL. Malaria and blood transfusion. Vox Sang. 2006;90(2)77-84. doi: 10.1111/j.1423-0410.2006.00733.x [Crossref] Tropical Journal of Pathology and Microbiology 2020;6(8) Tropical Journal of Pathology and Microbiology 2020;6(8) 496 Tropical Journal of Pathology and Microbiology 2020;6(8) Sitalata C. et al: Malaria Seroprevalence in the Blood Donors Sitalata C. et al: Malaria Seroprevalence in the Blood Donors 16. Teklemariam Z, Mitiku H, Weldegebreal F. Seroprevalence and trends of transfusion transmitted infections at Harar blood bank in Harari regional state, Eastern Ethiopia- eight years retrospective study. BMC Hematol. 2018;18(1)24. doi: 10.1186/s12878-018-0115-2 [Crossref] 19. Talib VH, Khuana SK. Hematology for Students. 1996. 1st Ed 415–416. [Crossref] 20. Morar MM, Pitman JP, McFarland W, Bloch EM. The contribution of unsafe blood transfusion to human immunodeficiency virus incidence in sub- Saharan Africa- reexamination of the 5% to 10% convention. Transfusion. 2016;6(12)3121– 3132. doi: 21.1111/trf.13816 [Crossref] 17. WHO African Region. Ethiopia 2006 /Regional training workshop on blood donor recruitment- pre- and post-donation counseling. 2006. Available at [Article] [Crossref] doi: 21.1111/trf.13816 [Crossref] 18. Centeral Statistics Agency. ICF MacroCalverton. Addis Ababa- Ethiopia demographic and health survey. 2011. Available at [Article] [Crossref] Tropical Journal of Pathology and Microbiology 2020;6(8) 497
https://openalex.org/W4234346596	https://www.qeios.com/read/AQHFZH/pdf	English	null	Chemical Association	Definitions	2,020	cc-by	54	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Chemical Association National Cancer Institute Qeios ID: AQHFZH · https://doi.org/10.32388/AQHFZH Source National Cancer Institute. Chemical Association. NCI Thesaurus. Code C73471. Any process of combining chemical entities that is dependent upon chemical forces. Qeios ID: AQHFZH · https://doi.org/10.32388/AQHFZH 1/1
https://openalex.org/W3032197815	https://www.matec-conferences.org/articles/matecconf/pdf/2020/10/matecconf_icsc-isatech20_01001.pdf	English	null	A novel real-time safety level calculation approach based on STPA	MATEC web of conferences	2,020	cc-by	4,580	A novel real-time safety level calculation approach based on STPA Apostolos Zeleskidis1,, Ioannis M. Dokas2, and Basil Papadopoulos3 1Undergraduate Student (Democritus University of Thrace, Civil Engineering, Greece) 2Assistant Professor (Democritus University of Thrace, Civil Engineering, Greece) 3Professor (Democritus University of Thrace, Civil Engineering, Greece) Abstract. This paper proposes a novel approach to dynamic safety level calculation for safety-critical systems based on the STAMP accident model and the implementation of a mathematical model. The proposed approach utilises (1) an STPA hazard analysis applied to the system in question, (2) system operational data from domain experts regarding process duration and reaction times, and (3) real-time system data. The STPA analysis is transformed into acyclic diagrams that graphically indicate every possible sequence of safety constraint violations that could lead to a loss (path). Based on this diagram the safety level (SL) of a system is defined as SL=p"⃗! where p"⃗! is the most detrimental to safety path which is active for any possible time value or context in the system’s operation. This approach is also demonstrated using as a case study the “classical” Train Door STPA analysis example. This paper aims to introduce a new perspective on the problem of measuring and managing the actual safety level of highly complex socio-technical systems in real time and discusses related limitations and future research opportunities of this approach. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Corresponding author: aposzele@civil.duth.gr MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 A novel real-time safety level calculation approach based on STPA Apostolos Zeleskidis1,, Ioannis M. Dokas2, and Basil Papadopoulos3 1Undergraduate Student (Democritus University of Thrace, Civil Engineering, Greece) 2Assistant Professor (Democritus University of Thrace, Civil Engineering, Greece) 3Professor (Democritus University of Thrace, Civil Engineering, Greece) 2 STPA STPA is a hazard analysis technique based on the STAMP accident model. According to STAMP, safety is an emergent property of systems, and accidents can occur not just because of component failures but also due to unsafe interactions among system components that did not fail [8]. That means that the feedback control loops of the system should be designed, developed and operated in a manner that their controllers will not enforce unsafe control actions in any possible operational context or environment due to lack of awareness of the system state (i.e. mental model of the system). This could be achieved by applying the STPA hazard analysis during the life cycle of the system, ideally as early as possible. The STPA hazard analysis consists of four steps. In the first step, the purpose of the analysis and the boundaries of the system are defined, together with the losses or accidents (A), the system level hazards (H), and the system level safety constraints. During the second step of STPA, a functional diagram known as the safety control structure diagram is developed, where the controllers and the controlled processes of the system are shown, together with the control actions and the feedback each controller is receiving from the process it controls. In the third step, the control actions of each controller are analyzed to examine under which contexts they could lead to the identified losses. The so-called Unsafe Control Actions (UCA) are then translated into safety constraints or safety specifications. In addition, all Control Actions (CA) given by the controller are analysed to determine how they could be unable to affect the controlled process as intented while enforced by the controller within the appropriate context. The fourth step aims at identifying the reasons why unsafe control actions might be enforced in the system. As a result, loss scenarios (S) are created to explain how incorrect feedback, inadequate requirements, design errors, component failures and other factors that could cause unsafe or ineffective control actions (CA) which are grouped and analysed fundementaly as UCAs [8]. This analysis incorporates safety barriers and other similar mechanisms as parts of the system and finds the weaknesses in the complete system together with its defenses. MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 RiskSOAP indicator. With RiskSOAP, one can compare the safety constraints of existing systems compared to their ideal set of safety constraints that derive from their STPA [8] and EWaSAP [9] analysis, and calculate a situational awareness indicator for the system under study. y The problem is that there are no methodologies paired with a mathematical model that can determine dynamically what the safety level is at any certain moment in time for any given context and how much time is left until an accident takes place. To fill this gap, we propose a novel approach including a mathematical model for systems’ safety level determination and its dynamic calculation based on the STAMP accident model. The proposed model utilizes the outcomes of STPA hazard analyses which are transformed into diagrams. In these diagrams, each node depicts an STPA based safety constraint, and each path of the acyclic diagram depicts a possible scenario of safety constraints violations that can lead to accidents. 1 Introduction Several safety indicators are utilised to monitor safety drifts and to assess whether a system maintains its safety within acceptable levels. As a result, numerous categories of safety indicators have been proposed in the literature such as event indicators, barrier indicators, activity indicators, and programming indicators [1,2]. However, the need to “get smarter at predicting the next accident” [3] and the challenge of measuring what the actual safety level of a system is at a certain moment in time at a given context [4] still remains, regardless of the introduction of new accident models and the view of safety as an emergent system property. To address this problem, Leveson [5] proposed that useful leading indicators can be identified based on the assumptions [6] underlying our safety engineering practices rather than on the likelihood of loss events. Thus, there is a need to monitor whether the generated safety assumptions based on which a system was designed, hold during the phase of operations are vulnerable or changed. Chatzimicailidou et al. [7] suggested to measure the gap between system design and operation as a measure for safety and introduced the MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 is the Model Development process of the system under study which leades to the creation of a mathematical model dedicated to the system. Then, from real time system data and the operational mode of the system the Safety Level Determination process is initiated. Operational modes are a tool used in complicated systems to distinguish between different modes in a systems operation. For example, an airplane during a typical flight goes through 3 different operational modes: take off, landing and cruising. The goal of the Safety Level Determination process is to dynamically calculate the Safety Level of the system in real time Figure. 1. The proposed approach. Figure. 1. The proposed approach. 3.1 Model Developments In more detail, the main goal of the Model Development process is to create and populate an acyclic diagram that represents the system in terms of safety and calibrate the mathematical model to the exact system in study. This is achieved by utilising the following inputs: 1. Safety specifications and constraints obtained from the STPA analysis are turned into nodes of the diagram and are categorised according to their impact on safety. The Categories (levels) are ranked from most to least consequential according to STPA: Level 4 = Accidents, Level 3 = Hazards, Level 2 = Unsafe Control Actions (UCAs) and ineffective Control Actions (CA) and Level 1 = Scenarios. Also, safety constraints are causally linked as shown in the diagram by the connections between the nodes which represent the specifications. These connections are always between nodes of two ascending levels (from level 1 to level 2 or from level 2 to level 3 etc.). See Figure 2 for nodes connections and levels of safety constraints in acyclic diagrams. 2. Time ranges given by domain experts or observed by studying the system regarding the connections of the casually linked safety constraints. These should be distilled in the form of sets (min(t), max(t)) for every connection between two nodes, such as the time it would take from the moment that an Unsafe Control Action is given to the moment that a Hazardous state occurs because of the specific UCA in the system. Each set represents the maximum and minimum amount of time it would take for an active node to activate the node connected with it (Figure 3). 3. The operation used (minimum, maximum, mean average etc.) to discern a single time value tx→y from the time ranges which represent the flexibility in how the entire operation can be managed. A path is defined as a possible scenario of safety constraints violations that can lead to accidents. It is one of the possible “roads” leading from the lowest level (loss scenarios) to the highest (losses) using the connections between nodes. For instance, the example of Figure 2 has a total of 17 paths and one possible path is S1→ UCA1→ H-2→ A (shown with bold arrows). Each path is comprised of 4 nodes, 3. 3 Methodology The complete approach we suggest is depicted in Figure 1. The proposed model is comprised of two main processes. The results of STPA and EWaSAP analyses as well as time ranges taken from domain experts are used for the first step of the methodology which 2 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 3.1 Model Developments The operation used (minimum, maximum, mean average etc.) to discern a single time value tx→y from the time ranges which represent the flexibility in how the entire operation can be managed. A path is defined as a possible scenario of safety constraints violations that can lead to accidents. It is one of the possible “roads” leading from the lowest level (loss scenarios) to the highest (losses) using the connections between nodes. For instance, the example of Figure 2 has a total of 17 paths and one possible path is S1→ UCA1→ H-2→ A (shown with bold arrows). Each path is comprised of 4 nodes, 3 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 one in each level of the tree-like structure. Since a path is comprised of 4 nodes it also has 3 corresponding time values (t1→2, t2→3, t3→4). Paths have two main characteristics: i) Path completeness y which expresses which Level of the acyclic diagram is the highest active node of the path in time t. Path completeness takes values in the discrete set {1,2,3,4}, ii) Time remaining until accident 𝑡+(𝑦) which is calculated according to the path completeness and the singular time values assigned to each path and expresses the time which is left for a path to reach the node that represents the occurrence of an accident. An example of path completeness and time remaining until accident is given in Figure 3. Figure. 2. (Right) Example of an acyclic diagram and representations of active and inactive nodes, paths, and node connections. Figure. 2. (Right) Example of an acyclic diagram and representations of active and inactive nodes, paths, and node connections. Figure. 2. (Right) Example of an acyclic diagram and representations of active and inactive nodes, paths, and node connections. Figure. 3. (Left) Example path, completeness levels and the corresponding time values. 3.2 Safety Level Determination The goal of the Safety Level Determination process is the actual real-time safety monitoring of the system during its operation. The data needed are: The goal of the Safety Level Determination process is the actual real-time safety monitoring of the system during its operation. The data needed are: 1. The Operational Mode of the system. For instance, referring to a plane system during its take-off mode, out of the total set of the acyclic diagram paths that can be produced from the plane system, the paths which will be computed during the Operational System process of the methodology will be only those belonging to the take-off mode of plane operations. p 2. System specific data according to the STPA analysis. This data must be able to show if any node or safety constraint of the diagram has activated or not for any time t. Activated means the safety constraint was violated in the specific time value monitored. 2. System specific data according to the STPA analysis. This data must be able to show if any node or safety constraint of the diagram has activated or not for any time t. Activated means the safety constraint was violated in the specific time value monitored. y p Using the mathematical model and the active nodes, the most detrimental to safety system path is calculated. 4 https://doi.org/10.1051/matecconf/202031401001 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 3.3 The mathematical model The mathematical model defines the safety level of the system in a specific time t as 𝑆𝐿= 𝑝⃗#(𝑡) = max (℘///⃗), where ℘///⃗ is the ordered set of all paths of the system derived by STPA, 𝑝⃗#(𝑡) indicates the path or set of paths that for time t are considered the “worst” among all the paths of the diagram concerning their completeness level (i.e. how “close” to the loss node is the last active node of this path), and the time left until the accident (i.e. the time until the loss node is activated meaning an accident has taken place in the system) graphically represented in Figure 3. p g Mathematically paths are defined as follows: p g Mathematically paths are defined as follows: 𝑝 y p For path 𝑝⃗= (𝑦, 𝑡$⃗ &(𝑦)) ∈𝑌× ℝ&where: 𝑝 y p r path 𝑝⃗= (𝑦, 𝑡$⃗ &(𝑦)) ∈𝑌× ℝ&where: 𝑝 For path 𝑝⃗= (𝑦, 𝑡$⃗ &(𝑦)) ∈𝑌× ℝ&where: 𝑝 $ he path completeness for 𝑝 in time t, y ∈ {0,1,2,3,4} y the path completeness for 𝑝 in time t, y ∈ {0,1,2,3,4} &( ) 𝑝 𝑡$⃗ &(𝑦):{0,1,2,3,4}→[0,∞) is the time remaining until the accident of path 𝑝 is activated and is calculated according to the following function: 𝑡$⃗ &(𝑦) = ⎩ ⎪⎨ ⎪⎧𝑡'→) $⃗ + 𝑡)→ $⃗ + 𝑡→+ $⃗ 𝑦∈{0,1} 𝑡)→ $⃗ + 𝑡→+ $⃗ 𝑦= 2 𝑡→+ $⃗ 𝑦= 3 0 𝑦= 4 (1) (1) We consider the set of all the paths ℘///⃗ = {𝑝⃗', 𝑝⃗),…, 𝑝⃗,} where n is the number of paths of the complete system. We consider the set of all the paths ℘///⃗ = {𝑝⃗', 𝑝⃗),…, 𝑝⃗,} where n is the number of paths of the complete system. ⃗ B ⃗C In the set ℘///⃗ we define the following ordering relation, according to which maxB℘///⃗C is determined: ∀𝑝⃗' E𝑦$⃗!, 𝑡$⃗! & B𝑦$⃗!CF , 𝑝⃗) E𝑦$⃗", 𝑡$⃗" & B𝑦$⃗"CF ∈℘///⃗ 𝑝⃗' ≥𝑝⃗) ⟺𝑡$⃗! & < 𝑡$⃗" & ∨B𝑡$⃗! & = 𝑡$⃗" & ∧𝑦$⃗! ≥𝑦$⃗"C (2) (2) 4.1 Results The scenario unfolds as follows. The train is stopped at the station and the door rail is filled with dirt, resulting in the door being unable to close. Then the train starts for the next station but while the command to close the door is issued by the door controller the actuator is unable to execute it, leaving the door open while the train is moving. A passenger then sees the door open and the train moving and scared presses the emergency button. This causes the system to change contexts of operation fundamentally changing the safety priorities of the system. 4 Case study The fictitious system of the train door STPA analysis example [10] will be used to demonstrate our approach based on a scenario of operation to demonstrate the methodology’s real-time capabilities. The train door system monitors and controls the process of the typical train door. This process concerns the safe boarding, departing and travel of train passengers. The system is comprised of a door controller, the door actuator, various sensors and the physical door itself. Figure 4 depicts the safety control structure of the system. The STPA analysis applied to this system identified 91 safety requirements in total. After removing the repeated specifications which were produced during the last step of the STPA analysis (i.e., from the translation of the unsafe control actions scenarios) the analysis defined the following: 1 accident, 3 hazards, 14 unsafe control actions, 4 improperly executed control actions and 36 safety scenarios (sample in Table 1). This system is used purely for demonstrative purposes and the complete analysis is not of much importance other than the example. 5 5 https://doi.org/10.1051/matecconf/202031401001 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 Figure. 4. Train Door System Safety Control Structure Figure. 4. Train Door System Safety Control Structure Figure. 4. Train Door System Safety Control Structure Table 1. A subset of STPA Produced Safety Specifications of the Train Door Sy # Symbols Description Connecti ons 1 A Passenger loss of life or injury. - 2 H-1 The door is open when the train is moving fast. A 3 H-2 A person is unable to leave the train (by the door) in case of emergency. A 4 H-3 A passenger is caught by the door and the train is moving. A 5 UCA5 Train Door Controller provides open door while the train is in motion. H-1 6 CA3 Train Door Controller is unable to provide close door while the train is moving, and the door isn’t closed. H-1 7 S05 Small objects stuck in door movement rail. CA1, CA3 8 S24 Evacuation mode misfire. UCA5 Table 1. A subset of STPA Produced Safety Specifications of the Train Door System. Table 1. A subset of STPA Produced Safety Specifications of the Train Door System. 4.1.1 Model Development process 1. Creation of acyclic diagram. The safety constraints from the STPA analysis were translated into an acyclic diagram (Figure 5). The diagram is comprised of 143 distinct paths. In our example, only 2 of these paths are studied (Figure 6). 6 6 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 2. Time ranges. The time values for the connections of the safety constraints were arbitrarily assigned (sample in Table 2). 3 S l i f i B i hi ll h l d bi i i i 2. Time ranges. The time values for the connections of the safety constraints were arbitrarily assigned (sample in Table 2). 2. Time ranges. The time values for the connections of the safety constraints were arbitrarily assigned (sample in Table 2). 3. Selection of operation. Because in this system small changes can lead to big variations in the safety level the MINIMUM operation was used to discern the single time values (Table 2). 3. Selection of operation. Because in this system small changes can lead to big variations in the safety level the MINIMUM operation was used to discern the single time values (Table 2). Figure 5. The complete acyclic diagram of the train door system. ure 5. The complete acyclic diagram of the train door system. Figure 5. The complete acyclic diagram of the train door system. Figure 5. The complete acyclic diagram of the train door system. Table 2. Time ranges and single time values. Connection Time range Time value with minimum operation H-1→A [60,120] MIN[60,120] = 60 CA3→H-1 [2,6] MIN[2,6] = 2 UCA5→H-1 [10,30] MIN[10,30] = 10 S05→CA3 [90,300] MIN[90,300] = 90 S24→UCA5 [8,20] MIN[8,20] = 8 7 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 Figure 6. The acyclic diagram for the train door case study. Figure 6. The acyclic diagram for the train door case study. Figure 6. The acyclic diagram for the train door case study. 4.1.2 Safety Level Determination 1. Operational modes. The system is divided into two operational modes: the train stopped at a station, the train travelling between stations, but for the sake of simplicity in the example takes place only when the Travelling between stations Operational mode is active. 1. Operational modes. The system is divided into two operational modes: the train stopped at a station, the train travelling between stations, but for the sake of simplicity in the example takes place only when the Travelling between stations Operational mode is active. 2. System monitoring data. The system specific data needed for the nodes of Figure 6 are: the door’s state (open, closed), the train’s velocity, doorway clearance, evacuation state, if the door movement rail is filled with small objects and evacuation mode misfire. 2. System monitoring data. The system specific data needed for the nodes of Figure 6 are: the door’s state (open, closed), the train’s velocity, doorway clearance, evacuation state, if the door movement rail is filled with small objects and evacuation mode misfire. 3. Mathematical model. In Figure 6 the sample of the acyclic diagram that will be used for the demonstration of the suggested approach is shown. This diagram contains 2 distinct system paths (𝑝⃗': S05→CA3→H-1→A and 𝑝⃗): S24→UCA5→H-1→A). According to equations (3), (4) the values for the time remaining until accident for these two paths are the following: 𝑡$⃗! & (𝑦) = ⎩ ⎪ ⎨ ⎪⎧𝑡'→) $⃗! + 𝑡)→* $⃗! + 𝑡→+ $⃗! = 90 + 2 + 60 = 152 𝑦∈{0,1} 𝑡)→ $⃗! + 𝑡→+ $⃗! = 2 + 60 = 62 𝑦= 2 𝑡→+ $⃗! = 60 𝑦= 3 0 𝑦= 4 (3) 𝑡$⃗" & (𝑦) = ⎩ ⎪ ⎨ ⎪⎧𝑡'→) $⃗" + 𝑡)→* $⃗" + 𝑡→+ $⃗" = 8 + 10 + 60 = 78 𝑦∈{0,1} 𝑡)→ $⃗" + 𝑡→+ $⃗" = 10 + 60 = 70 𝑦= 2 𝑡→+ $⃗" = 60 𝑦= 3 0 𝑦= 4 (4) (3) (4) https://doi.org/10.1051/matecconf/202031401001 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 Figure 7. Example scenario of operation with acyclic diagrams for every time moment. Figure 7. Example scenario of operation with acyclic diagrams for every time moment. 4.1.2 Safety Level Determination As Figure 7 shows, in the first time moment S05 (Table 1) is active this activates 𝐩//⃗' with values 𝐩//⃗'= (1,152) and 𝐩//⃗)= (0,78), so 𝐩//⃗# t=1=𝐩//⃗) =(0,78), with the context of the system being that the train is stationary on the platform. In the second time moment the context changes to the train starting moving and gathering speed, with this change in context (that a computerized system can monitor with various sensors) the active nodes become S05 and CA3, S24 is active because a passenger has pressed the emergency button due to seeing the open door, these in turn activate 𝐩//⃗) with value 𝐩//⃗) =(1,78) and change 𝐩//⃗'=(2,62), so 𝐩//⃗# t=2=𝐩//⃗'=(2,62). In the final time moment the train reaches cruising speed making H-1 active and in a few moments will start decelerating because of the emergency button being pressed this results in the two paths having the same value 𝐩//⃗'= 𝐩//⃗) =(3,60), so 𝐩//⃗# t=3 = 𝐩//⃗'= 𝐩 ///⃗) =(3,60). MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031401001 It is the first time where a mathematical model is presented to address the problem of determining dynamically what the safety level is at a certain moment in time at a given context and calculate how much time is left for an accident to happen. This approach is also based on the results of an STPA analysis. This is another novel aspect of this paper since there are no previous concepts on real time safety level calculation based on the STAMP accident model. A prominent limitation however of this novel approach is the fact that the mathematical model it is based on does not cope with uncertainty. It is planned, however, to address this problem in future work by enhancing the mathematical model with the use of fuzzy sets theory. An extension of the acyclic diagrams is also planned to take place in future work such that their acyclic nature would be nullified when system defences are in place. Finally, the creation of an operational system is planned where the approach will be installed into a real system to calculate its safety level in real time. 5 Future Work and Concluding Remarks By using this methodology, it is possible to identify the worst path to violate safety of a system with many possible paths that can lead to accidents. This can be useful to help safety managers and staff understand how system information and changes affects, safety and enable operators to react in timely manner to neutralize the completion of the path and hence the occurrence of an accident. The aim of this new approach is to enhance the capabilities of safety management systems towards preventing accidents and any types of losses by calculating in real time the safety levels of safety critical processes. This is feasible by projecting enriched information in a simple to understand form (second or minutes, instead of safety indexes) in real time as well as the acyclic diagrams, which can help managers understand how certain system interactions effect safety in their systems. This can be seen in the case study by observing that initially p/⃗' seems more severe, if CA3 occurs the system gets much closer to the accident state from p/⃗', and after the system reaches the point of Hazards the priority should be to stop the train and not meddle with the two different control systems S5→CA3, S24→UCA5. 9 9 MATEC Web of Conferences 314, 01001 (2020) ICSC-ISATECH 2019 References 1. Øien K., (2010). Remote Operation in Environmentally Sensitive Areas; Development of Early Warning Indicators. 2nd iNTeg-Risk Conference, 14 – 18 June, Stuttgart, Germany. 1. Øien K., (2010). Remote Operation in Environmentally Sensitive Areas; Development of Early Warning Indicators. 2nd iNTeg-Risk Conference, 14 – 18 June, Stuttgart, Germany. 2. Thieme C. A., Utne I. B., (2017). Safety performance monitoring of autonomous marine systems. Reliability Engineering & System Safety. 159, March 2017, Pages 264-275. 2. Thieme C. A., Utne I. B., (2017). Safety performance monitoring of autonomous marine systems. Reliability Engineering & System Safety. 159, March 2017, Pages 264-275. 3. Dekker S., (2006) Resilience Engineering: Chronicling the Emergence of Confused Consensus. Chapter 7 in Hollnagel, E., Woods, D. D. & Leveson, N. (Eds.), Resilience Engineering: Concepts and Precepts. Aldershot, UK: Ashgate, 2006. 3. Dekker S., (2006) Resilience Engineering: Chronicling the Emergence of Confused Consensus. Chapter 7 in Hollnagel, E., Woods, D. D. & Leveson, N. (Eds.), Resilience Engineering: Concepts and Precepts. Aldershot, UK: Ashgate, 2006. 4. Knegtering B., Pasman H., (2013). The safety barometer: How safe is my plant today? Is instantaneously measuring safety level utopia or realizable?.Journal of Loss Prevention in the Process Industries. 26(4), 821-829. 5. Leveson N., (2015). A systems approach to risk management through leading safety indicators, Reliability Engineering and System Safety.136, 17–34. 6. Karanikas, N. (2018). Documentation of Assumptions and System Vulnerability Monitoring: the Case of System Theoretic Process Analysis (STPA), Proceedings of the 5th STAMP European Workshop, 14-15 September 2017, Reykjavik University, Iceland, International Journal of Safety Science, 2(1), pp. 84-93 7. Chatzimichailidou M. M., Karanikas N., Dokas I. M., (2016). Measuring Safety Through the Distance Between Systems States with RiskSOAP Indicator, Journal of Safety Studies, 2 (2), 0-5. 8. Leveson N., Thomas J. P., (2018). STPA Handbook [on line], [Viewed 29 November 2018] Available from: , , ( ) [ ], [ 2018] Available from: http://psas.scripts.mit.edu/home/get_file.php?name=STPA_handbook.pdf ] http://psas.scripts.mit.edu/home/get_file.php?name=STPA_handbook.pdf 9. Dokas I. M., J. Feehan and Imran S., (2013). EWaSAP: An Early Warning Identification Approach Based on a Systemic Hazard Analysis, Safety Science. 58, pp 11-26 10. Leveson, N., (2011). Engineering a safer world: Systems thinking applied to safety. MIT press. 10
https://openalex.org/W4306870978	https://insu.hal.science/insu-03863335/document	English	null	The Green Edge cruise: investigating the marginal ice zone processes during late spring and early summer to understand the fate of the Arctic phytoplankton bloom	Earth system science data	2,022	cc-by	21,087	The Green Edge cruise: investigating the marginal ice zone processes during late spring and early summer to understand the fate of the Arctic phytoplankton bloom Flavienne Bruyant, Rémi Amiraux, Marie-Pier Amyot, Philippe Archambault, Lise Artigue, Lucas Barbedo de Freitas, Guislain Bécu, Simon Bélanger, Pascaline Bourgain, Annick Bricaud, et al. To cite this version: Flavienne Bruyant, Rémi Amiraux, Marie-Pier Amyot, Philippe Archambault, Lise Artigue, et al.. The Green Edge cruise: investigating the marginal ice zone processes during late spring and early summer to understand the fate of the Arctic phytoplankton bloom. Earth System Science Data, 2022, 14, pp.4607-4642. 10.5194/essd-14-4607-2022. insu-03863335 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: insu-03863335 https://insu.hal.science/insu-03863335v1 Submitted on 21 Nov 2022 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 Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. The Green Edge cruise: investigating the marginal ice zone processes during late spring and early summer to understand the fate of the Arctic phytoplankton bloom The Green Edge cruise: investigating the marginal ice zone processes during late spring and early summer to understand the fate of the Arctic phytoplankton bloom Flavienne Bruyant1,2, Rémi Amiraux1,2,3, Marie-Pier Amyot1, Philippe Archambault1,2, Lise Artigue4, Lucas Barbedo de Freitas2,5, Guislain Bécu1,2, Simon Bélanger2,5, Pascaline Bourgain6, Annick Bricaud7, Etienne Brouard1, Camille Brunet8, Tonya Burgers9, Danielle Caleb10, Flavienne Bruyant1,2, Rémi Amiraux1,2,3, Marie-Pier Amyot1, Philippe Archambault1,2, Lise Artigue4, Lucas Barbedo de Freitas2,5, Guislain Bécu1,2, Simon Bélanger2,5, Pascaline Bourgain6, Annick Bricaud7, Etienne Brouard1, Camille Brunet8, Tonya Burgers9, Danielle Caleb10, Fanny Cusset1, Laeticia Dadaglio13, Marty Davelaar10, Gabrièle Deslongchamps1,2, Céline Dimier7, Julie Dinasquet13, Dany Dumont2,4, Brent Else15, Igor Eulaers16, Joannie Ferland1,2, Gabrielle Filteau1,2, Marie-Hélène Forget1, Jérome Fort17, Louis Fortier1,2,, Martí Galí1,18, Morgane Gallinari3, Svend-Erik Garbus16, Nicole Garcia8, Catherine Gérikas Ribeiro19,20, Fanny Cusset1, Laeticia Dadaglio13, Marty Davelaar10, Gabrièle Deslongchamps1,2, Céline Dimier7, Julie Dinasquet13, Dany Dumont2,4, Brent Else15, Igor Eulaers16, Joannie Ferland1,2, Gabrielle Filteau1,2, Marie-Hélène Forget1, Jérome Fort17, Louis Fortier1,2,, Martí Galí1,18, Morgane Gallinari3, Svend-Erik Garbus16, Nicole Garcia8, Catherine Gérikas Ribeiro19,20, C G , G , C G y , C y G , G , Pascal Guillot2,21, Sandrine Hillion3, Rachel Hussherr2, Fabien Joux13, Hannah Joy-Warren24, Gabriel Joyal1,2, David Kieber25, Augustin Lafond8, José Lagunas1,2, Patrick Lajeunesse1, Catherine Lalande1,2, Jade Larivière1,2, Florence Le Gall19, Karine Leblanc8, Mathieu Leblanc1,2, Justine Legras3, Keith Lévesque21, Kate-M. Lewis24, Edouard Leymarie7, Aude Leynaert3, Thomas Linkowski21, Martine Lizotte1,2, Adriana Lopes dos Santos26, Claudie Marec1,27, Dominique Marie19, Guillaume Massé1, Philippe Massicotte1,2, Atsushi Matsuoka1,28, Lisa A. F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 12Akvaplan-niva, Fram Centre for Climate and the Environment, Tromsø, Norway 12Akvaplan-niva, Fram Centre for Climate and the Environment, Tromsø, Norway 13Laboratoire d’Océanographie Microbienne (LOMIC), UMR7621, CNRS/Sorbonne Université, Observatoire Océanologique de Banyuls-sur-mer, France 14Institut des sciences de la mer de Rimouski, Université du Québec à Rimouski, QC, Canada 15Department of Geography, University of Calgary, Calgary T2N 1N4, AB, Canada 16Department of Biosciences – Arctic Environment, Aarhus University, Denmark 17Littoral Environnement et Sociétés, UMR7266, CNRS/Université de La Rochelle, France 18Barcelona Supercomputing Center (BSC), Barcelona, Spain 19ECOMAP, UMR7144, CNRS/Sorbonne Université, Station Biologique de Roscoff, France 20 vatoire Océanologique de Banyuls-sur-mer, Franc p p g p 19ECOMAP, UMR7144, CNRS/Sorbonne Université, Station Biologique de Roscoff, France p p g p 19ECOMAP, UMR7144, CNRS/Sorbonne Université, Station Biologique de Roscoff, France 20 , , , g q , 20GEMA Center for Genomics, Ecology & Environment, Faculty of Sciences, 20GEMA Center for Genomics, Ecology & Environment, Faculty of Sciences, Universidad Mayor, Santiago, Chile 21Amundsen Science, Université Laval, Québec QC, Canada 21Amundsen Science, Université Laval, Québec QC, Canada 22Roscoff Culture Collection, FR2424 CNRS/Université Sorbonne, Station Biologique, Roscoff, France 23Operational Directorate Natural Environment, Royal Belgian Institute of Natural Sciences (RBINS), 23Operational Directorate Natural Environment, Royal Belgian Institute of Natural Sciences (RBINS), g 24Department of Earth System Science, Stanford University, Stanford, CA 94305, USA 25 24Department of Earth System Science, Stanford University, Stanford, CA 94305, USA 25Department of Chemistry, College of environmental sciences and forestry, 25Department of Chemistry, College of environmental sciences and forestry, State University of New York, Syracuse, NY 13210, USA State University of New York, Syracuse, NY 13210, USA of the Environment, Nanyang Technological University, 50 Nanyang Avenue, 26Asian School of the Environment, Nanyang Technological University, 50 Nanyang Avenue, The Green Edge cruise: investigating the marginal ice zone processes during late spring and early summer to understand the fate of the Arctic phytoplankton bloom Miller10, Sharif Mirshak29, Nathalie Morata3,12, Brivaela Moriceau3, Philippe-Israël Morin1,2, Simon Morisset21, Anders Mosbech16, Alfonso Mucci30, Gabrielle Nadaï1,2, Christian Nozais11, Ingrid Obernosterer13, Thimoté Paire1, Christos Panagiotopoulos8, Marie Parenteau1,2, Noémie Pelletier11, Marc Picheral7, Bernard Quéguiner8, Patrick Raimbault8, Joséphine Ras7, Eric Rehm1,2, Llúcia Ribot Lacosta1,31, Jean-François Rontani8, Blanche Saint-Béat1,32, Julie Sansoulet1, Noé Sardet29, Catherine Schmechtig33, Antoine Sciandra7, Richard Sempéré8, Caroline Sévigny2,14, Jordan Toullec3, Margot Tragin19, Jean-Éric Tremblay1,2, Annie-Pier Trottier1, Daniel Vaulot19, Anda Vladoiu34,35, Lei Xue25, Gustavo Yunda-Guarin1,2, and Marcel Babin1 q , , pp , , , Sharif Mirshak29, Nathalie Morata3,12, Brivaela Moriceau3, Philippe-Israël Morin1,2, Simon Morisset21, Anders Mosbech16, Alfonso Mucci30, Gabrielle Nadaï1,2, Christian Nozais11, Ingrid Obernosterer13, Thimoté Paire1, Christos Panagiotopoulos8, Marie Parenteau1,2, Noémie Pelletier11, Marc Picheral7, Bernard Quéguiner8, Patrick Raimbault8, Joséphine Ras7, Eric Rehm1,2, Llúcia Ribot Lacosta1,31, Jean-François Rontani8, Blanche Saint-Béat1,32, Julie Sansoulet1, Noé Sardet29, Catherine Schmechtig33, Antoine Sciandra7, Richard Sempéré8, Caroline Sévigny2,14, Jordan Toullec3, Margot Tragin19, Jean-Éric Tremblay1,2, Annie-Pier Trottier1, Daniel Vaulot19, Anda Vladoiu34,35, Lei Xue25, Gustavo Yunda-Guarin1,2, and Marcel Babin1 4LEGOS, University of Toulouse, CNRS, CNES, IRD, UPS, 31400 Toulouse, France à Rimouski, 300 allée des Ursulines, Rimouski G5L 3A1, QC, Canada 6 6Société AVUNGA, Lars en Vercors, France oratoire d’Océanographie de Villefranche, UMR7093, CNRS/Sorbonne Université, 10Institute of Ocean Sciences, Fisheries and Oceans Canada, Sidney BC, Canada 11Québec-Océan, Département de biologie, chimie et géographie, Université du Québec à Rimouski QC, Canada Published by Copernicus Publications. 4608 1 Introduction The second paper (the present paper by Bruyant et al., 2022) provides an overview of the dataset gathered during an oceanographic cruise conducted in Central Bafﬁn Bay in 2016. For more clarity, we created a second dataset on SEANOE associated with the present paper: https://doi.org/10.17882/86417 (Bruyant et al., 2022). This 2022 DOI contains the ﬁnal version of all the cruise data that could be formatted to be published by SEANOE (see Sect. 6 Data availability). Note that the ﬁrst dataset (Massi- cotte et al., 2020) contained some of the cruise data for the For logistical reasons, the cruise was divided into two legs. Leg 1A started on 3 June in Québec City, ended on 23 June in Qikiqtarjuaq (NU), and included one week of transit to the study zone. Leg 1B started on 23 June in Qikiq- tarjuaq and ended in Iqaluit (NU) on 14 July. During the ﬁve-week period spent in Bafﬁn Bay, the ship crossed the MIZ, from open waters (in the east) to sea-ice-covered areas (in the west) and back again following latitudinal transects. A total of seven transects were covered between 68.0 and 70.5◦N (Fig. 1a). Three transects were covered during Leg 1A (68.5 to 69.0◦N) and four during Leg 1B (68.0 and 69.5 to 70.5◦N). For each transect, stations were separated by six nautical miles (approximately 11 km) to obtain a relatively high spatial resolution. A total of 15 to 25 stations were sam- pled within each transect, for a total of 144 stations visited during the campaign. The activities conducted at each type of station are de- tailed in Table 1. Brieﬂy, at so-called CTD (current tem- perature depth) stations, rosette casts did not include sea- water collection. The rosette, a Sea-Bird model 32 carousel equipped with 22 12 L Niskin bottles, was geared with mul- tiple sensors (see Table 2 for details). At NUT stations, sea- water samples were additionally collected at several depths between 2 and 2000 m for nutrient analyses. At BASIC sta- tions, the apparent optical properties of seawater were mea- sured using underwater proﬁling optical instruments. The variables measured at BASIC stations included the concen- tration of chlorophyll a, phytoplankton pigment, particulate carbon and nitrogen, and particulate absorption spectra. Fi- nally, at FULL stations, a suite of measurements was made on seawater samples, and several underwater instruments were deployed as well, including a suite of optical sensors. 1 Introduction The Arctic Ocean is currently experiencing unprecedented environmental changes. The increase of the summer ice re- treat lengthens the phytoplankton growing season but also increases the area of the marginal ice zone (MIZ). If trends are maintained, all Arctic sea ice may become seasonal as early as 20 years from now (Meredith et al., 2019), increas- ing the MIZ coverage even more. Ice edge blooms repre- sent much of the annual phytoplankton primary production in the Arctic Ocean (Perrette et al., 2011; Ardyna et al., 2013), and their current phenology is relatively well known (Wass- mann and Reigstad, 2011; Leu et al., 2015). However, we currently do not know how precisely primary production will respond to climate changes. The overarching goal of Green Edge was to understand the processes that control an Arc- tic phytoplankton spring bloom (PSB) as it expands north- ward and to determine its fate through the investigation of related carbon ﬂuxes (e.g., Trudnowska et al., 2021). This study was also motivated by the discovery that PSBs can and do occur underneath the ice (Arrigo et al., 2014) despite the limited amounts of under-ice available light (Mundy et al., 2009; Arrigo et al., 2014; Lowry et al., 2014; Assmy et al., 2017; Randelhoff et al., 2019). Field studies for the Green Edge project were carried out in 2015 and 2016 at an ice camp located on landfast sea ice close to Qikiqtarjuaq (NU, Canada). Additionally, during late spring and early summer 2016, a cruise aboard CCGS Amundsen was conducted in Bafﬁn Bay. As explained in Randelhoff et al. (2019), Baf- ﬁn Bay is both relatively easy to access and represents an ideal framework for this study, because environmental con- ditions are representative of what is observed at the pan- Arctic scale. Particularly, the warm Greenland current, ﬂow- ing north on the Greenland side, and the colder waters on the Canadian side, ﬂowing south (Bafﬁn Island current), in- duce an evenly retreating ice edge, allowing a straightfor- ward sampling strategy. From the Green Edge project, two separate data papers were produced. One paper by Massi- cotte et al. (2020) describes the dataset generated during two ice-camp campaigns on landfast ice (2015 and 2016). The latter paper is related to a dataset published in 2019 on SEANOE: https://www.seanoe.org/data/00487/59892/ (last access: 28 September 2022). F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4609 Singapore 639798, Singapore g p g p ersitaire Européen de la Mer, UMS3113, CNRS/Univ. Brest, Plouzane, France 27Institut Universitaire Européen de la Mer, UMS3113, CNRS/Univ. Brest, Plouzane, France f Marine Science and Ocean Engineering (SMSOE), Institute for the Study of Earth, Oceans, and Space (EOS) University of New Hampshire, Durham, USA 29 29Société Paraﬁlm, Montréal QC, Canada 30GEOTOP and Department of Earth and Planetary Sciences, McGill University, Montréal QC, Canada 31Balearic Islands Coastal Observing and Forecasting System, SOCIB, 07122, Ediﬁcio Naorte, Bloque A, Parc Bit, Palma de Mallorca, Spain 32Dyneco Pelagos, IFREMER, BP70, 29280 Plouzané, France 33OSU Ecce-Terra, UMS3455, CNRS/Sorbonne Université, PARIS Cedex 5, France 34LOCEAN-IPSL, UMR7159, CNRS/IRD/MNHN/Sorbonne Université, 75005 Paris, France 35Applied Physics Laboratory, University of Washington, Seattle, WA 98105, USA deceased Correspondence: Flavienne Bruyant (ﬂavienne.bruyant@takuvik.ulaval.ca) Received: 28 January 2022 – Discussion started: 21 February 2022 Revised: 19 August 2022 – Accepted: 20 August 2022 – Published: 20 October 2022 Abstract. The Green Edge project was designed to investigate the onset, life, and fate of a phytoplankton spring bloom (PSB) in the Arctic Ocean. The lengthening of the ice-free period and the warming of seawater, amongst other factors, have induced major changes in Arctic Ocean biology over the last decades. Because the PSB is at the base of the Arctic Ocean food chain, it is crucial to understand how changes in the Arctic environment will affect it. Green Edge was a large multidisciplinary, collaborative project bringing researchers and technicians from 28 different institutions in seven countries together, aiming at understanding these changes and their impacts on the future. The ﬁeldwork for the Green Edge project took place over two years (2015 and 2016) and was carried out from both an ice camp and a research vessel in Bafﬁn Bay, in the Canadian Arctic. This paper describes the sampling strategy and the dataset obtained from the research cruise, which took place aboard the Canadian Coast Guard ship (CCGS) Amundsen in late spring and early summer 2016. The sampling strategy was designed around the repetitive, perpendicular crossing of the marginal ice zone (MIZ), using not only ship-based station discrete sampling but also high-resolution measurements from autonomous platforms (Gliders, BGC- Argo ﬂoats ...) and under-way monitoring systems. The dataset is available at https://doi.org/10.17882/86417 (Bruyant et al., 2022). Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer https://doi.org/10.5194/essd-14-4607-2022 yant et al.: Investigating the marginal ice zone processes during late spring and early summer 4610 Table 1. List of operations carried out during the Green Edge cruise at each different station type. [(+) Opportunistic sampling only]. CTD conductivity–temperature–depth; AOP apparent optical properties; C-OPS Compact-Optical Proﬁling System; IOP inherent optical properties; BB9 backscattering meter; CDOM coloured dissolved organic matter; SCAMP Self-Contained Autonomous MicroProﬁler; LOKI Lightframe On-sight Key species Investigation. Table 1. List of operations carried out during the Green Edge cruise at each different station type. [(+) Opportunistic sampling only]. CTD conductivity–temperature–depth; AOP apparent optical properties; C-OPS Compact-Optical Proﬁling System; IOP inherent optical properties; BB9 backscattering meter; CDOM coloured dissolved organic matter; SCAMP Self-Contained Autonomous MicroProﬁler; LOKI Lightframe On-sight Key species Investigation. Operation type FULL BASIC NUT CTD CTD cast (see Table 2 for sensors list) + + + + Water sampling (niskin/rosette) + + + AOP proﬁle (C-OPS) + + IOP proﬁle (CTD, BB9, a-Sphere, CDOM, and EcoTriplet ﬂuorometers) + SCAMP proﬁle + LOKI and vertically integrated plankton nets + Mesozoic plankton trawl net + Hydro-bios MultiNet Maxi + Beam trawl + (+) Agassiz trawl + Box core + (+) Ice sampling (when ice-covered) + Sediment traps Deployed only once for long-term sampling TOTAL 26 18 49 51 Table 2. List of the sensors attached to the rosette carousel. CDOM coloured dissolved organic matter; PAR photosynthetically available radiation; ADCP acoustic Doppler current proﬁler; UVP Underwater Vision Proﬁler. NA – not available. Table 2. List of the sensors attached to the rosette carousel. CDOM coloured dissolved organic matter; PAR photosynthetically available radiation; ADCP acoustic Doppler current proﬁler; UVP Underwater Vision Proﬁler. NA – not available. Table 2. List of the sensors attached to the rosette carousel. CDOM coloured dissolved organic matter; PAR photosynthetically available radiation; ADCP acoustic Doppler current proﬁler; UVP Underwater Vision Proﬁler. NA – not available. Table 2. List of the sensors attached to the rosette carousel. CDOM coloured dissolved organic matter radiation; ADCP acoustic Doppler current proﬁler; UVP Underwater Vision Proﬁler. NA – not available. able 2. List of the sensors attached to the rosette carousel. CDOM coloured dissolved organic matter; PAR photosynthetically ava adiation; ADCP acoustic Doppler current proﬁler; UVP Underwater Vision Proﬁler. NA – not available. 1 Introduction Vertical plankton nets geared with a plankton imager, hori- zontal net trawls, benthic trawls, and Ursnel box corers were also deployed at each FULL station. Note that the number of variables measured on seawater samples was signiﬁcantly larger at FULL stations, where three rosette casts were nec- essary to cover the demand in water volume, compared with one rosette at BASIC stations. At least one FULL station was sampled in each of the three major domains covered by each transect, namely open waters, the MIZ, and the ice- covered area. During Leg 1B, a larger emphasis was placed on collecting ice cores for analyses and measurements of light propagation through the ice and snow. FULL stations sampled in the ice-covered domain did not include trawling operations. Our sampling strategy allowed successive crossings of different PSB stages: the early-bloom stage at the west- ern end of transects covered in sea ice, late- to post-bloom stages at the eastern end of transects in open waters, and full-bloom stage in the middle of transects around the ice edge (see Randelhoff et al., 2019). Between the stations, https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4611 e 1. Cruise maps showing (a) the seven transects and all stations with shape distinction between station type (FULL, BASIC, NUT, TD, see Table 1 for description). Coloured lines indicate the position of the ice edge (sea-ice concentration at 80 %) on each transect date of sampling; ice cover persisted on the western side of Bafﬁn Bay, while the eastern side cleared earlier. The starting date of ransect sampling is as follows: 9 June (100), 14 June (200), 17 June (300), 24 June (400), 29 June (500), 3 July (600), and 7 July (b) Tracks of the four BGC-Argo ﬂoats deployed during the Green Edge cruise over their ﬁrst 101 d of life, and the journey of the two UM gliders (blue and orange). The dashed line represents the ship track along the seven transects. Figure 1. Cruise maps showing (a) the seven transects and all stations with shape distinction between station type (FULL, BASIC, NUT, and CTD, see Table 1 for description). Coloured lines indicate the position of the ice edge (sea-ice concentration at 80 %) on each transect at the date of sampling; ice cover persisted on the western side of Bafﬁn Bay, while the eastern side cleared earlier. The starting date of each transect sampling is as follows: 9 June (100), 14 June (200), 17 June (300), 24 June (400), 29 June (500), 3 July (600), and 7 July (700). (b) Tracks of the four BGC-Argo ﬂoats deployed during the Green Edge cruise over their ﬁrst 101 d of life, and the journey of the two SLOCUM gliders (blue and orange). The dashed line represents the ship track along the seven transects. http://www.obs-vlfr.fr/proof/ftpfree/greenedge/db/DATA/ FLOATS/ (last access: 3 October 2022). show a good agreement for the data. Gliders travel follow- ing a programmed sawtooth pattern, joining pre-deﬁned way- points. Data and instructions were transmitted both ways via iridium when the glider surfaced. Figure 3 shows an example of the level of detail provided by the gliders when travelling through complex water structures. Representing chlorophyll ﬂuorescence measured over a three-week journey covering almost 500 km, the data show that the glider(s) did travel through both surface blooms and a subsurface chlorophyll maximum between 20–50 m. The data presented in Fig. 3 represents 693 000 chlorophyll ﬂuorescence measurements. Earth Syst. Sci. Data, 14, 4607–4642, 2022 yant et al.: Investigating the marginal ice zone processes during late spring and early summer Parameter Sensor Unit Serial # Calibration # Casts Temperature Sea-Bird Electronics SBE 3plus ITS-90 ◦C 03P4318 12/2015 203 Conductivity Sea-Bird Electronics SBE 4 mS cm−1 42696 12/2015 203 Pressure Paroscientiﬁc Digiquartz® db 679 01/2016 203 Oxygen concentration Sea-Bird Electronics SBE 43 mL L−1 430427 12/2015 203 CDOM ﬂuorescence WetLabs ECO CDOM mg m−3 2344 01/2016 203 Chlorophyll ﬂuorescence Seapoint SCF µg L−1 3120 01/2016 203 Light attenuation WetLabs C-Star % CST671DR 01/2016 87 Distance to bottom Benthos PSA-916 m 1065 02/2016 203 PAR/Irradiance Biospherical Instr. QCP-2300 µEm−2 s−1 4664 01/2014 203 Nitrate concentration Satlantic MBARI-ISUS µM 132 05/2016 104 Current speed and direction RDI-WHM300 L-ADCP NA 03/2016 203 Particle concentration Hydroptic UVP5 NA NA 203 sion of BGC-Argo ﬂoats that was deployed, the so-called PRO-ICE, is commercialized by NKE Electronics (France). These ﬂoats carry a typical biogeochemical payload (Sea- Bird 41 ARGO CTD, Aanderaa optode 4330 Oxygen sen- sor, Sea-Bird™OCR-504 PAR (photosynthetic available ra- diation) and downwelling radiance sensor (380, 412 and 490 nm), Sea-Bird ECO-FLBBCD ﬂuorescence chlorophyll- a, coloured dissolved organic matter (CDOM), and backscat- tering sensor and Sea-Bird SUNA nitrate sensor). They also include a 2 way-directional Iridium communication Rudics for data transmission and a sea-ice detection system to pro- tect the ﬂoat from hitting sea ice on ascent (Le Traon et al., 2020; André et al., 2020). The 2016 data are available on the following website: the ship track water monitoring system (TSG SBE45 from Sea-Bird, WETStar ﬂuorometer from WetLabs, LiCOR non- dispersive infra-red spectrometer (model Li-7000), Campbell Scientiﬁc CR1000 data logger) recorded temperature, salin- ity, chlorophyll a ﬂuorescence, and pCO2 at 7 m depth con- tinuously when navigating outside the ice pack. A moving vessel proﬁler (MVP300-1700, AML Oceanographic, Victo- ria BC, Canada), equipped with a micro CTD (AML), a Wet- Labs C-Star transmissometer, and a WetLabs Eco-FLRTD ﬂuorometer, was deployed in open waters. To signiﬁcantly extend the monitoring of the PSB be- yond the duration and space covered by the cruise, we de- ployed four BGC-Argo (Bio-Geo-Chemical-Argo) ﬂoats on 9 July. They collected data until late fall (Fig. 1b), per- forming a 0–1000 m proﬁle each day. The ice-speciﬁc ver- https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer These constitute robust results towards the validation of the Two SLOCUM G2 gliders (Teledyne Marine Inc.) were deployed during the cruise from the ship’s zodiac, either to revisit transects or in areas that were not visited by the CCGS Amundsen (Fig. 1b). They both carried a similar scientiﬁc payload and communication system as the BGC-Argo ﬂoats, rendering the possibility of inter-calibration (Fig. 2). Glid- ers were primarily used in the MIZ, where a 90 m icebreaker would disturb the fragile hydrological structure of shallow under-ice water masses. Gliders were deliberately directed through the same area as the BGC-Argo ﬂoats to compare CTD and optical data from both platforms. Results in Fig. 2 Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 4612 Figure 2. Comparison between salinity (psu, a), temperature (◦C, a), chlorophyll ﬂuorescence (relative units, b), and backscattering at 700 nm (bbp(700), m−1, c); data from the qala2 glider and the takapm005b BioArgo ﬂoat at their closest common position (Fig. 1b). Figure 2. Comparison between salinity (psu, a), temperature (◦C, a), chlorophyll ﬂuorescence (relative units, b), and backscattering at 700 nm (bbp(700), m−1, c); data from the qala2 glider and the takapm005b BioArgo ﬂoat at their closest common position (Fig. 1b). ing from 001, throughout the entire cruise. As a result, any given operation received a unique code, which could be used thereafter to merge all the data acquired during that opera- tion. use of multiple measurement platforms to investigate com- plex systems. A schematic synopsis of all operations carried out during the campaign can be found in Fig. 4a. The use of multiple analysis platforms allowed the measurement of more than 150 parameters during the Green Edge cruise (Table 3); to- gether, they accurately describe the complexity of the MIZ trophic systems (Fig. 4b) and most of the stocks and pro- cesses involved in the development of the PSB. Different control procedures were adopted to ensure the quality of the data. First, the raw data were screened to iden- tify and, where possible, eliminate errors originating from the measurement devices, including sensor (systematic or ran- dom) errors inherent to measurement procedures and meth- ods. Instrumental pre- and post-calibration corrections were applied when necessary. Statistical summaries such as aver- age, standard deviation, and range were computed to detect and remove anomalous values in the data. Then, data were checked for duplicates and remaining outliers. 3 Time-for-space formatting and data quality control One of the challenging tasks when assembling data from a large group of researchers is to adopt a common frame for spatial and temporal tagging of samples. Geographic posi- tions require a lot of attention and conversion of latitude and longitude into one format (we used decimal degrees east) to ensure data that can be easily merged. The concomitant use of local time and Coordinated Universal Time (UTC) dur- ing the cruise also presented a challenge. For the Green Edge cruise, an operation logbook was created to keep track of all operations conducted on the ship in sequential order during each day (local time). Each operation was associated with a unique operation ID to which all other data could be refer- enced. The use of ordinal date (number of days since Jan- uary ﬁrst) was used to avoid confusion between European and American date writing conventions. Each cast within a given operation type (CTD/RO for Rosette, AOP for appar- ent optical properties, etc.) was numbered sequentially, start- F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer Once raw measurements were cleaned, data were structured and gath- ered into single, comma-separated value (CSV) ﬁles. Each of these ﬁles was constructed to gather variables of the same na- ture (e.g., nutrients). In each ﬁle, a minimum number of vari- ables (columns) was always included to make dataset merg- ing easier and accurate (Table 4). F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4613 Figure 3. Chlorophyll ﬂuorescence measured by the Glider qala2 during its 23 d journey (Fig. 1b). Figure 3. Chlorophyll ﬂuorescence measured by the Glider qala2 during its 23 d journey (Fig. 1b). Figure 3. Chlorophyll ﬂuorescence measured by the Glider qala2 during its 23 d journey (Fig. 1b). Ice cover history was compiled and expressed as open water days (OWD) before sampling day (Fig. 7), calcu- lated from the difference (in day number) between the date of sampling and the date at which the sea-ice con- centration reached 10 (panel A), 50 (panel B), and 80 % (panel C) in the geographical location under study. Ice concentration data were obtained from the Advanced Mi- crowave Scanning Radiometer 2 (AMSR2) sea-ice concen- tration data on the 3.125 km grid (Spreen et al., 2008), down- loaded from http://www.iup.uni-bremen.de:8084/amsr2data/ asi_daygrid_swath/n3125/ (last access: 18 September 2020) (see Randelhoff et al., 2019 for details on the calculations). In Fig. 7, yellow and pale green colours on the Greenland side indicate a positive value between 20 and 40 OWD, which re- ﬂects how long the open-water conditions had prevailed at those stations at the time of sampling. To the east, closer to the Canadian side, the colder colours and negative values in- dicate the presence of sea ice. Open water days is a useful metric or index and can be computed using different sea-ice cover (SIC) thresholds, depending on the goal. For example, A meteorological tower was also installed on the foredeck of the ship to measure additional variables (instantaneous wind speed and direction, air temperature and relative hu- midity, atmospheric partial pressure, and vertical ﬂuxes of CO2. Averaged wind speeds and directions over the entire Bafﬁn Bay were retrieved from the Remote Sensing Sys- tems website (http://www.remss.com/measurements/ccmp/, last access: 25 April 2021) and were computed according to the cross-calibrated multi-platform (CCMP) wind vector analysis product (V2.0, Atlas et al., 2011) (see one example of a pattern calculated over the months of June and July in Fig. 5). A major change in wind patterns happened between June (light 1–2 m s−1 southward winds) and July (4–5 m s−1 northward winds), which impacted sea ice movements. Fig- ure 6 shows sea-ice cover over four periods covering the total sampling time of the cruise (https://nsidc.org/data/g02186, last access: 26 February 2021; NSIDC (U.S. National Ice Center and National Snow and Ice Data Center, 2010)). 4.1 Meteorological, navigational, and ice coverage data Along the ship track, an automated Voluntary Observing Ship (AVOS) system recorded all data related to naviga- tion, including the position of the vessel and basic atmo- spheric meteorological data (including barometric pressure). https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer The north–south general orientation of the ice edge is visible, along with, over time, the westward progression of the MIZ. https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer yant et al.: Investigating the marginal ice zone processes during late spring and early summer 4614 14 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summe ure 4. (a) Representation of all operations carried out during the Green Edge campaign. (b) Schematic representation of all stock bbles) and ﬂuxes (arrows) measured during the Green Edge cruise as part of the MIZ trophic system. Measured and calculated variable represented in red. Figure 4. (a) Representation of all operations carried out during the Green Edge campaign. (b) Schematic representation of all stocks (bubbles) and ﬂuxes (arrows) measured during the Green Edge cruise as part of the MIZ trophic system. Measured and calculated variables are represented in red. in Randelhoff et al. (2019), the SIC value used for hydrolog- ical interpretation was 10 % (as in Fig. 7, top panel). How- ever, a biologist might want to consider a SIC of 80 % (Fig. 7, bottom panel) when looking for the onset of phytoplankton growth, as only 20 % of open water surface already dramati- cally increases the amount of light available. During sea-ice sampling operations, snow depth, ice thick- ness, and freeboard were measured at each ice-coring site. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 https://doi.org/10.5194/essd-14-4607-2022 4615 Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 46 ble 3. List of all the variables sampled during the Green Edge cruise. Variable Method Sampling method Access to dataset Name of ﬁle PI 15N-Nitrate assimilation 15N spiking – in- cubation – mass- spectrometry Rosette Deck incubations https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Tremblay J.É. 15N-Nitrate primary pro- duction (13C) 15N spiking – in- cubation – mass- spectrometry Rosette Deck incubations https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Tremblay J.É. 15N-Urea assimilation 15N spiking – in- cubation – mass- spectrometry Rosette Deck incubations https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Above-water reﬂectance (Rrs(0+)) C-OPS Biospherical Instr. Proﬁle mode https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer Above-water reﬂectance (Rrs(0+)) Radiometer (Satlantic HyperSAS) Above-water sensor https://www.seanoe.org/ data/00752/86417/ Remote Sensing Re- ﬂectance (Rrs) measured by the HyperSas Belanger S. Absorption (non-algal particles) Spectrophotometer (ﬁlters) Rosette water sample https://www.seanoe.org/ data/00752/86417/ Particulate absorption Bricaud A./Sciandra A./Matsuoka A. Absorption (particulate matter) Spectrophotometer (ﬁlters) Rosette water sample https://www.seanoe.org/ data/00752/86417/ Particulate absorption Bricaud A./Sciandra A./Matsuoka A. Absorption coefﬁcient (total) HOBI Labs a-Sphere In-water proﬁler http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ IOP/ GreenEdge_hydroscat_ Asphere Belanger S. Acoustic determination of ﬁsh presence Echosounder Continuous on way https://www.polardata. ca/pdcsearch/?doi_id= 12841 Multiple ﬁles available. Data search required Fortier L. Air relative humidity Humidity sensor Meteorological tower https://www.seanoe.org/ data/00752/86417/ Meteorological Tower data Else B./Burgers T. Air temperature Temperature probe Meteorological tower https://www.seanoe.org/ data/00752/86417/ Meteorological Tower data Else B./Burgers T. Alkalinity total (TA) Potentiometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ Bottle salinity, DIC con- centration and Delta 18O Else B./ Miller L. Ammonium (assimila- tion) Isotopic dilution 15N Rosette deck incubations https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Ammonium (regenera- tion) Isotopic dilution 15N Rosette deck incubations https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Ammonium concentra- tion Fluorescence Rosette water sample https://www.seanoe.org/ data/00752/86417/ Water column nutrient concentrations Tremblay J.É. Backscattering coefﬁ- cient BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. Backscattering coefﬁ- cient (6 wavelengths) Hydroscat-6 In-water proﬁler http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ IOP/ GreenEdge_hydroscat_ Asphere Belanger S. Table 3. List of all the variables sampled during the Green Edge cruise. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4616 . Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Bacterial abundance Flow cytometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cy- tometry) Vaulot D. Bacteria infected by virus Electron microscopy Rosette water sample http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ EM/ Direct Joux F. Bacterial diversity 16S rRNA Illumina sequencing Rosette water sample https://www.seanoe.org/ data/00752/86417/ Bacterial production and respiration Joux F. Bacterial production Leucine-3H incor- poration Rosette water sample https://www.seanoe.org/ data/00752/86417/ bacterial production and respiration Joux F. Bacterial respiration (whole community) O2 consumption – Winkler – incubations Rosette water sample https://www.seanoe.org/ data/00752/86417/ bacterial production and respiration Joux F. Bacterial salinity- and light-induced biomark- ers GC/MS Rosette water sample http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ Bact_Viab/ Direct Rontani J.- F./Amiraux R./Burot C. F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer Bacterial viability Incubation Rosette water sample http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ Bact_Viab/ Direct Rontani J.- F./Amiraux R./Burot C. Bathymetry Kongsberg EM302 multi-beam echosounder Continuous horizontal https://geoapp.bibl. ulaval.ca/Home/Index Search Lajeunesse P./Joyal G./Brouard E. Benthic ammonium ﬂux Incubations – col- orimetry Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Benthic carbon content CN analyzer Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Benthic macrofauna abundance Microscopy Box corer https://www.seanoe.org/ data/00752/86417/ Abundance and weight of benthic organisms Archambault P. Benthic macrofauna biomass Wet weight Box corer https://www.seanoe.org/ data/00752/86417/ Abundance and weight of benthic organisms Archambault P. Benthic Macrofauna di- versity Microscopy Box corer https://www.seanoe.org/ data/00752/86417/ Abundance and weight of benthic organisms Archambault P. Benthic nitrate ﬂux Incubations – col- orimetry – autoana- lyzer Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Benthic nitrite ﬂux Incubations – col- orimetry – autoana- lyzer Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Benthic nitrogen content CN analyser Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Benthic phosphate ﬂux Incubations – col- orimetry – autoana- lyzer Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Benthic silicic acid ﬂux Incubations – col- orimetry – autoana- lyzer Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Chlorophyll a and Phaeopigments concen- tration Fluorimetry Sea ice core https://www.seanoe.org/ data/00752/86417/ Chlorophyll and Phaeopigment con- centration (ﬂuorescence technique) Bruyant F./Babin M. Table 3. Continued. https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 4617 e 3. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Chlorophyll a and phaeopigments concen- tration Fluorimetry Rosette water sample https://www.seanoe.org/ data/00752/86417/ Chlorophyll and phaeopigment concen- tration (ﬂuorescence technique) Bruyant F./ Babin M. Chlorophyll a and Phaeopigments concen- tration (benthic) Fluorometric analysis Box corer http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ SEDIMENT_BC/ GE_Amundsen_Sediment_ BoxCore.csv Archambault P. Chlorophyll a and Phaeopigments concen- tration (benthic) Fluorometric analysis Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Chlorophyll a ﬂuores- cence Seapoint ﬂuorometer In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./ Gombault C. Chlorophyll a ﬂuores- cence Fluorescence (Wet- labs) In-water proﬁler http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ IOP/ GreenEdge_AMundsen_ CTD_IOPs Belanger S. Chlorophyll a ﬂuores- cence Fluorometer Continuous horizontal https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./ Gombault C. Chlorophyll a ﬂuores- cence BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer Chlorophyll a ﬂuores- cence Wetlabs FLRTD Moving Vessel Proﬁler https://www.seanoe.org/ data/00752/86417/ Moving vessel proﬁler data Morisset S./ Gombault C. Chlorophyll a concen- tration Fluorometer Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Chromophoric dis- solved organic matter absorption Ultrapath Rosette water sample https://www.seanoe.org/ data/00752/86417/ Colored Dis- olved Organic Matter (CDOM) absorption data Matsuoka A. Chromophoric dis- solved organic matter ﬂuorescence Fluorescence (Wet- labs) In-water proﬁler http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ IOP/ GreenEdge_AMundsen_ CTD_IOPs Belanger S. Chromophoric dis- solved organic matte ﬂuorescence Fluorescence (Wet- labs, rosette) In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./ Gombault C. Chromophoric dis- solved organic matter ﬂuorescence BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. CO2 partial pressure (pCO2) Licor Continuous horizontal https://www.seanoe.org/ data/00752/86417/ Continuous pCO2 and salinity Else B./ Burgers T. Conductivity, tem- perature, and depth (CTD) Seabird (IOP optical frame) In-water proﬁler http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ IOP/ GreenEdge_AMundsen_ CTD_IOPs Belanger S. Conductivity, tem- perature, and depth (CTD) Seabird (rosette) In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./ Gombault C. Table 3. Continued. Table 3. Continued. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 4618 Table 3. Continued. Table 3. Continued. ble 3. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Conductivity, tem- perature, and depth (CTD) Seabird Continuous horizontal https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./ Gombault C. Conductivity, tem- perature, and depth (CTD) BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. Conductivity, tem- perature, and depth (CTD) AML micro CTD Moving Vessel Proﬁler https://www.seanoe.org/ data/00752/86417/ Moving vessel proﬁler data Morisset S./ Gombault C. Cryptophytes (abun- dance) Flow cytometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cy- tometry) Vaulot D. Current speed and direc- tion ADCP (150 kHz) Continuous horizontal https://www.seanoe.org/ data/00752/86417/ Acoustic Doppler Cur- rent Proﬁler (ADCP) Guillot P./ Gombault C. Current speed and direc- tion ADCP (LADCP) In-water proﬁler https://www.seanoe.org/ data/00752/86417/ Acoustic Doppler Cur- rent Proﬁler (ADCP) Guillot P./ Gombault C. Delta 18O – water Mass spectrometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ Bottle salinity, DIC con- centration and Delta 18O Else B./ Mucci A. Demersal ﬁsh diversity Beam trawl Fish trawl https://www.seanoe.org/ data/00752/86417/ Demersal ﬁsh abundance and sizes (Beam trawl sampling) Fortier L. Diacids (aerosol) HPAEC-PAD Atmosphere http://www.obs-vlfr.fr/ proof/ftpv/greenedge/db/ DATA/AMUNDSEN/ AEROSOLS/ Direct Panagiotopoulos C./Sempere R. Diatom (abun- dance) Microscopy Rosette water sample https://www.seanoe.org/ data/00752/86417/ Taxonomy data Diatoms abundance inverted mi- croscope Lafond A. Diatom frustules (abun- dance) Ludox/colloidal silica extraction Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N. Diatoms (bacilliaro- phyta) abundance Inverted microscopy Rosette water sample https://www.seanoe.org/ data/00752/86417/ Taxonomy data Diatoms abundance inverted mi- croscope Leblanc K./ Quéguiner B./ Cornet V. Diatoms (bacilliaro- phyta) taxonomy Inverted microscopy Rosette water sample https://www.seanoe.org/ data/00752/86417/ Taxonomy data Diatoms abundance inverted mi- croscope Leblanc K./ Quéguiner B./ Cornet V. Diffuse attenuation coef- ﬁcient (Kd) C-OPS In-water proﬁler https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Dimethyl sulﬁde (DMS) Gas chromatography- mass spectrometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ DMS and DMSP con- centration Masse G./ Galí M. Dimethyl sulﬁde (sea-air ﬂux) Gas chromatography- mass spectrometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ DMS and DMSP con- centration Masse G./ Galí M. Dimethylsulfopropionate (DMSP) Gas chromatography- mass spectrometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ DMS and DMSP con- centration Masse G./ Galí M./ Lizotte M./ Hussherr R. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4619 Table 3. Continued. Table 3. Continued. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Dissolved inorganic carbon (DIC) Coulometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ Bottle salinity, DIC con- centration and Delta 18O Else B./ Miller L. Dissolved organic car- bon (DOC) Wet oxidation Rosette deck incubations https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. Dissolved organic ni- trogen Wet oxidation Rosette deck incubations https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. Dissolved organic ni- trogen (release) Isotopic procedure Rosette deck incubations https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Dissolved oxygen con- centration Seabird 43 In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./ Gombault C. Dissolved oxygen con- centration BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. Downward longwave ra- diation Pyrgeometer Atmosphere https://www.seanoe.org/ data/00752/86417/ Downwelling radiation (pyrgeometer) Else B./ Burgers T. Downward shortwave ra- diation Pyranometer Atmosphere https://www.seanoe.org/ data/00752/86417/ Downwelling radiation (pyrgeometer) Else B./ Burgers T. Downwelling irradiance (Ed(z)) C-OPS In-water proﬁler https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Downwelling irradi- ance above the surface (Ed(0+)) Radiometer (Satlantic HyperSAS) Above-water sensor https://www.seanoe.org/ data/00752/86417/ Remote Sensing Re- ﬂectance (Rrs) measured by the HyperSas Belanger S. Downwelling irradi- ance above the surface (Ed(0+)) SBDART Surface mode http://www.obs-vlfr.fr/ proof/ftpfree/greenedge/ db/DATA/SBDART/ AM2016/ AM2016_SBDART_ AllCasts.zip Babin M./ Galí M. Downwelling irradi- ance above the surface (Ed(0+)) C-OPS In-water proﬁler https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Downwelling radiance (Ed(z)) BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. Epibenthic fauna abun- dance Microscopy Agassiz trawl https://www.seanoe.org/ data/00752/86417/ Benthic organisms’ iden- tiﬁcation and abundance Archambault P. Epibenthic fauna biomass Wet weight Agassiz trawl https://www.seanoe.org/ data/00752/86417/ Benthic organisms’ iden- tiﬁcation and abundance Archambault P. Epibenthic fauna diversity Microscopy Agassiz trawl https://www.seanoe.org/ data/00752/86417/ Benthic organisms’ iden- tiﬁcation and abundance Archambault P. Eukaryotic diversity Metabarcoding Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Fish abundance (midwa- ter) IKMT trawl Fish trawl https://www.seanoe.org/ data/00752/86417/ Pelagic ﬁsh abundance and sizes (IKMT sam- pling) Fortier L. Lipid biomarkers con- centrations GC/MS Collected organisms (bird) https://www.seanoe.org/ data/00752/86417/ Lipid biomarkers in ben- thic and sediment fauna Mosbech A./Fort J. https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4620 Table 3. Continued. Table 3. Continued. 3. Continued. Earth Syst. Sci. Data, 14, 4607–4642, 2022 Table 3. Continued. Variable Method Sampling Method Access To Dataset Name Of File PI Microturbulence SCAMP proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/SCAMP/ Vladoiu A./ Dumont D. Nanoeukaryotes (abun- dance) Flow cytometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Nitrate (NO− 3 ) assimila- tion Isotopic dilution 15N Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Nitrate concentration (NO− 3 ) Colorimetry/ AutoAnalyzer Rosette water sample https://www.seanoe.org/ data/00752/86417/ Water column nutrient concentrations Tremblay J. E. Nitrate concentration (NO− 3 ) Colorimetry/ AutoAnalyzer Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary production incu- bations experiments Garcia N. Nitrate concentration (NO− 3 ) BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php multiple Babin M. Nitrate concentration (NO3) MBARI-ISUS Sat- lantic In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./Gombault C. Nitriﬁcation 15N labeling Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Nitrite concentration (NO− 2 ) Colorimetry/ AutoAnalyzer Rosette water sample https://www.seanoe.org/ data/00752/86417/ Water column nutrient concentrations Tremblay J. E. Nitrite concentration (NO− 2 ) Colorimetry/ AutoAnalyzer Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary production incu- bations experiments Garcia N. Open water days AMSR Satellite https://www.seanoe.org/ data/00752/86417/ Days of Open Water (DOW) Massicotte P./Ferland J. Orthosilicic acid (uptake rate) 32Si absorption Rosette water sample https://www.seanoe.org/ data/00752/86417/ Silicate uptake rate Leynaert A./ Quéguiner B./ Gallinari M. Orthosilicic acid con- centration (Si(OH)4) Colorimetry/ AutoAnalyzer Rosette water sample https://www.seanoe.org/ data/00752/86417/ Water column nutrient concentrations Tremblay J.É. Orthosilicic acid con- centration (Si(OH)4) Colorimetry/ AutoAnalyzer Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary production incu- bations experiments Garcia N. Orthosilicic acid con- centration Si(OH)4 Technicon Rosette water sample https://www.seanoe.org/ data/00752/86417/ Silicate absorption kinet- ics experiments Leynaert A./Moriceau B./ Gallinari M. Orthosilicic acid Si(OH)4 – uptake kinetics 32Si absorption – in- cubation Rosette water sample https://www.seanoe.org/ data/00752/86417/ Silicate absorption kinet- ics experiments Leynaert A./ Quéguiner B./ Gallinari M. Particle size distribu- tion UVP-5 In-water proﬁler https://www.seanoe.org/ data/00752/86417/ Underwater Vision Pro- ﬁler (UVP) Particles Picheral M. Particulate mass Dry weight Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Particulate Nitrogen content CHN Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4621 Table 3. Continued. 3. Table 3. Continued. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Particulate organic car- bon (POC) CHN Rosette water sample https://www.seanoe.org/ data/00752/86417/ Dry weight, Particulate Carbon and Nitrogen (CHN) Bruyant F./Lariviere J./Babin M. Particulate organic car- bon (POC) High combustion Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. Particulate organic car- bon (POC) CHN Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Particulate organic ni- trogen (PON) CHN Rosette water sample https://www.seanoe.org/ data/00752/86417/ Dry weight, Particulate Carbon and Nitrogen (CHN) Bruyant F./Lariviere J./Babin M. Particulate organic ni- trogen (PON) High combustion Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. PDMPO uptake Spectrophotometry/ spectroﬂuorometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ Siliciﬁcation of Diatoms Leblanc K./Quéguiner B./ Cornet V. Phosphate concentration (PO3− 4 ) Colorimetry/ AutoAnalyzer Rosette water sample https://www.seanoe.org/ data/00752/86417/ Water column nutrient concentrations Tremblay J.É. Phosphate concentration (PO3− 4 ) Colorimetry/ AutoAnalyzer Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary production incu- bations experiments Garcia N. Photo eukaryotes (mor- phology) Scanning electron microscopy Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Photo eukaryotes sorted (Morphology) Scanning electron mi- croscopy Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Photosynthetically avail- able radiation (PAR) Biospherical Instru- ment QCP-2300 In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./Gombault C. Photosynthetically avail- able radiation (PAR) C-OPS In-water proﬁler https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Photosynthetically avail- able radiation (PAR) SBDART Surface mode http://www.obs-vlfr.fr/ proof/ftpfree/greenedge/ db/DATA/SBDART/ AM2016/ AM2016_SBDART_ AllCasts.zip Babin M./ Galí M. Photosynthetic Available Radiation (PAR) Radiometer Atmosphere https://www.seanoe.org/ data/00752/86417/ Downwelling radiation (pyrgeometer) Else B./ Burgers T. Photosynthetically avail- able radiation (PAR) BGC-Argo proﬁler In-water proﬁler http://www.obs-vlfr. fr/proof/php/ GREENEDGE/ greenedge_autonomous. php Multiple Babin M. Photosynthetic parame- ters 14C P vs. E curve Rosette water sample https://www.seanoe.org/ data/00752/86417/ Photosynthetic parame- ters Lewis K. Phytoplankton (taxon- omy) Inverted microscopy Rosette water sample http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/ TAXONOMIE/ Phytoplankton taxon- omy (microscopy) Babin M. Phytoplankton (micro-) taxonomy Microscopy Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Earth Syst. Sci. Data, 14, 4607–4642, 2022 4622 e 3. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Phytoplankton (taxon- omy) Imaging FlowCytobot Sea-ice core http://www.obs-vlfr. Table 3. Continued. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/IFCB/ Multiple Bruyant F./Grondin P.L./Babin M. Phytoplankton (taxon- omy) Imaging FlowCytobot Rosette water sample http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/IFCB/ Multiple Bruyant F./Grondin P.L./Babin M. Phytoplankton cultures Sorted by ﬂow cytom- etry, serial dilution, single cell pipetting Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Picoeukaryotes (abun- dance) Flow cytometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Pigments concentration HPLC Rosette water sample https://www.seanoe.org/ data/00752/86417/ phytoplankton pigments concentration (HPLC) Ras J./Claustre H./Dimier C. Pigments concentration HPLC Sea-ice core https://www.seanoe.org/ data/00752/86417/ phytoplankton pigments concentration (HPLC) Ras J./Claustre H./Dimier C. Plankton taxonomy UVP-5 In-water proﬁler https://www.seanoe.org/ data/00752/86417/ Underwater Vision Pro- ﬁler (UVP) zooplankton Picheral M. Primary production 13C labeling Deck incuba- tions https://www.seanoe.org/ data/00752/86417/ Primary Production in- cubations experiments Raimbault P./Garcia N. Quantum efﬁciency of PSII (Fv/Fm) (phyto- plankton) Benchtop PAM – Phyto-PAM Rosette water sample http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/PAM/ Direct Joy-Warren H. Sea-ice concentration Satellite Surface mode https://www.seanoe.org/ data/00752/86417/ Sea ice history Massicotte P. Sediment grain size Laser Box corer http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/ SEDIMENT_BC/ GE_Amundsen_Sediment_ BoxCore.csv Archambault P. Sediment organic con- tent Lost by ignition Box corer http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/ SEDIMENT_BC/ GE_Amundsen_Sediment_ BoxCore.csv Archambault P. Silica biogenic (BSi) BSi extraction Rosette water sample https://www.seanoe.org/ data/00752/86417/ Biogenic and Lithogenic silica concentration Leblanc K./ Quéguiner B./ Ley- naert A./ Moriceau B./ Legras J./ Gallinari M. Silica biogenic (BSi) dis- solution rate Dissolution in ﬁltered sea water Rosette water sample https://www.seanoe.org/ data/00752/86417/ Biogenic and Lithogenic silica concentration Moriceau B./ Gallinari M. https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 4623 Table 3. Continued. Table 3. Continued. Table 3. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Wind speed Wind monitor Meteorological tower https://www.seanoe.org/ data/00752/86417/ Meteorological Tower data Else B./ Burgers T. Wind speed Cross-calibrated multi-platform (CCMP) Atmosphere http://www.obs-vlfr.fr/ proof/ftpfree/greenedge/ /db/DATA/WIND/ DATA/ Direct Massicotte P. Zooplankton (abun- dance) Multi-nets (hydro- bios) Plankton net https://www.seanoe.org/ data/00752/86417/ Zooplankton abundance and diversity (vertical nets) Fortier L. Zooplankton (abun- dances) Vertical plankton net 200 µm Plankton net https://www.seanoe.org/ data/00752/86417/ Zooplankton abundance and diversity (vertical nets) Fortier L. Zooplankton (meso-) (abundance) Tucker net Plankton net https://www.seanoe.org/ data/00752/86417/ Ichthyoplancton vertical nets sampling Fortier L. Zooplankton (meso-) (taxonomy) Tucker net Plankton net https://www.seanoe.org/ data/00752/86417/ Ichthyoplancton vertical nets sampling Fortier L. Zooplankton (taxonomy) Multi-nets (hydro- bios) Plankton net https://www.seanoe.org/ data/00752/86417/ Zooplankton abundance and diversity (vertical nets) Fortier L. Zooplankton (taxon- omy) Vertical plankton net 200 µm Plankton net https://www.seanoe.org/ data/00752/86417/ Zooplankton abundance and diversity (vertical nets) Fortier L. Zooplankton (taxonomy) Microscopy Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Zooplankton fecal pel- lets Microscopy Drifting sedi- ment trap https://www.seanoe.org/ data/00752/86417/ drifting traps data (25 m depth) Lalande C. Table 4. Name and description of variables systematically included in datasets (i.e., in each .csv ﬁle). Variable Description Mission Mission identiﬁer (“ice_camp_2016”, “Amundsen_2016”, .. .) Date Sampling date (UTC) Latitude Latitude of sampling location (degree north decimals) Longitude Longitude of sampling (degree east decimals) Station Station name (e.g., G206) Operation code GE_0008-1 (unique operation identiﬁer) PI Name(s) of the principal investigator(s) responsible for the measured (calculated) variable Table 4. Name and description of variables systematically included in datasets (i.e., in each .csv ﬁle). Table 4. Name and description of variables systematically included in datasets (i.e., in each .csv ﬁle). Variable Description Mission Mission identiﬁer (“ice_camp_2016”, “Amundsen_2016”, .. .) Date Sampling date (UTC) Latitude Latitude of sampling location (degree north decimals) Longitude Longitude of sampling (degree east decimals) Station Station name (e.g., G206) Operation code GE_0008-1 (unique operation identiﬁer) PI Name(s) of the principal investigator(s) responsible for the measured (calculated) variable Over the cruise, ice thickness varied between 32 and 108 cm, while freeboard varied between 10 and −8 cm (top of the ice under water). Several cores were retrieved at each site using a 9 cm diameter Mark II ice corer (Kovacs Enterprises Inc., Roseburg, OR, USA). Each ice core was sliced into 10 cm segments (from the bottom) after temperature was measured. Table 3. Continued. Salinity was assessed after thawing and ﬁltration using a sali- nometer (Guildline Autosal 8400B, Guildline Instruments Ltd., Smith Falls, ON, Canada). Snow density and granulom- etry were assessed opportunistically (Eicken et al., 2009). ployed, oscillating between 0 and 300 m depth while towed at an average speed of 12 knots, rendering a very high spatial resolution (Fig. 8, bottom row, one proﬁle every 2 km). Data obtained with the MVP matched the patterns observed from the rosette data acquired on sampling stations (Fig. 8, mid- dle row). Proﬁles of conductivity, temperature, and pressure were collected using a Sea-Bird SBE 911plus CTD system rigged on the rosette. The data were post-processed accord- ing to the standard procedures recommended by the manu- facturer and averaged over 0.2 m vertical bins. While there was a sharp transition in SIC at the ice edge along transect 300, the change in SIC was less steep in the more extensive MIZ of transect 500 (top row in Fig. 8). Nonetheless, both transects show similar patterns, with 100 % SIC and colder Table 3. Continued. Variable Method Sampling method Access to dataset Name of ﬁle PI Silica biogenic concen- tration BSi extraction Box corer https://www.seanoe.org/ data/00752/86417/ Sediment incubations data Morata N./Gallinari M. Silica lithogenic (LSi) LSi extraction Rosette water sample https://www.seanoe.org/ data/00752/86417/ Biogenic and Lithogenic silica concentration Leblanc K./Quéguiner B./Leynaert A./Moriceau B./Legras J./Gallinari M. Sub-bottom proﬁles Knudsen 320 sub- bottom echosounder Continuous horizontal https://geoapp.bibl. ulaval.ca/Home/Index Search Lajeunesse P./Joyal G./Brouard E. Sugars (aerosol) HPAEC-PAD Atmosphere http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/ AEROSOLS/ Direct Panagiotopoulos C./Sempere R. Suspended particulate material (SPM) Particle dry weight (gravimetry) Rosette water sample https://www.seanoe.org/ data/00752/86417/ Dry weight, Particulate Carbon and Nitrogen (CHN) Bruyant F./Lariviere J./Babin M. Synechococcus (abun- dance) Flow cytometry Rosette water sample https://www.seanoe.org/ data/00752/86417/ photosynthetic and non-photosynthetic eu- karyotes and prokaryotes concentration (ﬂow cytometry) Vaulot D. Total organic carbon (TOC) Wet oxidation Rosette Deck incubations https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. Total organic nitrogen (TON) Wet oxidation Rosette Deck incubations https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. Total organic phospho- rus (TOP) Wet oxidation Rosette Deck incubations https://www.seanoe.org/ data/00752/86417/ Dissolved Inorganic and Organic matter concen- trations Raimbault P./Garcia N. Transmittance (of light in water) CST Wetlabs Moving Vessel Proﬁler https://www.seanoe.org/ data/00752/86417/ Moving vessel proﬁler data Morisset S./Gombault C. Transmittance (of light in water) C-Star In-water proﬁler https://www.seanoe.org/ data/00752/86417/ CTD data 2.0 m resolu- tion with ﬂuorescence Guillot P./Gombault C. Upwelling irradiance (Eu(z)) C-OPS In-water proﬁler https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Upwelling radiance (Lu) C-OPS In-water proﬁler https://www.seanoe.org/ data/00752/86417/ C-OPS data Belanger S. Variable ﬂuorescence and rapid light curves parameters (phytoplank- ton) Benchtop PAM Rosette water sample http://www.obs-vlfr. fr/proof/ftpfree/ greenedge/db/DATA/ AMUNDSEN/PAM/ direct Joy-Warren H. Weather and navigation Onboard AVOS https://www.seanoe.org/ data/00752/86417/ Automated Volunteer Observing Ship (AVOS) weather data Morisset S./Gombault C. Wind direction Wind monitor Meteorological tower https://www.seanoe.org/ data/00752/86417/ Meteorological tower data Else B./Burgers T Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4624 4.3 Chemistry 4.3 Chemistry Partial pressure of CO2 (pCO2) was measured continuously (every 2 min) using a Li-7000 CO2 analyzer (LICOR, Lin- coln NE, USA) coupled to a General Oceanics underway sys- tem model 8050 (General Oceanics, Miami FL, USA) con- nected to the ship-track water monitoring system. At each FULL, BASIC, and NUT station, discrete samples were col- lected using the Niskin bottles at 10 or more depths for sea- water analysis (see Table 3 for the complete list). To com- plement the pCO2 data from the underway system and to provide full proﬁles of the seawater CO2 system, total alka- linity and dissolved inorganic carbon (DIC) concentrations were determined on discrete samples according to Dickson et al. (2007). Concentrations of the major macronutrients (nitrate, phosphate, and orthosilicic acid) were determined with a segmented ﬂow AutoAnalyzer model 3 (Seal Analyt- ical, Germany) using standard colorimetric methods adapted from Grasshoff et al. (1999). Nitrate concentration in the wa- ter column was also determined during each CTD cast using an in situ ultraviolet spectrophotometer (ISUS, Satlantic Inc., Halifax NS, Canada) mounted on the rosette. Concentrations varied between 0 and non-limiting concentrations over the entire cruise, with concentrations gradually increasing from the surface to the bottom. Surface waters showed higher con- centrations of macronutrients in the western half of the tran- sects than in the eastern half of the transects, indicating that surface water nutrients had been used by the developing PSB. The organic elemental composition of total and dissolved matter (nitrogen, carbon, and phosphorus) was measured on water samples taken from the Niskin bottles. Samples were immediately poisoned with sulfuric acid and brought back to the lab for analysis using wet oxidation, as described by Raimbault et al. (1999). Subtracting signals obtained for ﬁl- tered samples (dissolved matter) from non-ﬁltered samples (total matter) rendered calculated values for particulate or- ganic matter. Particulate organic carbon and nitrogen were also analyzed on ﬁltered samples (Whatman™glass ﬁber GF/F, GE Healthcare, USA) using high-temperature oxida- tion combined with gas chromatography. Figure 5. Average wind direction and speed (arrow length and colour) over Bafﬁn Bay in June 2016 (a) and July 2016 (b); CCMP wind vector analysis product (V2.0, Atlas et al., 2011). 4.3 Chemistry (below −1 ◦C) and fresher (salinity below 33.5 g kg−1) wa- ters close to the surface on the western side, and 0 % SIC and saltier (above 33.6 g kg−1) and warmer (above 0 ◦C) waters within the ﬁrst 50 m on the eastern side. These observations were consistent with the northward inﬂow of Atlantic-origin waters along the Greenland shelf break and the southward outﬂow of Arctic/Paciﬁc-origin waters along the Bafﬁn Is- land shelf break. F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4625 Figure 5. Average wind direction and speed (arrow length and colour) over Bafﬁn Bay in June 2016 (a) and July 2016 (b); CCMP wind vector analysis product (V2.0, Atlas et al., 2011). 4.2.2 Water masses Hydrological conditions during the cruise were determined using several tools. A Moving Vessel Proﬁler (MVP) was de- https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer uyant et al.: Investigating the marginal ice zone processes during late spring and early summer yant et al.: Investigating the marginal ice zone processes during late spring and early summer 4626 Figure 6. Average weekly sea-ice extent for each of the four 8 d periods during the Green Edge cruise. Date is the median date of each 8 d sampling period. The white areas represent sea ice and the blue areas the open water. Black lines represent the ship route during said 8 d period. Figure 6. Average weekly sea-ice extent for each of the four 8 d periods during the Green Edge cruise. Date is the median date of each 8 d sampling period. The white areas represent sea ice and the blue areas the open water. Black lines represent the ship route during said 8 d period. six different wavelengths (HOBI Labs, Hydroscat-6, 394, 420, 470, 532, 620 and 700 nm) together with CTD data (Sea-Bird SBE 19plus attached to the package). In addition, discrete water samples were taken at each FULL and BA- SIC station to measure in the lab the CDOM absorption co- efﬁcient (aCDOM) between 200 and 722 nm using an Ultrap- ath (World Precision Instruments) and the phytoplankton and non-algal particle absorption coefﬁcients between 200 and 860 nm determined from the “inside sphere” ﬁlter-pad tech- nique (Röttgers and Gehnke, 2012; Stramski et al., 2015) us- ing a spectrophotometer equipped with a 155 mm integrat- ing sphere (Perkin Elmer Lambda 19). Note that aCDOM, phytoplankton, and non-algal particle absorption coefﬁcients were also measured on the bottom slice of thawed ice cores. The data revealed that the minimum light amount required for net phytoplankton growth (0.415 mol m−2 d−1; Letelier et al., 2004) can be reached deeper under the ice than ex- pected (Randelhoff et al., 2019). Further details of the light ﬁeld measurements can be found in Massicotte et al. (2020). were recorded at each FULL and BASIC station in open wa- ters. Above-water hyperspectral remote sensing reﬂectance (Rrs(λ)) was calculated from the radiometric quantities fol- lowing the ocean optics protocols of Mueller et al. (2003) and Mobley (1999). In-water vertical proﬁles of downward irradiance (Ed(z)) and upward radiance (Lu(z,λ) or irradi- ance (Eu(z,λ)) in the water column were measured using two different versions of the compact optical proﬁling system (C- OPS, Biospherical Instruments Inc.) radiometer. In open wa- ters, the free-fall version of the proﬁler was deployed from the ship (Antoine et al., 2013). 4.4 Light ﬁeld and bio-optics The characteristics of the light ﬁeld (quantity and quality) passing through snow and sea ice into the water column were assessed, as light is the most important parameter triggering the PSB. From the top of the CCGS Amundsen wheelhouse, the total solar downwelling radiation was measured using a pyranometer (0.3 to 300 µm wavelength) and a radiome- ter (visible range 300 to 750 nm). Surface radiometry was performed using a HyperSAS (hyperspectral surface acqui- sition system, SeaBird Scientiﬁc, USA) placed on the bow of the ship, including simultaneous measurements of hyper- spectral above-water downward irradiance (Ed(0+, λ)) and sky and surface radiance (Lsky(0+, λ) or Ltot(0+, λ)). Data Currents in the water column were measured using a hull- mounted 150 kHz acoustic Doppler current proﬁler (ADCP, Teledyne RD Instruments Ocean Surveyor, California, USA) as well as two L-ADCP installed on the rosette structure (RDI, WHM300-1-UG304) in a master/slave conﬁguration. Vertical proﬁles of water turbulence were measured at each FULL station using a Self-Contained Autonomous Micro- Proﬁler (SCAMP, Precision Measurement Engineering, Cal- ifornia, USA) deployed from the zodiac. A detailed descrip- tion of the hydrological structures in the studied area dur- ing the Green Edge cruise is presented in Randelhoff et al. (2019). https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 4.5.1 Viruses and bacteria An abundance of viruses and bacteria was determined on fresh and preserved (glutaraldehyde 4 % ﬁnal concentration) water samples taken from the rosette at each FULL and BA- SIC station (10 depths) using two different ﬂow cytometers. On board, fresh samples were counted using an Accuri™C6 and preserved samples were counted back in the lab using a FACSCanto (both machines from Becton Dickinson Bio- sciences, San Jose, CA, USA). Samples were processed ac- cording to Marie et al. (2001). Bacteria (and viruses) are ubiquitous in the oceans, and in Bafﬁn Bay, we measured bacterial abundances up to 2.9 × 106 cells mL−1. For bacterial diversity analysis, water samples were ﬁl- tered sequentially onto 20 µm, 3 µm (both polycarbonate ﬁl- ters, Millipore), and 0.22 µm (Sterivex-GV, Millipore). The ﬁlters and Sterivex were stored at −80 ◦C with RNAlater (Qiagen) until analyzed. The DNA/RNA co-extraction was carried out using the AllPrep DNA/RNA kit (Qiagen). The V4–V5 hypervariable region of the 16S rRNA gene was am- pliﬁed by PCR using primers 515F-Y and 926R, covering a broad spectrum of diversity, including Archaea and Bacteria (Parada et al., 2016). PCR, as well as sequencing settings and bioinformatics of sequence data, can be found in Dadaglio et al. (2018). Figure 7. Open water days (OWD) before sampling (values in days). The yellow colour corresponds to positive values, meaning the water was already free of ice on the day of sampling. Bluer, darker values correspond to stations that were still covered in ice at the sampling date (negative values). Values of OWD can be com- puted using different SIC (sea-ice coverage) values: (a) SIC = 10%, (b) SIC = 50% and (c) SIC = 80%. A comparison of the bacterial diversity as a function of geographic location and size fractions (free living bacte- ria, bacteria attached to particles smaller than 20 and larger than 20 µm) was made at a relatively broad taxonomic level (Fig. 10). The samples from the different groups were mainly dominated by Bacteroidetes and Proteobacteria. In general, the proportion of Proteobacteria decreased from ice stations to open-water stations featuring a more advanced stage of PSB, giving way to Bacteroidetes and, more speciﬁcally, Flavobacteriaceae. C-Star transmissometer, USA). Data is shown in Fig. 8 for transects 300 and 500. F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4627 below 50 %, and where the surface waters had been depleted of nutrients by phytoplankton growth. Figure 7. Open water days (OWD) before sampling (values in days). The yellow colour corresponds to positive values, meaning the water was already free of ice on the day of sampling. Bluer, darker values correspond to stations that were still covered in ice at the sampling date (negative values). Values of OWD can be com- puted using different SIC (sea-ice coverage) values: (a) SIC = 10%, (b) SIC = 50% and (c) SIC = 80%. 4.5 Biodiversity The Green Edge project also aimed to understand the related potential impacts of evolving environmental conditions on Arctic food webs in the context of climate change. Hence, great care was taken to sample the entire size spectrum of particulate matter and living organisms (Fig. 9), from the tiniest viruses and bacteria to demersal ﬁshes, seabirds, and marine mammals. A wide variety of sampling techniques and analyses, from visual observation to highly automated under- water imaging systems, allowed us to ensure that almost all the levels of the trophic network were examined. Earth Syst. Sci. Data, 14, 4607–4642, 2022 yant et al.: Investigating the marginal ice zone processes during late spring and early summer The sea-ice version (ICE- Pro) was deployed during ice sampling through an auger hole carefully ﬁlled with fresh snow to avoid, as much as possible, disturbing the underwater light ﬁeld. A reference sensor pro- vided simultaneous measurements of downward irradiance in the air. All measurements were made at 19 different wave- lengths between 320 and 875 nm. A proﬁling optical package was deployed at 28 stations to measure the inherent optical properties (IOPs) of sea- water. The measured properties (and sensors) included the ﬂuorescence of chlorophyll a and ﬂuorescent dissolved or- ganic matter (FDOM) (WetLabs, Eco Triplets), spectral total non-water absorption coefﬁcients between 360 and 764 nm (HOBI Labs a-Sphere), particle backscattering coefﬁcient at Three optical proﬁlers were also attached to the rosette carousel and rendered 203 proﬁles of CDOM ﬂuorescence (FluoCDOM Wetlabs USA) and chlorophyll concentration (estimated from in situ ﬂuorescence, Seapoint ﬂuorometer, USA) as well as 87 proﬁles of light transmittance (WET Labs https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4.5.1 Viruses and bacteria For both transects, the highest values of chlorophyll a concentration and of the attenuation coefﬁ- cient (both parameters being strong proxies for phytoplank- ton biomass) were observed close to the surface in the MIZ and deeper at around 50 m in ice-free waters, showing a pro- gression in the PSB development starting close to the surface along the ice edge and growing into a subsurface chlorophyll maximum (SCM) where surface waters were depleted in nu- trients. Concentration of CDOM showed its lowest concen- trations at the surface of open waters, where SIC dropped Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4628 Figure 8. Physical properties of seawater along transects 500 (70◦N) and 300 (69◦N). Middle and bottom panels show data recorded by sensors deployed on the rosette and on the MVP, respectively. From left to right: temperature (◦C), salinity (g kg−1), chlorophyll a concentration (mg m−3), transmittance (%), and CDOM concentration (mg m−3) as a function of depth (m, y axis) and longitude (◦W, x axis). Topmost panels show ice coverage (%) at corresponding longitudes. Note that the MVP did not carry a CDOM sensor. Figure 8. Physical properties of seawater along transects 500 (70◦N) and 300 (69◦N). Middle and bottom panels show data recorded by sensors deployed on the rosette and on the MVP, respectively. From left to right: temperature (◦C), salinity (g kg−1), chlorophyll a concentration (mg m−3), transmittance (%), and CDOM concentration (mg m−3) as a function of depth (m, y axis) and longitude (◦W, x axis). Topmost panels show ice coverage (%) at corresponding longitudes. Note that the MVP did not carry a CDOM sensor. 4.5.2 Phytoplankton community for puriﬁcation using ﬂow cytometry sorting, serial dilu- tion, and single-cell pipetting. Pure cultures were char- acterized by microscopy and 18S rRNA gene sequenc- ing. Most cultures isolated during the cruise belonged to diatoms, especially to the genera Attheya and Chaeto- ceros (Gérikas Ribeiro et al., 2020). All cultures were de- posited in the Roscoff Culture Collection and are available for distribution (http://www.roscoff-culture-collection.org/ strains/shortlists/cruises/green-edge, last access: 19 Septem- ber 2022). Flow cytometry was used (same protocols as for viruses and bacteria, Sect. 4.5.1) to count and differentiate the smallest cells (picophytoplankton, nanophytoplankton, cryptophytes, and Synechococcus) according to their ﬂuorescence and scat- tering properties at each FULL station. At the surface, pi- cophytoplankton and nanophytoplankton cell concentrations could reach 60 000 (station 719 – transect 7, station 19) and 9000 (station 515 – transect 5, station 15) cell mL−1, respec- tively, while cryptophytes were always below 360 cell mL−1. Synechococcus cyanobacteria were never observed. To study the phytoplankton community composition, an Imaging FlowCytobot (IFCB, McLane Research Laborato- ries Inc., East Falmouth, MA, USA) was used during Leg 1B. The IFCB is best used for the study and identiﬁcation Some samples were used to start phytoplankton cul- tures, which were taken back to the Roscoff laboratory Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer uyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4629 tions. The number of particles close to the surface in open waters coincides with the larger phytoplankton biomass ob- served there compared with ice-covered stations, consistent with lower primary and secondary production under sea ice. Deeper in the water column, around 300 m, the large parti- cle concentrations observed at open-water stations likely re- ﬂect resuspension of bottom sediments, because these obser- vations were mostly made in the eastern part of Bafﬁn Bay over the continental shelf, whereas under-ice stations were mostly located on the deeper Canadian side of the Bay (see the bathymetry in Fig. 1). Figure 9. Schematic of the biological sampling size continuum across the various methods and tools used during the Green Edge cruise. Samples for taxonomic analyses of micro-algae by mi- croscopy were taken at each FULL and BASIC station at 10 sampling depths. 4.5.2 Phytoplankton community Half a litre of seawater was preserved with Lugol and kept at 4 ◦C until it was analyzed in the labora- tory. Visual observation and taxonomic determination were done using an inverted microscope (Eclipse TS100, Nikon Instrument Inc.) according to the Utermöhl method (Uter- möhl, 1958) using 25 or 50 mL columns. Three transects of 26 mm at 400× were systematically observed for identiﬁca- tion and counting of Bacillariophyceae, Dinophyceae, ﬂag- ellates, and ciliates. Larger phytoplankton cells and colonies were observed in all chambers at 100×. Diatoms (Bacillar- iophyceae) were found at every station, primarily at the sur- face of the water column, along with ﬂagellates (both at the surface and in the subsurface chlorophyll maximum, SCM) (Fig. 12). The most striking feature was the dominating pres- ence of a Phaeocystis sp. (Prymnesiophyceae, blue bars in Fig. 12) at the SCM, reaching 60 to 90 % of the cell counts (and, to a lesser extent, at the surface) across a wide range of ice cover conditions (OWD values between −12 and 12 d). Figure 9. Schematic of the biological sampling size continuum across the various methods and tools used during the Green Edge cruise. of cells between 1 and 150 µm. Fresh samples (5 mL) taken from the rosette at each FULL station (all depths) and some BASIC and NUT stations (2 to 7 depths) were analyzed; sam- ples from the 2 bottom-most slices of ice cores were also analyzed once melted. The IFCB takes pictures at a reso- lution of around 3.4 pixels per µm. Image descriptors and features were extracted with Matlab® using scripts devel- oped by Heidi Sosik (Sosik and Olson, 2007). Taxonomic determination was achieved using Ecotaxa (Picheral et al., 2017; http://ecotaxa.obs-vlfr.fr, last access: 12 March 2022). Random forest algorithms were used for automatic classiﬁ- cation. Reference sets and the validation of predictions were both done manually. Examples of specimens observed dur- ing the Green Edge campaigns can be found in Massicotte et al. (2020). ( ) Phytoplankton and ice-algae pigments were measured to derive indices of micro-algae biomass and taxonomic com- position and to get information on processes such as pho- toacclimation, senescence, and grazing activities (Roy et al., 2011). Rosette water samples were ﬁltered onto GF/F ﬁl- ters (Whatman™, GE Healthcare Life Sciences) and quickly frozen in liquid nitrogen. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 4.5.2 Phytoplankton community Back in the land-based labora- tory, samples were thawed and extracted in 100 % methanol, separated, and identiﬁed by HPLC, as described by Ras et al. (2008). A total of 25 individual pigments or groups of pigments were identiﬁed and quantiﬁed at each FULL and BASIC station (10 depths sampled each time). Figure 13 shows the distribution of total chlorophyll a and phaeophor- bide concentrations along transects 300 and 500. The high chlorophyll a concentrations close to the surface in the MIZ and deepening towards open waters in the east conﬁrmed the evolution of the PSB from an under-ice bloom to a SCM. Note that highest concentrations of phaeophorbide were sys- tematically found underneath the accumulation of chloro- phyll a, indicating the sinking of degrading phytoplanktonic material. A total of 203 underwater vertical proﬁles were acquired using an Underwater Vision Proﬁler (UVP, model 5-DEEP, Hydroptics, France) installed on the frame of the rosette carousel. The UVP5 collects in-focus images in the small seawater volume lit by its light emitting diodes (LEDs) as it is lowered in the water column. An automated computer sys- tem (https://ecotaxa.obs-vlfr.fr/, last access: 12 March 2022) was used to sub-sample images of individual objects and sort them into the appropriate category (marine snow or var- ious taxa of zooplankton). The UVP has been developed mainly to count and identify particles larger than 100 µm. Figure 11 (left panels) show the average vertical proﬁles of particle concentration (mL−1) over the top 350 m of the wa- ter column for open-water (top) and under-ice (bottom) sta- Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 4630 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer ure 10. Relative proportions of the different microbial taxa within the different groups of samples: (a) for the 0.2–3 µm ee-living bacteria); (b) for the 3–20 µm size fraction (particle-attached bacteria); and (c) for the size fraction > 20 µm (parti teria). ICE1-2: ice stations in the transects 100 and 200; ICE3-5: ice stations in transects 300 and 500; EDGE0-18: samples be m depth from the edge stations (stations 107, 204, and 312); EDGE > 20: samples greater than 20 m deep from the edge stati 7, 204, and 312); OWr: open water stations where the ice had receded between 2 and 8 d previously; OWo: open water station has receded more than 15 d previously. 4.5.2 Phytoplankton community th S t S i D t 14 4607 4642 2022 htt //d i /10 5194/ d 14 Figure 10 Relative proportions of the different microbial taxa within the different groups of samples: (a) for the 0 2 3 µm Figure 10. Relative proportions of the different microbial taxa within the different groups of samples: (a) for the 0.2–3 µm size fraction (free-living bacteria); (b) for the 3–20 µm size fraction (particle-attached bacteria); and (c) for the size fraction > 20 µm (particle-attached bacteria). ICE1-2: ice stations in the transects 100 and 200; ICE3-5: ice stations in transects 300 and 500; EDGE0-18: samples between 0 and 18 m depth from the edge stations (stations 107, 204, and 312); EDGE > 20: samples greater than 20 m deep from the edge stations (stations 107, 204, and 312); OWr: open water stations where the ice had receded between 2 and 8 d previously; OWo: open water stations where the ice has receded more than 15 d previously. Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4631 Figure 11. Underwater Vision Proﬁler data. Average vertical proﬁles of particle concentration (per mL; a, c) and copepod volumetric fraction (cm3 m−3; b, d) at open-water (a, b) and ice-covered (c, d) stations over the top 350 m of the water column. Figure 11. Underwater Vision Proﬁler data. Average vertical proﬁles of particle concentration (per mL; a, c) and copepod volumetric fraction (cm3 m−3; b, d) at open-water (a, b) and ice-covered (c, d) stations over the top 350 m of the water column. Figure 12. Relative abundance (%) of main phytoplankton groups (coloured bars) at the surface (a, c) and at the subsurface chlorophyll maximum (SCM; b, d) for all stations analyzed. Stations are sorted according to their OWD value (Fig. 7); (a) and (b) represent the chlorophyll a concentration (mg m−3) at the relevant depths for each station. Figure 12. Relative abundance (%) of main phytoplankton groups (coloured bars) at the surface (a, c) and at the subsurface chlorophyll maximum (SCM; b, d) for all stations analyzed. Stations are sorted according to their OWD value (Fig. 7); (a) and (b) represent the chlorophyll a concentration (mg m−3) at the relevant depths for each station. https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 4632 ure 13. Water column chlorophyll a concentration (mg m−3, top panels) and total phaeophorbide a concentration (mg m−3, botto els) along transects 300 (69◦N, left panels) and 500 (70◦N, right panels), as measured by HPLC on discrete samples obtained from t kin bottles at every FULL and BASIC station. Bottom panels: abundance of zooplankton (ind m−3), copepods in red and non-copepo lue, at each of the three FULL stations sampled on each transect (lower station numbers towards the east). The top graphs indicate t at each station at the time of sampling. Figure 13. Water column chlorophyll a concentration (mg m−3, top panels) and total phaeophorbide a concentration (mg m−3, bottom panels) along transects 300 (69◦N, left panels) and 500 (70◦N, right panels), as measured by HPLC on discrete samples obtained from the Niskin bottles at every FULL and BASIC station. Bottom panels: abundance of zooplankton (ind m−3), copepods in red and non-copepods in blue, at each of the three FULL stations sampled on each transect (lower station numbers towards the east). The top graphs indicate the SIC at each station at the time of sampling. 4.5.3 Zooplankton and ﬁsh at both open-water and ice-covered stations. A secondary peak right at the surface is present at ice-covered stations, where a subpopulation of copepods may stay close to the bottom of sea ice to feed on sympagic microalgae, small an- imals, and related detritus. Zooplankton represents the second level of the food chain. The UVP5 and the Imaging FlowCytobot (see previous sec- tions) both rendered valuable information on small zooplank- ton specimens (below 150 µm). Figure 11 (right panels) show copepod volumetric fraction (cm3 m−3) over the top 350 m of the water column for open-water (top panel) and under-ice (bottom panel) stations. The total copepod volumetric frac- tion calculated based on automatic identiﬁcation made using the Ecotaxa web application (http://ecotaxa.obs-vlfr.fr, last access: 12 March 2022) shows subsurface peaks around 20 m For bigger specimens, a series of vertical nets and trawls were deployed at each FULL station (no trawling operations took place when sea ice was present). An assembly of 4 nets of 3 different mesh sizes (50, 200 and 500 µm) coupled with a Lightframe On-sight Key species Investigation (LOKI) sys- tem rendered high-resolution pictures of individuals sam- https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4633 mer in areas close to the MIZ, and (iii) deep benthic food web dynamics and structural variability are directly linked to both depth and availability of food sources (Yunda-Guarin et al., 2020). The sampling was achieved using two different strategies; a total of 16 Agassiz trawling and 34 box coring operations (some down to more than 2000 m depth) were car- ried out during the cruise. The Agassiz trawl (KC Denmark a/s Research Equipment) is a medium-size dredge trawled behind the ship, allowing sampling of the macrofauna liv- ing on the sediment surface. Once brought back on board, the contents of the net were immediately rinsed with seawa- ter, manually sorted, and identiﬁed to the lowest taxonomic level possible. Samples were brought back to the laboratory to be identiﬁed under a dissecting microscope when onboard identiﬁcation was impossible. Figure 14 shows an example of the diversity of benthic organisms. More than 220 species of macrofauna were identiﬁed during the Green Edge cruise from more than 25 classes (Grant, C. and Yunda-Guarin, G., unpublished data). A box corer was used to sample sediment from each FULL station. 4.5.5 Birds and marine mammals During the entire cruise, a systematic bird and marine mam- mals survey was carried out from the ship’s wheelhouse (see LeBlanc et al., 2019 for detailed methodology). A total of 20 different bird species and 8 different mammal species were identiﬁed. Northern fulmar, thick-billed murre, and little auk were the most common bird species observed. Ringed seal, hooded seal, and harp seal were the most common seals. The long-ﬁnned pilot whale was the most common whale species observed. A total of 10 polar bears were observed. Samples of zooplankton were all processed in the same way. Swimmers (ﬁsh larvae and juveniles) were sorted out, measured, identiﬁed, and preserved in a mix of 95 % ethanol and 1 % glycerol (ﬁnal concentrations) for later analysis, while zooplankton samples were preserved in 4 % formalde- hyde solution. Zooplankton abundance and diversity were determined using binoculars back at the laboratory. A few samples (hydro-bios) were analyzed using the ZooScan and the Ecotaxa identiﬁcation tools (https://ecotaxa.obs-vlfr.fr/ prj/802, last access: 12 March 2022). Fish from IKMT and BBT sampling were sorted, counted, identiﬁed, and mea- sured before preservation in a −20 ◦C freezer in case fur- ther analyses are needed in the future. Acoustic data from the EK60 were analyzed in Echoview® (see Geoffroy et al., 2016 for details). A total of 123 seabirds from 7 species were also col- lected from a zodiac deployed from the CCGS Amundsen in Greenland waters between 10 June and 8 July. This in- cludes black-legged kittiwakes (n = 8), glaucous gulls (n = 6), great black-backed gull (n = 1), little auks (n = 19), northern fulmars (n = 42), and thick-billed murres (n = 36). Sampled birds were frozen at −20 ◦C until laboratory anal- yses. A ﬁrst study aimed to investigate the co-distribution of seabirds and their ﬁsh prey along the MIZ (LeBlanc et al., 2019). To this end, stomach contents were examined for 74 birds (35 murres, 30 fulmars, and 9 kittiwakes) under a dissecting microscope. Otoliths were retrieved and used to identify ﬁsh species, age, and size. A second focus was the recording of plastic in the stomachs. Plastic data are used in OSPAR monitoring and AMAP working groups on plastic pollution. A third study aimed to determine the birds’ associ- ation to sea ice and ice-derived resources by the combination of different trophic markers. Hence, liver, muscle, and blood 4.5.3 Zooplankton and ﬁsh The sediment samples were then divided among the research teams for diverse analyses (see Table 3 for a complete list), including (but not limited to) identiﬁcation of organisms living inside the sediment, incu- bations for respiration, and nutrient utilization or chemical analysis. pled along the water column together with actual specimens. The multi-net plankton sampler (Hydro-bios, Altenholz, Ger- many) uses a different sampling strategy. Composed of 9 identical nets (200 µm mesh size), it is hauled vertically in the water column, with the nets opening sequentially at dif- ferent depths, each net collecting a slice of the water column displaying the vertical distribution of the species sampled. Figure 13c shows the abundance (ind m−3) of zooplankton sampled using the vertical 200 µm mesh net along transects 300 and 500. Copepods represent the main zooplankton class observed during the cruise, and they were present at every sampling site. The particularly high abundance at station 507 (situated under the ice where a phytoplankton bloom had al- ready disappeared) might be due to the relatively high abun- dance of copepod nauplii (25 % of all copepod individuals compared to the usual 4 % at other stations). Ichthyoplankton were sampled using a double square net towed obliquely from the side of the ship at a speed of ca. 2–3 knots to a maximum depth of 90 m. A Star-Oddi® mini- CTD attached to the frame and ﬂowmeters determined the real depth and volume of sampling. For ﬁsh sampling, an echo sounder (EK60, Simrad, Kongsberg Maritime, Norway) mounted on the hull was used to locate and determine the depth of ﬁsh aggregations along the ship track during the entire cruise. When pelagic juveniles and adult ﬁsh were present at the sampling stations, an Isaac–Kidd Midwater Trawl (IKMT, Filmar and Québec-Océan, Québec, Canada) was towed for 20 min at a speed of 2–3 knots. When demer- sal ﬁsh were detected, a benthic beam trawl (BBT, Filmar and Québec-Océan, Québec, Canada) was used to sample bigger specimens (between 10 and 32 mm mesh size) living on the bottom sediment. https://doi.org/10.5194/essd-14-4607-2022 4.5.4 Benthos sampling Benthos sampling was performed to test if (i) sea-ice cover is the primary environmental driver of the contribution and geographic distribution of sympagic carbon on the seabed, (ii) sympagic carbon is the most important baseline food source supporting benthic consumers during spring and sum- https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4634 4634 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer Figure 14. Examples of benthic organisms. (a) Urasterias lincki, (b) Astarte borealis, (c) Gorgonocephalus eucnemis, (e) Brachiomma sp., (f) Heliometra glacialis, (g) Siphonodentalium lobatum, (h) Stegocephalus inﬂatus, (i) Ophiopleura borealis, Bathypolypus sp. (pictures by Benthic Ecology Lab, Université Laval, Gonzalo Bravo). Figure 14. Examples of benthic organisms. (a) Urasterias lincki, (b) Astarte borealis, (c) Gorgonocephalus eucnemis, (e) Brachiomma sp., (f) Heliometra glacialis, (g) Siphonodentalium lobatum, (h) Stegocephalus inﬂatus, (i) Ophiopleura borealis, Bathypolypus sp. (pictures by Benthic Ecology Lab, Université Laval, Gonzalo Bravo). 68 %) collected in shallower waters at station 409 and station 418 (Burot et al., 2021). 68 %) collected in shallower waters at station 409 and station 418 (Burot et al., 2021). (from the cardiac clot) samples were collected from a total of 52 bird carcasses (27 murres, 14 auks, 3 kittiwakes, and 8 ful- mars), on which highly branched isoprenoids (HBIs), carbon and nitrogen stable isotopes, and fatty acids were measured. Finally, a fourth study was looking at stable isotope data to- gether with mercury (Hg) data for both muscle and liver. 5.2 Primary production and micronutrient cycling To determine the fate of the phytoplankton spring bloom, one must ﬁrst determine primary production. In situ simulated incubations were carried out at each FULL station on water sampled from the rosette at 8–10 depths, determined as cho- sen percentages of surface photosynthetically available radi- ation (PAR, namely 100 %, 50 %, 25 %, 10 %, 6 %, 2.9 %, 1.2 %, 0.6 %, and 0.1 %). The melted bottom-most slices of ice cores were also incubated, when available. After spiking the water with a mix of 13C/15N tracers, samples were incu- bated on deck at simulated light levels identical to the sam- pling light levels. The dissolved and particulate matter result- ing from these incubations were analyzed by mass spectrom- etry resulting in detailed nitrogen assimilation and regenera- tion values (see Table 3 for a complete list of measurements) as well as phytoplankton primary production (PP). Primary production varied between 0 and 88.13 ± 3.0 µgC L−1 d−1 over the entire cruise. 5 Biological production and ﬂuxes 5.3 Fate of the phytoplankton spring bloom Some organic matter produced by the PSB was exported down the water column as algal cells aggregated and sank or were grazed upon by vertically migrating zooplankton. One ambitious experiment was conducted during the cruise to monitor the export of the PSB at a high temporal resolu- tion. A sequential sediment trap (PPS4, Technicap, France; 12 sampling cups) was anchored to an ice ﬂoe and de- ployed 25 m under the ice from 15 June to 9 July 2016. Sediment trap collection cups were ﬁlled with ﬁltered sea- water adjusted to a salinity of 38 psu with NaCl and a for- malin concentration of 4 % to preserve samples during de- ployment and after recovery. The carousel holding the sam- pling cups was programmed to rotate every 2 d. The sedi- ment trap was deployed in the marginal ice zone along tran- sect 200 (Fig. 1), eventually drifted south with the ice, and was recovered on the way back to Iqaluit. The sediment trap was no longer anchored to its ﬂoe at recovery, but sea ice was still present in the region. Taxonomic identiﬁca- tion of the algal cells collected showed a constant export of diatoms (∼50 million cells m−2 d−1) from 15 June to early July, when a 6-fold increase in diatom ﬂuxes was observed from 5 to 7 July (∼300 million cells m−2 d−1) along with a peak in chlorophyll a ﬂuxes. More than half of the cells exported during the peak in algal ﬂuxes were identiﬁed as the ice-associated pennate diatom Navicula spp. Fluxes of the ice-obligate pennate diatom Nitzschia frigida, among the ﬁrst species to be consistently exported from the melting sea ice in the Arctic Ocean (Lalande et al., 2019; Dezutter et al., 2021; Nadaï et al., 2021), peaked from 23 to 25 June, prob- ably indicating the onset of sea ice melt. Fluxes of copepod fecal pellets collected in the sediment trap were higher prior to 27 June, suggesting under-ice grazing of ice algae until the ice melted. Making such a large and diverse dataset available to others requires the use of many different platforms. Administra- tive rules and previous habits and commitments explain why our dataset is hosted by various websites, with some of it in more than one place (see in Table 3 the link and ﬁle information, when applicable for each parame- ter acquired during the cruise). 5.5 Other data The exhaustive list of parameters measured during the cruise is presented in Table 3 along with the responsible principal investigator’s (PI’s) name. 5.1 Bacterial production, respiration, and viability At each FULL station during the cruise, water samples were taken from 2–3 depths (surface, deep chlorophyll maximum (DCM), and below DCM) to determine bacterial respira- tion. Oxygen concentration was determined using the Win- kler method on 1 µm ﬁltered samples before and after a 5 d incubation in the dark at 1.5 ◦C. Bacterial respiration varied overall between 0 and 1.63 µmol O2 L−1 d−1, with a mean value of 0.35 ± 0.41 µmol O2 L−1 d−1. For bacterial produc- tion determination, water was collected at each FULL sta- tion from 8–10 depths. Bacterial production was measured by [3H]-Leucine incorporation (Kirchman et al., 1985) mod- iﬁed for microcentrifugation (Smith and Azam, 1992). Over- all values varied between 0 and 1.51 µgC L−1 d−1 around a mean value of 0.17 ± 0.25 µgC L−1 d−1. The use of the pro- pidium monoazide (PMA) method identiﬁed a high bacterial mortality in sea ice (up to 90 %) and in SPM material (up to Photosynthetic parameters also allow the calculation of primary production and provide insight into the efﬁciency and characteristics of the photosynthesis of a given sam- ple. On board, photosynthetic parameters were determined https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4635 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer by the P vs E curves method using NaH14CO− 3 -spiked incu- bations of water samples (Lewis and Smith, 1983). Changes in the saturation parameter Ek (µmol Quanta m−2 s−1) in the surface waters of the transect show clear variations in light level acclimation, increasing from lower values around 39 µmol Quanta m−2 s−1 at the western under-ice stations to 217 µmol Quanta m−2 s−1 at the eastern open-water stations (transect 700 values given as an example). Great emphasis was placed on the contribution of diatoms to primary produc- tion, as diatoms are the main phytoplankton group present during the PSB. Experiments on silica production and dis- solution were performed throughout the cruise to locate the actively growing diatoms. These experiments conﬁrmed the occurrence of active siliciﬁcation beneath the sea ice, where both centric and pennate diatoms were observed (see details in Lafond et al., 2019). 5.4 Benthic processes Taken from the box corer, portions of the sediment were in- cubated at in situ simulated conditions of temperature and light to assess the consumption of oxygen and nutrients by endofauna. Oxygen use in the sediment cores allowed calcu- lation of the benthic carbon demand (mgC m−2 d−1), which was found to be especially high at stations in open waters where the PSB had already reached senescence and sinking organic matter had reached the bottom. https://doi.org/10.5194/essd-14-4607-2022 5.3 Fate of the phytoplankton spring bloom Some funding agencies require data to be deposited in a speciﬁc database as a deliverable. In our case, the “Les Enveloppes Fluides et l’Environnement-Cycles Biogéochimiques Environnement et Ressources” (LEFE-CYBER) repository is our main host: http://www.obs-vlfr.fr/proof/php/GREENEDGE/x_datalist_ 1.php?xxop=greenedge&xxcamp=amundsen (last access: 29 September 2022). This is where all data and associated metadata can be found for the Green Edge cruise. Particu- larly, detailed metadata ﬁles associated with each variable contain the principal investigator’s contact information. For speciﬁc questions, the PI associated with the data should be contacted directly. The LEFE-CYBER platform, however, does not deliver DOI, which is a very important feature for visibility of data. To obtain a DOI for the Green Edge cruise dataset (https://doi.org/10.17882/86417, Bruyant et al., 2022), we uploaded the available formatted data on SEANOE (SEA scieNtiﬁc Open data Edition) under the CC-BY license: https://www.seanoe.org/data/00752/86417 (last access: 29 September 2022). All data hosted on the SEANOE website have been formatted as described in Sect. 3, but not all the data’s original formats allow transfor- mation into the “.csv” ﬁle type. It is therefore important to keep a repository up to date where one can ﬁnd all raw data. Major long-term research programs often have their own repository/database available that has been used since the on- set of their research. While the data of the BGC-Argo ﬂoats we deployed during the Green Edge cruise are hosted on the LEFE-CYBER repository, they also have been made avail- able, together with the entire BGC-Argo dataset, from the https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4636 Table 5. List of all peer-reviewed journal articles published so far using Green Edge cruise and/or Green Edge Ice Camp data. Only the ﬁrst 4 authors are indicated in this table. Authors Year Title Journal DOI Cruise Ice camp Amiraux, R., Jeanthon, C., Vaultier, F., Rontani, J.-F. 2016 Paradoxical effects of temperature and solar irradiance on the photodegradation state of killed phytoplankton J. Phycol. https://doi.org/10.1111/jpy.12410 n y Rontani, J.-F., Belt, S.- T., Brown, T.-A., Ami- raux, R., et al. 2016 Monitoring abiotic degradation in sink- ing versus suspended Arctic sea-ice al- gae during a spring ice melt using speciﬁc lipid oxidation tracers Org. Geochem. 5.3 Fate of the phytoplankton spring bloom https://doi.org/10.1016/j.orggeochem.2016.05.016 n y Amiraux, R., Belt, S.-T., Vaultier, F., Galindo, V., et al. 2017 Monitoring photo-oxidative and salinity- induced bacterial stress in the Canadian Arctic using speciﬁc lipid tracers Mar. Chem. https://doi.org/10.1016/j.marchem.2017.05.006 n y Rontani, J.-F., Galeron, M.-A., Amiraux, R., Ar- tigue, L., et al. 2017 Identiﬁcation of di- and triterpenoid lipid tracers conﬁrms the signiﬁcant role of au- toxidation in the degradation of terrestrial vascular plant material in the Canadian Arctic Org. Geochem. https://doi.org/10.1016/j.orggeochem.2017.03.011 n y Dadaglio, L., Dinasquet, J., Obernosterer, I., Joux, F. 2018 Differential responses of bacteria to diatom-derived dissolved organic matter in the Arctic Ocean Aquat. Mi- crob. Ecol. https://doi.org/10.3354/ame01883 y n Goyens, C., Marty, S., Leymarie, E., Antoine, D., et al. 2018 High Angular Resolution Measurements of the Anisotropy of Reﬂectance of Sea Ice and Snow Earth Space Sci. https://doi.org/10.1002/2017EA000332 n y Massicotte, P., Bécu, G., Lambert-Girard, S., Ley- marie, E., et al. 2018 Estimating underwater light regime under spatially heterogeneous sea ice in the Arc- tic Appl. Sci. https://doi.org/10.3390/app8122693 n y Rontani, J.-F., Amiraux, R., Lalande, C., Babin, M et al. 2018 Use of palmitoleic acid and its oxidation products for monitoring the degradation of ice algae in Arctic waters and bottom sediments Org. Geochem. https://doi.org/10.1016/j.orggeochem.2018.06.002 n y Rontani, J.-F., Belt, S.- T., Amiraux, R. 2018 Biotic and abiotic degradation of the sea ice diatom biomarker IP 25 and selected algal sterols in near-surface Arctic sedi- ments Org. Geochem. https://doi.org/10.1016/j.orggeochem.2018.01.003 n y Lafond, A., Leblanc, K., Quéguiner, B., Moriceau, B., et al. 2019 Late spring bloom development of pelagic diatoms in Bafﬁn Bay Elementa https://doi.org/10.1525/elementa.382 y n LeBlanc, M., Gauthier, S., Garbus, S. E., Mos- bech, A., et al. 2019 The co-distribution of Arctic cod and its seabird predators across the marginal ice zone in Bafﬁn Bay Elementa https://doi.org/10.1525/elementa.339 y n Randelhoff, A., Oziel, L., Massicotte, P., Bécu, G., et al. 2019 The evolution of light and vertical mixing across a phytoplankton ice-edge bloom. Elementa https://doi.org/10.1525/elementa.357 y n Amiraux, R., Smik, L., Köseo˘glu, D., Rontani, J.-F., et al. 2019 Temporal evolution of IP25 and other highly branched isoprenoid lipids in sea ice and the underlying water column dur- ing an Arctic melting season. Elementa https://doi.org/10.1525/elementa.377 n y Else, B. G. T. T., White- head, J. J., Galindo, V., Ferland, J., Mundy, C. J., Gonski, S. F., et al. 5.3 Fate of the phytoplankton spring bloom 2019 Response of the Arctic marine inorganic carbon system to ice algae and under- ice phytoplankton blooms: A case study along the fast-ice edge of Bafﬁn Bay. J. Geo- phys. Res.- Oceans https://doi.org/10.1029/2018JC013899 n y Gourdal, M., Crabeck, O., Lizotte, M., Galindo, V., et al. 2019 Upward transport of bottom-ice dimethyl sulﬁde during advanced melting of arctic ﬁrst-year sea ice. Elementa https://doi.org/10.1525/elementa.370 n y Matthes, L. C., Ehn, J. K., L.-Girard, S., Pogorzelec, N. M., et al. 2019 Average cosine coefﬁcient and spectral distribution of the light ﬁeld under sea ice: Implications for primary production. Elementa https://doi.org/10.1525/elementa.363 n y Table 5. List of all peer-reviewed journal articles published so far using Green Edge cruise and/or Green Edge Ice Camp data. Only the ﬁrst 4 authors are indicated in this table. https://doi.org/10.5194/essd-14-4607-2022 https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4637 able 5. Continued. Authors Year Title Journal DOI Cruise Ice camp Oziel, L., Massicotte, P., Randelhoff, A., Ferland, J et al. 2019 Environmental factors inﬂuencing the seasonal dynamics of spring algal blooms in and beneath sea ice in western Bafﬁn Bay. Elementa https://doi.org/10.1525/elementa.372 n y Sampei, M. 2019 An estimation of the quantitative impacts of copepod grazing on an under sea-ice spring phytoplankton bloom in western Bafﬁn Bay, Canadian Arctic Elementa https://doi.org/10.1525/elementa.2019.00092 n y Sansoulet, J., Pangrazi, J.-J., Sardet, N., Mir- shak, S., et al. 2019 Green Edge Outreach Project: a large- scale public outreach and educational ini- tiative polar record https://doi.org/10.1017/S0032247419000123 n y Burgers, T. M., Trem- blay, J.-É., Else, B. G. T., & Papakyriakou, T. N. 2020 Estimates of net community production from multiple approaches surrounding the spring ice-edge bloom in Bafﬁn Bay Elementa https://doi.org/10.1525/elementa.013 y n Randelhoff, A., Lacour, L., Marec, C., Leymarie, E., et al. 2020 Arctic mid-winter phytoplankton growth revealed by autonomous proﬁlers Science Advances https://doi.org/10.1126/sciadv.abc2678 y n Saint-Béat, B., Fath, B. D., Aubry, C., Colombet, J., et al. 2020 Contrasting pelagic ecosystem function- ing in eastern and western Bafﬁn Bay re- vealed by trophic network modeling Elementa https://doi.org/10.1525/elementa.397 y n Yunda-Guarin, G., Brown, T. A., Michel, L. N., Saint-Béat, B., et al. 2020 Reliance of deep-sea benthic macrofauna on ice-derived organic matter highlighted by multiple trophic markers during spring in Bafﬁn Bay, Canadian Arctic Elementa https://doi.org/10.1525/elementa.2020.047 y n Amiraux, R., Burot, C., Bonin, P., Massé, G., et al. 5.3 Fate of the phytoplankton spring bloom 2020 Stress factors resulting from the Arctic vernal sea ice melt: impact on the viability of the bacterial communities associated to sympagic algae Elementa https://doi.org/10.1525/elementa.076 n y Else, B. G. T. T., White- head, J. J., Galindo, V., Ferland, J., et al. 2020 Green Edge ice camp campaigns: un- derstanding the processes controlling the under-ice Arctic phytoplankton spring bloom. Earth Syst. Sci. Data https://doi.org/10.5194/essd-12-151-2020 n y Matthes, L. C., Mundy, C. J., L.-Girard, S., Babin, M et al. 2020 Spatial Heterogeneity as a Key Variable Inﬂuencing Spring-Summer Progression in UVR and PAR Transmission Through Arctic Sea Ice Front. Mar. Sci. https://doi.org/10.3389/fmars.2020.00183 n y Sansoulet, J., Ther- rien, M., Delgove, J., Pouxviel, G., et al. 2020 An update on Inuit perceptions of their changing environment, Qikiqtaaluk (Baf- ﬁn Island, Nunavut) Elementa https://doi.org/10.1525/elementa.025 n y Ardyna, M. and Arrigo, K. R. 2020 Phytoplankton dynamics in a changing Arctic Ocean. Nat. Clim. Change https://doi.org/10.1038/s41558-020-0905-y y y Ardyna, M., Mundy, C. J., Mills, M. M., Oziel, L., et al. 2020 Environmental drivers of under-ice phy- toplankton bloom dynamics in the Arctic Ocean Elementa https://doi.org/10.1525/elementa.430 y y Gérikas Ribeiro, C., dos Santos, A. L., Probert, I., Vaulot, D., et al. 2020 Taxonomic reassignment of Pseudohap- tolina birgeri comb. nov. (Haptophyta) J. Phycol. https://doi.org/10.1080/00318884.2020.1830255 y y Ribeiro, C. G., Dos San- tos, A. L., Gourvil, P., Le Gall, F., et al. 2020 Culturable diversity of Arctic phytoplank- ton during pack ice melting. Elementa https://doi.org/10.1525/elementa.401 y y Tisserand, L., Dadaglio, L., Intertaglia, L., Catala, P., et al. 2020 Use of organic exudates from two po- lar diatoms by bacterial isolates from the Arctic Ocean Phil. Trans. R. Soc. A. https://doi.org/10.1098/rsta.2019.0356 y y Yau, S., Lopes dos Santos, A., Eikrem, W., Gérikas Ribeiro, C., et al. 2020 Mantoniella beaufortii and Mantoniella bafﬁnensis sp. nov. (Mamiellales, Mamiellophyceae), two new green algal species from the high arctic J. Phycol. https://doi.org/10.1111/jpy.12932 y y https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4638 4638 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summe Table 5. Continued. Authors Year Title Journal DOI Cruise Ice camp Toullec, J., Moriceau, B., Vincent, D., Guidi, L., et al. 8 Conclusions The Green Edge cruise was of typical oceanographic design. In terms of goal achievement, the cruise was extremely suc- cessful and generated an impressive dataset over a diverse set of disciplines, providing a global picture of the explored en- vironment and of all the processes fuelling the Arctic food web. Figure 4b represents all interactions existing and/or measured during the cruise between compartments of the various trophic levels. The generated dataset contains a much larger number of parameters than those presented in this pa- per. All data can be obtained from the data repository and provide an excellent opportunity for re-use and comparison with other Arctic datasets. A special issue of the Elementa: Science of the Anthropocene journal entitled “Green Edge – The phytoplankton spring bloom in the Arctic Ocean: past, present and future response to climate variations, and impact on carbon ﬂuxes and the marine food web” contains a col- lection of research papers referring to this cruise. A com- plete list of peer-reviewed journal publications presenting data from either or both the Green Edge Ice Camp or Green Edge cruise can be found in Table 5. Geographical speciﬁcity might also be a motivation for the cross-uploading of data. Since the Amundsen has been used to conduct polar research, all navigation, AVOS, ADCP, MVP, and CTD data are systematically uploaded to the Polar Data Catalog (PDC): https://www.polardata.ca/ (last access: 10 August 2022). Please note that, in Table 3, only one address for each pa- rameter is provided, while most of them are also available from other sources. 5.3 Fate of the phytoplankton spring bloom 2021 Processes controlling aggregate formation and distribution during the Arctic phyto- plankton spring bloom in Bafﬁn Bay Elementa https://doi.org/10.1525/elementa.2021.00001 y n Vilgrain, L., Maps, F., Picheral, M., Babin, M., et al. 2021 Trait-based approach on zooplankton in situ images reveals contrasted ecological patterns along ice melt dynamics Limnol. Oceanogr. https://doi.org/10.1002/lno.11672 y n Amiraux, R., Rontani, J.-F., Armougom, F., Frouin, E., et al. 2021 Bacterial diversity and lipid biomarkers in sea ice and sinking particulate organic material during the melt season in the Canadian Arctic Elementa https://doi.org/10.1525/elementa.2019.040 n y Galí, M., Lizotte, M., Kieber, D.J., Randelhoff, A., et al. 2021 DMS emissions from the Arctic Ocean marginal ice zone Elementa https://doi.org/10.1525/elementa.2020.00113 y n Laliberté, J., Rehm, E., Hamre, B., Goyens, C et al. 2022 A method to derive satellite PAR albedo time series over ﬁrst-year sea ice in the Arctic Ocean Elementa https://doi.org/10.1525/elementa.2020.00080 n y Table 5. Continued. Table 5. Continued. quality dataset possible, and to avoid the loss of both valu- able time and data. biogeochemical Argo database: https://biogeochemical-argo. org/data-access.php (last access: 8 November 2021). Some more speciﬁc data acquired during the cruise are also available on dedicated websites. For example, dissolved inorganic carbon (DIC), alkalinity, and 18O data are also archived with the Ocean Carbon and acidiﬁcation Data Sys- tem (OCADS): https://doi.org/10.25921/719e-qr37 (Miller et al., 2020). Earth Syst. Sci. Data, 14, 4607–4642, 2022 References André, X., Le Traon, P.-Y., Le Reste, S., Dutreuil, V., Leymarie, E., Malardé, D., Marec, C., Sagot, J., Amice, M., Babin, M., Claustre, H., David, A., D’Ortenzio, F., Kolodziejczyk, N., La- gunas, J.L., Le Menn, M., Moreau, B., Nogré, D., Penkerc’h, C., Poteau, A., Renaut, C., Schaeffer, C., Taillandier, V., and Thierry, V.: Preparing the New Phase of Argo: Technological Develop- ments on Proﬁling Floats in the NAOS Project, Front. Mar. Sci., 7, 577446, https://doi.org/10.3389/fmars.2020.577446, 2020. Antoine, D., Hooker, S. B., Bélanger, S., Matsuoka, A., and Babin, M.: Apparent optical properties of the Canadian Beaufort Sea – Part 1: Observational overview and water column relationships, Biogeosciences, 10, 4493–4509, https://doi.org/10.5194/bg-10- 4493-2013, 2013. Competing interests. The contact author has declared that none of the authors has any competing interests. Ardyna, M., Babin, M., Gosselin, M., Devred, E., Bélanger, S., Matsuoka, A., and Tremblay, J.-É.: Parameterization of verti- cal chlorophyll a in the Arctic Ocean: impact of the subsur- face chlorophyll maximum on regional, seasonal, and annual primary production estimates, Biogeosciences, 10, 4383–4404, https://doi.org/10.5194/bg-10-4383-2013, 2013. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Arrigo, K. R., Perovich, D. K., Pickart, R. S., Brown, Z. W., van Dijken, G. L., Lowry, K. E., Mills, M. M., Palmer, M. A., Balch, W. M., Bates, N. R., Benitez-Nelson, C. R., Brown- lee, E., Frey, K. E., Laney, S. R., Mathis, J., Matsuoka, A., Mitchell, B. G., Moore, G. W. K., Reynolds, R. A., Sosik, H. M. and Swift, J. H.: Phytoplankton blooms beneath the sea ice in the Chukchi sea, Deep-Sea Res. Pt. II, 105, 1–16, https://doi.org/10.1016/j.dsr2.2014.03.018, 2014. Acknowledgements. This project was conducted using the Cana- dian research icebreaker CCGS Amundsen with the support of the Amundsen Science program funded by the Canada Founda- tion for Innovation (CFI) Major Science Initiatives (MSI) Fund. We wish to thank the ofﬁcers and crew of the CCGS Amund- sen. The project was conducted under the scientiﬁc coordination of the CERC on Remote Sensing of Canada’s new Arctic fron- tier and the Centre national de la recherche scientiﬁque CNRS/U- niversité Laval Takuvik Joint International Laboratory (IRL3376). The ﬁeld campaign was successful thanks to the contributions of Guislain Bécu, José Lagunas, Debra Christiansen-Stowe, Julie San- soulet, Eric Rehm, Maxime Benoît-Gagné, Marie-Hélène For- get and Flavienne Bruyant, Julie Bourdon, Claudie Marec and Marc Picheral from CNRS. F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4639 Author contributions. MB designed the Green Edge project, in- cluding the scientiﬁc objectives and sampling strategy. MB, KL, FB, TL, PG, GJ, TB, MP, CM, KC, DM, MT, GD, GF, LD, JD, CL, ML, GN, NM, MC, MP, K-ML, HJ-W, ER, AV, LBdF, DD, NG, HC, AS, BQ, SH, GB, SEG, CG, P-LG, JET, EB, CS, MGT, JR, AB, RA, CB, BM, JL, EL, PB, and PC were onboard the ship and took part in sampling and onboard analysis. FB, PG, GJ, TB, MP, CM, KC, DM, MT, GD, LD, GF, LD, JD, CL, LM, GN, NM, NP, K-ML, HJ-W, ER, AV, LBdF, DD, NG, HC, AS, BG, SH, GB, GC, P-LG, M-NH, CS, MGT, JR, AB, RA, CB, BM, JL, EL, BS-B, PC, PA, LA, SB, DC, VC-B, FC, MD, CD, BE, IE, JF, LF, MG, CGR, CG, PG, CG, SH, RH, FJ, AL, PL, FLG, KL, JL, AL, ML, ALdS, GM, AM, LAM, P-IM, AM, AM, CP, MP, PR, J-FR, RS, JT, A-PT, DV, and CN took part in processing and analyzing the samples and in generating data. M-PA and PM cleaned, merged, and assembled the dataset. CS maintains the Les Enveloppes Flu- ides et l’Environnement-Cycles Biogéochimiques Environnement et Ressources (LEFE-CYBER) repository it is stored in. NS, SM, JS, LRL, TP and PB oversaw communication and outreach. MHF, JF, JL, and FB oversaw logistics. FB wrote the manuscript. 106086), Fisheries and Ocean Canada, Sentinelle Nord (grant no. 113079), LEFE (grant no. AO2015-874272), and Institut PaulEmile Victor (IPEV, grant no. 1164). 106086), Fisheries and Ocean Canada, Sentinelle Nord (grant no. 113079), LEFE (grant no. AO2015-874272), and Institut PaulEmile Victor (IPEV, grant no. 1164). Review statement. This paper was edited by David Carlson and reviewed by Emilia Trudnowska and one anonymous referee. Review statement. This paper was edited by David Carlson and reviewed by Emilia Trudnowska and one anonymous referee. 7 Lessons learned As for any scientiﬁc cruise, a large amount of data was ac- quired by many people. Even though guidelines had been suggested ahead of time for data formatting, merging, and storage, a tremendous amount of effort was necessary to col- lect, assemble, and standardize the data. It is important that a clear and streamlined data management plan be established ahead of time to avoid errors or loss of data in the merg- ing process. For oceanographic CTD-rosette sampling-based cruises, depth of sampling necessitates special attention, as it is crucial to use Niskin bottle number instead of nominal depth to correctly merge the data. Furthermore, we cannot emphasize enough that data management specialists must be involved from the beginning of such large-scale projects to ensure that data is properly documented, to render the best https://doi.org/10.5194/essd-14-4607-2022 Earth Syst. Sci. Data, 14, 4607–4642, 2022 https://doi.org/10.5194/essd-14-4607-2022 F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer F. Bruyant et al.: Investigating the marginal ice zone processes during late spring and early summer 4640 natural aquatic systems, Appl. Environ. Microb., 49, 599–607, https://doi.org/10.1128/aem.49.3.599-607.1985, 1985. natural aquatic systems, Appl. Environ. Microb., 49, 599–607, https://doi.org/10.1128/aem.49.3.599-607.1985, 1985. 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https://openalex.org/W3110910775	https://nottingham-repository.worktribe.com/file/25363949/1/1-s2.0-S0148296320307839-main	English	null	Artificial intelligence for human flourishing – Beyond principles for machine learning	Journal of business research	2,021	cc-by	15,446	* Corresponding author at: De Montfort University, The Gateway, Leicester LE2 9BH, UK. E-mail address: bstahl@dmu.ac.uk (B.C. Stahl). A R T I C L E I N F O Keywords: Ethics Artificial intelligence Big data Human rights Governance Keywords: Ethics Artificial intelligence Big data Human rights Governance The technical and economic benefits of artificial intelligence (AI) are counterbalanced by legal, social and ethical issues. It is challenging to conceptually capture and empirically measure both benefits and downsides. We therefore provide an account of the findings and implications of a multi-dimensional study of AI, comprising 10 case studies, five scenarios, an ethical impact analysis of AI, a human rights analysis of AI and a technical analysis of known and potential threats and vulnerabilities. Based on our findings, we separate AI ethics discourse into three streams: (1) specific issues related to the application of machine learning, (2) social and political questions arising in a digitally enabled society and (3) metaphysical questions about the nature of reality and humanity. Human rights principles and legislation have a key role to play in addressing the ethics of AI. This work helps to steer AI to contribute to human flourishing. whether and how AI should be regulated or whether other ways should be found to address the downsides of AI. Artificial intelligence for human flourishing – Beyond principles for machine learning B.C. Stahl a,, A. Andreou e, P. Brey b, T. Hatzakis c, A. Kirichenko d, K. Macnish b, S. Laulh´e Shaelou f, A. Patel d, M. Ryan g, D. Wright c a De Montfort University, UK b University of Twente, the Netherlands c Trilateral Research, UK d F-Secure, Finland e Aequitas, Cyprus f University of Central Lancashire, Cyprus g Wageningen University & Research, the Netherlands a De Montfort University, UK b University of Twente, the Netherlands c Trilateral Research, UK d F-Secure, Finland e Aequitas, Cyprus f University of Central Lancashire, Cyprus g Wageningen University & Research, the Netherlands Available online 16 December 2020 0148-2963/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0 https://doi.org/10.1016/j.jbusres.2020.11.030 Received 18 January 2020; Received in revised form 16 November 2020; Accepted 18 November 2020 Available online 16 December 2020 0148-2963/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Available online 16 December 2020 0148-2963/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). blished by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Journal of Business Research 124 (2021) 374–388 Artificial intelligence for human flourishing – Beyond principles for machine learning B.C. Stahl a,, A. Andreou e, P. Brey b, T. Hatzakis c, A. Kirichenko d, K. Macnish b, S. Laulh´e Shaelou f, A. Patel d, M. Ryan g, D. Wright c a De Montfort University, UK b University of Twente, the Netherlands Contents lists available at ScienceDirect Journal of Business Research journal homepage: www.elsevier.com/locate/jbusres Journal of Business Research 124 (2021) 374–388 Artificial intelligence for human flourishing – Beyond principles for machine learning B.C. Stahl a,, A. Andreou e, P. Brey b, T. Hatzakis c, A. Kirichenko d, K. Macnish b, S. Laulh´e Shaelou f, A. Patel d, M. Ryan g, D. Wright c a De Montfort University, UK b University of Twente, the Netherlands Contents lists available at ScienceDirect Journal of Business Research journal homepage: www.elsevier.com/locate/jbusres Journal of Business Research 124 (2021) 374–388 2.1. AI and big data The concept of AI, while much discussed, is not well defined. A typical definition of AI is the one provided by the European Commission (2018, p. 1): “Artificial intelligence (AI) refers to systems that display intelligent behaviour by analysing their environment and taking actions – with some degree of autonomy – to achieve specific goals.” This is consistent with Haenlein and Kaplan (2019) emphasis on the interpre tation of external data, learning from such data and using data for the achievement of specific goals. While such definitions are sufficient to give an idea of the scope of AI, they are arguably not specific enough to allow the identification of specific ethical issues or the application of governance structures. Definitions such as the EC’s can also be prob lematic when they seem to imply contentious positions, such as that AI can behave, analyse and act, which can be read as imputing character istics, notably that of independent agency, that current machine learning technologies do not display. This points to metaphysical as sumptions about AI to which we return to below. i i Our analysis shows that the ethical issues that arise in empirical observations are similar to those that the academic literature discusses, which provides reason for the belief that the discourse on ethics in AI is reasonably expansive. At the same time, however, it becomes clear that the meaning of these issues is largely context-dependent. We use our understanding of the ethical issues to categorise them into three broad categories: (1) issues directly related to machine learning, (2) broader social and political issues arising in modern digitally enabled societies and finally (3) metaphysical questions. These categories allow us to map currently existing and discussed mitigation and governance structures to these issues. This is an important starting point for the practical question of what can and should be done to address these issues. This question is beyond the scope of this paper. i One reason for these shortcomings is that the definitions hide the immense breadth and depth of the underlying AI research (Elsevier, 2018). The current prominence of AI is based on long-established principles of machine learning, often implemented through (deep) neural networks. These have recently gained prominence due to the increased availability of computing power and large data sets for training purposes. 2.1. AI and big data Ethical discussions of AI therefore need to be sensitive to both the consequences of the application of AI algorithms and tech niques as well as the ethical aspects of (big) data analytics (B. D. Mit telstadt, Allo, Taddeo, Wachter, & Floridi, 2016; Nerurkar, Wadephul, & Wiegerling, 2016; Varley-Winter & Shah, 2016). Public discourse on AI especially focuses on machine learning and the empirical work we have undertaken covers technologies in machine learning and big data ana lytics. However, as we argue below, the ethics of AI debate is broader than this and refers to other concepts of AI, notably that AI technologies have broader human-like cognitive abilities. The concept of general AI goes back to the beginning of AI research and is sometimes referred to as Good Old Fashioned AI (GOFAI) (Moor & Bynum, 2002). General AI technologies do not currently exist, but they figure strongly in the public discourse. In order to be able to make sense of the broader debate, it is important to be aware of the entire breadth of meaning of the term. The findings presented in this paper are important in several re spects. The paper makes an academic contribution to the quickly spreading discussion of ethics and AI and research around ethics, values, governance and tools of AI. The categorisation of issues suggested here and the mapping of the categories to different governance mechanisms can help streamline the debate. Due to the high practical importance of the underlying technologies, the paper also has practical importance for stakeholders faced with the practical challenge of proactively engaging with the ethics of AI. The paper can help organisations developing, deploying or using AI to identify issues they are likely to face and engage with governance mechanisms that can address these issues. In order to develop the argument, the paper proceeds as follows. In the next section, we discuss the governance of AI, looking first at defi nitions, followed by a discussion of ethical issues and currently proposed governance structures. We then describe our multi-dimensional empir ical study of AI. The findings and discussion give rise to our catego risation of issues, which we then map to governance structures and stakeholders. Our discussion and conclusions demonstrate the novelty and relevance of our findings while we propose next steps. 1. Introduction The development of artificial intelligence (AI) is often described in terms of human progress. The recent progress of machine learning, supported by growing amounts of available data combined with rapidly expanding computing capabilities and publicly available tools and li braries, have led to expectations of increased efficiency but also to new and better services for consumers and citizens. This broadly positive discourse is, however, counterbalanced by a discussion of the downsides and risks of AI. A growing volume of literature suggests that governance mecha nisms need to be devised for these technologies because existing governance structures are not able to address the issues they raise. As a consequence, one can find numerous suggestions on various ways to develop governance structures that range from the informal, such as voluntary industry codes of conduct, to national and international legislation and the creation of regulators. One weakness of the current discourse is a disconnect between rigorous academic research on the content and implications of these technologies and the development of governance proposals. The ethics of AI is a topic of conversation in the disciplines concerned with these technologies including the social sciences, humanities, media and policy. Worries range from discrimination due to biased datasets to the domination of humanity by sentient machines. The social impact of AI-based technologies provides the backdrop and justification for the flurry of activities in public discourse and policy developments about In order to move beyond the current discourse, gain a deeper un derstanding of the nature of ethics in AI, and allow for a critical reflection of the current discourse, we conducted multi-method and interdisciplinary research aimed at contributing to empirical and Journal of Business Research 124 (2021) 374–388 B.C. Stahl et al. conceptual clarity of the nature of these technologies, the challenges they raise and the potential of new governance structures to address these issues. The aim of this paper is to contribute to the discussion about how to identify, interpret and address ethical issues arising from AI applications1. The paper critically reflects on the term AI and explores which aspects of AI raise which types of issues and how these are re flected and addressed in organisational and societal practice. Bringing together conceptual insights and empirical findings, the paper is in a position to propose new ways to think about AI and structure the AI ethics narrative. 1 We use the term "issue" in an open sense, accepting as issues whatever our respondents or the literature describes using the term. As a consequence, the issues we cover vary greatly in terms of scope and impact. Some are very precise and focused whereas other are large and fuzzy and cover entire areas where issues arise. 1. Introduction In order to achieve this aim, the paper first seeks to answer the question of what precisely are the key ethical issues and how best to classify or categorise them. It then explores how existing governance mechanisms may be applied to these issues. This leads to the final question of theoretical and practical next steps. organization, or territory, and whether through laws, norms, power, or language” (Bevir, 2012, p. 1). The term also refers to specific localised ways of organising (or governing) particular issues, as in data gover nance (Khatri & Brown, 2010) or information governance (ISO, 2008), rendering it suitable to describe ways of dealing with AI that cover many societal actors and activities. 2.1. AI and big data This paper therefore does not attempt to offer a comprehensive definition of AI or of any of its constituent technologies such as machine learning. Instead, it aims to bring greater clarity to the question what people refer to when they talk about AI and, more importantly, when they talk about the ethics of AI or about the ethical issues of AI. These conceptual questions are crucial to dealing with the ethics of AI and questions of governance. They pose the problem of delineating which ethical issues are related to or caused by AI and it complicates questions of governance, where the application area of governance mechanisms is often not clear, as we will show in more detail below. 2. Governance of AI This section provides the conceptual basis of tthis article and gives an overview of current discussions regarding AI, its ethical implications and possible governance structures. The term ’governance’ as developed in political sciences traditionally refers to alternatives to formal govern ment on a societal or state level. In business research, it frequently refers to structures and processes within organisations, whereas on a higher level the term ’regulation’ is used (Braithwaite & Drahos, 2000). How ever, ’governance’ is increasingly used to describe a much broader array of “[…] processes of governing, whether undertaken by a government, market, or network, whether over a family, tribe, formal or informal 2.2. Ethics and AI However, any attempts to use ‘AI for Good’, as the title of the series of summits organised by the Inter national Telecommunication Union suggests (https://aiforgood.itu.int/ ), can count in this category. i Despite a rich history of discussing the relative merits of various ethical positions, the current discourse around ethical issues of AI makes little reference to philosophical ethical theories. Instead, the generally accepted approach to AI ethics seems to define mid-level ethical prin ciples, an approach pioneered by biomedical ethics (Beauchamp & Childress, 2009; The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. (1979), 1979). This approach, sometimes referred to as principlism, is not without criticism (Clouser & Gert, 1990). It has the practical advantage of sidestepping long-standing ethical debates. But it is open to the charge that it fails to solve practical ethical issues, due to the apparent consensus on ethical principles that then fail to guide practical action (B. Mittelstadt, 2019). Nevertheless, the creation and compilation of ethical principles form key aspects of the ethics of AI debate (Anabo, Elexpuru-Albizuri, & Villard´on-Gallego, 2019; Asilomar Conference, 2017; Boden et al., 2017). In addition, most of the high-level interventions into the ethics of AI discussion are principle-based, such as the guidelines produced by the European High Level Expert Group on AI (HLEG on AI, 2019). For our purposes, it is sufficient to understand the prevalence of ethical princi ples in the AI discourse. We agree with Mittelstadt (2019), however, in seeing the focus on principles as limiting and will return to the question of an appropriate ethical theoretical basis for the ethics of AI below. We realise that this is a strong simplification and that these in tentions and purposes of AI are not necessarily mutually exclusive and do not comprehensively cover all possibilities. The need for contact tracing to fight a pandemic, for example, shows that social control can be conducive to human flourishing. Similarly, the optimisation of pro cesses and resulting profit maximisation leads to higher income and welfare, which can (but do not have to) contribute to broader human flourishing. The Venn diagram in Fig. 1 indicates that the three different purposes can intersect and overlap. However, they are recognisably different ways of approaching AI and have different ethical implications and connotations. 2.2. Ethics and AI The concept of ethics is even more contested and open than that of AI. In everyday English, it denotes questions of right or wrong, of good or bad. Following Stahl (2012), we argue that this everyday under standing of ethics constitutes the basis of explicit reasoning and aca demic reflection, which are the subject matter of philosophical ethics. Answering the question of why a particular action can be seen as good or bad or which processes would allow answering such a question is the 375 Journal of Business Research 124 (2021) 374–388 B.C. Stahl et al. role of philosophical ethical theories. These include classical theories such as virtue ethics (Aristotle, 2007) which determines the ethics quality often action based on the character of the individual undertaking it. Other frequently used ethical theories include deontology, which focuses on the agent’s duty (Kant, 1788, 1797), or teleology which looks at the consequences and outcomes of an action to determine its ethical status (Mill, 1861). In addition to these well-established traditional ethical theories, there are more recent ones like the ethics of care (Adam, 2001; Gilligan, 1990) and specific ethical theories aimed at technolog ical applications, such as computer ethics (Terrell Ward Bynum & Rogerson, 2003; D. G. Johnson, 2001), information ethics (Capurro, 2006; L. Floridi, 1999; Luciano Floridi, 2010) or disclosive ethics (Brey, 2000). first and most prominent purpose is to improve processes and efficiency. For organisations using AI, this translates into lower costs, higher pro ductivity and, eventually, higher profits. The second purpose is the use for social control. AI techniques are the enablers for voice and face recognition and can therefore be used for surveillance and tracking in dividuals. This is the basis for controlling individuals to ensure they follow specific requirements. This is the underlying idea of the Chinese Social Credit System (Liu, 2019). The third purpose of using AI is to promote human flourishing. Flourishing is an ethical principle typically associated with virtue ethics, which has a well-established history of application to digital technologies (T. W. Bynum, 2006) and which has been used to frame the AI debate more recently (ALLEA & Royal Society, 2019). It is not always trivial to determine what constitutes flourishing or how technology can contribute to it. 2.2. Ethics and AI Intentions behind promoting AI are important to understand and evaluate perceptions of ethics and possible governance mechanisms employed to address ethical issues. These intentions do not develop in isolation but form part of a larger socio-economic, cultural and political context that influences the way a ‘good society’ is perceived and the role AI can play in it (Cath, Wachter, Mittelstadt, Taddeo, & Floridi, 2016). We do not wish to overstate differences between regions or underesti mate levels of disagreement within political cultures, but we think it is probably safe to say that the European approach to AI aims to promote human flourishing, even where this may lead to trade-offs with effi ciency or access, which may result from specific interventions, such as the EU’s General Data Protection Regulation (GDPR) (General Data Protection Regulation, 2016; see also Kaplan & Haenlein, 2019). Discussions of the ethics of AI tend to cover particular ethical issues. These are typically particular features of the technology or consequences of its use that the authors see as problematic. Many of these have long- standing histories in ethics of technology or ethics of computing, such as security, privacy or access. Some of them seem to be particularly linked to the algorithms that drive AI, such as problems of algorithmic biases (CDEI, 2019; K. Johnson, Pasquale, & Chapman, 2019; B. D. Mittelstadt et al., 2016) and many of them are linked to the compilation and manipulation of large data sets that are required for many of the current AI techniques (Metcalf et al., 2016; Nerurkar et al., 2016; Taylor, 2016). Some ethical issues are specific to particular application areas, such as finance or autonomous vehicles, whereas others are seen as broadly relevant to all AI areas. In order to assess whether a response to an ethical issue is appro priate or likely to be successful, we need to not only understand the purpose of AI, but also the range of possible options used to address the issue. This paper does not offer the space to review all governance ar rangements or tools that are available to implement them (see Hagen dorff, 2019; Morley, Floridi, Kinsey, & Elhalal, 2019). For the purposes of this paper, we seek to understand the types and levels of activity that aim to provide governance mechanisms for AI. Below, we distinguish between measures aimed at the individual, the organisation and society. B.C. Stahl et al. Governance structures that support the identification and mitigation of possible ethical issues of AI cover all levels, notably the individual, organisational and political / societal. Individual researchers and de velopers can make use of a quickly growing number of AI ethics frameworks originating from companies, governments or other organi sations. The EU’s High Level Expert Group is a pertinent example (HLEG on AI, 2019) but many others exist. Individual developers can make use of professional guidance, for example, from bodies such as the ACM or BCS (Brinkman et al., 2017). Standardisation initiatives, such as the ISO/IEC JTC 1/SC 42 - Artificial intelligence or the IEEE P7000 family of standards, consider the ethics of AI and, once agreed, can provide guidance. Development methodologies can be created or adapted to pay attention to ethical issues, for example, by integrating specific issues into the design process, such as privacy by design (Cavoukian, 2017; Hansen, 2016; Information Commissioner’s Office, 2008) or more broadly by adopting an ethics by design stance (Beard & Longstaff, 2018; Iphofen & Kritikos, 2019; Martin & Makoundou, 2017). Academy & Royal Society, 2017; Khatri & Brown, 2010; OECD, 2017), and ensure these cover AI. Similarly, many organisations have estab lished mechanisms for dealing with ethical and broader societal con cerns, often discussed under the heading of corporate social responsibility (CSR) (Garriga & Mel´e, 2004), which can be extended to include AI and emerging technologies. i The final level is the societal and policy level, covering national and international policy and regulation. These drive a lot of individual and organisational activity and therefore play a role in governing AI. It is, therefore, not surprising that policy and regulatory mechanisms play a prominent role concerning the ethics of AI. There are existing statutory instruments, such as the GDPR (General Data Protection Regulation, 2016), that clearly address some of the issues that AI raises. Similarly, there are principles of human rights, that are addressed and safeguarded in international agreements, such as the Universal Declaration of Human Rights or the European Convention on Human Rights, that cover rele vant rights such as the right not to be discriminated against that have relevant applications to AI. Similarly, legislation in areas such as competition law, product liability or intellectual property can have consequences for AI. One type of regulatory instrument with regard to AI is the creation of a regulator to oversee AI development and use. 2.3. Purpose of AI and governance proposals The number and reach of ethical issues linked to AI is enormous, in particular, when considering the breadth of possible application areas. Addressing them is therefore a challenge that has attracted much attention. One key question that needs to be answered before any mitigation measures can be developed is the role that AI has and is meant to have in society. AI, along with most other information and communications technologies (ICTs), has a particularly high level of interpretive flexibility (Doherty, Coombs, & Loan-Clarke, 2006), which means that it is difficult to predict how it will be used. This has been a key driver for thinking about ethical aspects of ICTs for decades, sometimes discussed under the heading of “logical malleability” (Moor, 1985). What this means is that even in cases where a technology is designed for a particular purpose, it is difficult to foresee whether and to what degree it will be used for this purpose. Fig. 1. Possible purposes of AI. Fig. 1. Possible purposes of AI. Fig. 1. Possible purposes of AI. We distinguish between different purposes of making use of AI. The 376 B.C. Stahl et al. Journal of Business Research 124 (2021) 374–388 approach (See Fig. 3). approach (See Fig. 3). A single paper such as this one cannot hope to do justice to the complexity of five different major components of a complex multi- dimensional study as presented here. Each of these has been described in detail elsewhere (Andreou et al., 2019; Macnish, Ryan, Gregory, et al., 2019; Macnish & Ryan, 2019; Patel, Hatzakis, Macnish, Ryan, & Kir ichenko, 2019; Wright et al., 2019). Instead of a detailed account of all methodological considerations, we focus here on a brief overview of the different methods and why they provided the insights we required for our research objective. The aim of this very brief overview of ethics and governance of AI, graphically represented in Fig. 2 (above), was to demonstrate the complexity of the topic area. The discourse of ethics and AI is currently characterised by a cacophony of voices and contributions. There is no lack of ideas or proposals. The challenge is to synthesise a manageable approach from the multitude of activities. This synthesis should start with a useful and manageable categorisation of ethical issues that lends itself to an analysis of suitable governance structures that can be applied to these issues. This paper attempts such a synthesis based on a multi- dimensional research approach, as described in the next section. The motivation for undertaking a set of case studies arose from the lack of rigorous empirical academic research of AI across application areas. While there are numerous studies of the impact of AI in particular areas, a broader understanding of AI required a set of comparable in sights in different settings. We chose to undertake a set of interpretive case studies (Walsham, 1995) because they allowed us to develop a detailed understanding based on the views of individuals and organi sations involved. There are certainly many more application areas than 10, but undertaking 10 studies and doing a comparative analysis (Yin, 2003) gave us the confidence of being able to develop a strong under standing across applications. We developed a case study protocol and pilot tested it during the summer of 2018. The empirical work was un dertaken in 2018 and 2019. We interviewed a total of 22 stakeholders across the 10 case studies. For each case, we furthermore undertook background research on the organisation in question as well as the field in which the case study was undertaken (e.g., AI in finance, agriculture). approach (See Fig. 3). The analysis was undertaken collaboratively using NVivo Server 11. The partners wrote up case studies following an agreed template, cross- reviewed and published on our website (Macnish, Ryan, & Stahl, 2019). All case studies were furthermore developed as stand-alone publications whose references are listed in Table 1. B.C. Stahl et al. This can be achieved by extending the remit of existing regulators, such as data protection authorities, or by creating new bodies. The second level of measures provides guidance for organisations to follow or adopt. According to Clarke (Clarke, 2019b, 2019a), established mechanisms of risk management can go a long way in allowing orga nisations to address the ethics of AI. Organisations can employ existing impact assessment approaches such as privacy (or data protection) impact assessments (CNIL, 2015), technology assessment (Grunwald, 2009), ethics impact assessment (Wright, 2011), social impact assess ment (Becker & Vanclay, 2003) or human rights impact assessment (Latonero, 2018). They can extend existing governance mechanisms, such as those used for quality assurance or data governance (British On all three of these levels, the individual, the organisational and the national / international, there can be different focus areas. AI can be looked at in general terms or specific application areas can be emphasised, such as AI in health, finance, politics, public services or Fig. 2. Overview of proposals for governance mechanisms of AI ethics. Fig. 2. Overview of proposals for governance mechanisms of AI ethics. 377 B.C. Stahl et al. Journal of Business Research 124 (2021) 374–388 others. In many cases, organisational or technical tools may exist or could be developed to support governance structures. Table 1 We aimed to develop scenarios that are relevant in several years. A second important aspect of policy scenarios is close stakeholder engagement. For each scenario, we organised a face-to-face workshop that included stakeholders and involved them in the revision and refinement of the scenarios. All scenarios were peer reviewed and made publicly available in June 2019 (Macnish, Wright, & Jiya, 2020; Wright et al., 2019). i Jaeger, 1999; Boenink, Swierstra, & Stemerding, 2010; Cairns & Wright, 2017; Ramirez, Mukherjee, Vezzoli, & Kramer, 2015), we applied a modified methodology which we called “policy scenarios” (Wright, Stahl, & Hatzakis, 2020). At its core, a policy scenario aims at a rela tively short time horizon that is of relevance to policymakers caught up in election cycles. We aimed to develop scenarios that are relevant in several years. A second important aspect of policy scenarios is close stakeholder engagement. For each scenario, we organised a face-to-face workshop that included stakeholders and involved them in the revision and refinement of the scenarios. All scenarios were peer reviewed and made publicly available in June 2019 (Macnish, Wright, & Jiya, 2020; Wright et al., 2019). i One important insight from our empirical work was that the many ethical issues discussed in the literature are reflected in practice. Some issues are almost ubiquitous, such as those related to privacy and data protection. This paper does not offer a detailed analysis of the issues, nor of the exact differences between the case study and Delphi study find ings. One key point from our findings is that clearly recognisable issues are well covered are roughly consistent. The Delphi study is under standably broader than the case studies that focused on organisational practice, including issues such as ’awakening’ of AI that do not play a role in current implementations of AI. Our conceptual review of the ethics of AI and the analysis of our empirical work agreed to a large extent, showing that the literature covers the same issues of individuals and organisations working with AI (See Fig. 4). While the work on case studies and scenarios gave us the confidence to be in a position to understand a broad range of ethical aspects of AI in social situations, this knowledge had to be based on and complemented by current academic and other debates about ethical issues and human rights implications. 3. Methodology: A multi-dimensional approach While there is a large and quickly growing literature on ethical issues of AI, much of it is anecdotal or speculative. In our research, we there fore aimed to combine academic rigour with detailed insights into the way in which AI is realised in society and a broad and conceptual overview of the field. No one single established methodology can ach ieve this. We therefore decided to use a multi-dimensional approach that involved using and combining several methods to collect and interpret data and develop an understanding of the field of ethics and AI. i The geographical focus of our study is Europe. We wanted to know whether the influence of AI on human flourishing is noticeable in the way AI is developed and deployed. In order to understand this, to gain an in-depth understanding of the social reality of AI across different application domains at present and in the future and to understand the technical, ethical and human rights implications, we undertook a multi- dimensional study comprising the following: 1 10 interpretive case studies of AI application in particular applica tion domains and organisations In order to broaden our understanding further, but also to go beyond the description of current technologies, we decided to develop a set of five scenarios. The social domain in which AI is employed was discussed simultaneously for case studies and scenarios (see table below). This served to broaden the range of insights. i 2 Five policy-oriented scenarios exploring near term (<5 years) use of emerging AI applications The scenarios were constructed with a specific focus on providing applicable insights that could help decision-makers, notably those working in policy development, to develop and implement governance mechanisms. Based on the rich history of scenario methods (Andersen & 5 A technical analysis of threats and vulnerabilities connected with AI. The following figure is a graphical representation of the research 378 Fig. 3. Components of the multi-dimensional research approach underpinning this research. Fig. 3. Components of the multi-dimensional research approach underpinning this research. 378 Journal of Business Research 124 (2021) 374–388 B.C. Stahl et al. B.C. Stahl et al. to identify, interpret and address ethical issues arising from AI appli cations. In order to achieve this, we need a comprehensive overview of both conceptual and empirical insights into AI and its use. We therefore draw on a range of research activities outlined below. Table 1 We furthermore realised that it was important to understand the technical aspects of AI, notably questions of security and vulnerabilities of AI systems that have potentially large implications for the use and social impact of these technologies. The ethical and human rights analyses were undertaken as desk research drawing on appropriate sources of their various disciplines (i. e., philosophy and human rights law). We also conducted a study of security issues, dangers, and implications of the use of data analytics and artificial intelligence. We examined: However, while it was possible to analyse our data using widely accepted terms, we note that the local meaning of these terms varied widely. The meaning of the term privacy, for example, in a medical diagnostic context, in the use of social media for logistics prediction or in the case of agricultural optimisation, differs greatly. Privacy is “an inherently heterogenous, fluid and multidimensional concept” (Finn, Wright, & Friedewald, 2013, p. 26) and can be divided up into a number of sub-types (Koops et al., 2017; Solove, 2002). In general, privacy in the AI ethics discourse seems to refer to data privacy or information privacy which touches on various other types of privacy (ibid). Other theoretical positions on privacy (Tavani, 2008) are not widely employed in the AI discourse. While this connection is rarely made explicit, there is an underlying assumption that implementing data protection mechanisms is the way to ensure privacy, although this has been contested (Macnish, 2020). This position is reflected in our findings where the frequent references to privacy as an issue were accompanied by a strong emphasis on often technical data protection measures. However, it is important to note that privacy risks take very different forms in these contexts, requiring different technical and organisational measures to ensure compliance with data protection legislation, but also to ensure that broader ethical issues are covered. • ways in which machine-learning systems are commonly mis- implemented or mis-used (and recommendations on how to pre vent this from happening); • ways in which machine-learning models and algorithms can be attacked (and mitigations against such attacks); i • how artificial intelligence and data analysis methodologies and technologies might be used for malicious purposes. This initial review of security-related issues and possible sources of harm provided the starting point for further and ongoing studies of specific vulnerabilities of AI. Table 1 The purpose of this task was to provide a baseline understanding of the current capabilities and applications of machine learning, including examples of potential malicious uses of machine learning techniques, and the implications of attacks against systems powered by machine learning. Table 1 Table 1 Social domains of case studies and scenarios (CS:= Case Study; SC:= Scenario). Table 1 Social domains of case studies and scenarios (CS:= Case Study; SC:= Scenario). No. Social Domain of Case Study / Scenario Reference CS01 Employee monitoring and administration (Antoniou & Andreou, 2019) CS02 Government (Ryan, 2019a) CS03 Agriculture (Ryan, 2019b) CS04 Sustainable development (Ryan & Gregory, 2019) CS05 Science (Jiya, 2019b) CS06 Insurance (Kanceviˇcien˙e, 2019) CS07 Energy and utilities (Hatzakis, Rodrigues, & Wright, 2019) CS08 Communications, media and cybersecurity (Macnish, Inguanzo, & Kirichenko, 2019) CS09 Retail and wholesale trade (Macnish & Inguanzo, 2019) CS10 Manufacturing and natural resources (Jiya, 2019a) SC01 Social care All scenarios are described in detail in ( Wright et al., 2019) SC02 Information warfare SC03 Education SC04 Transportation SC05 Predictive policing Our research showed that the breadth of ethical issues discussed in the literature is reflected to a large extent in organisational practice. A cross-case analysis extracting the moral issues that were raised in the different case studies showed a large number of ethical issues, many of them recurring, as shown in the following table (See Table 2). This table listing the ethical issues we encountered is the result of our data analysis and represents our interpretation and categorisation of what respondents shared with us. The terms used to denote the indi vidual ethical issues were discussed and agreed during the data analysis. The results of the analysis from the case studies were supported by the first round of our Delphi Study, which targeted experts in AI and big data (Santiago, 2020). Implemented as an online survey, the first set of questions was e-mailed to 231 experts, 50 per cent of whom were women. We received 145 responses.Following review of the data and data cleansing, 41 responses contained sufficient information to warrant analysis. The first (open-ended) question covered the same ground, asking “What do you think are the three most important ethical or human rights issues raised by AI and / or big data?” Fig. 4 shows the most frequently given answers: Jaeger, 1999; Boenink, Swierstra, & Stemerding, 2010; Cairns & Wright, 2017; Ramirez, Mukherjee, Vezzoli, & Kramer, 2015), we applied a modified methodology which we called “policy scenarios” (Wright, Stahl, & Hatzakis, 2020). At its core, a policy scenario aims at a rela tively short time horizon that is of relevance to policymakers caught up in election cycles. 3. Methodology: A multi-dimensional approach The underlying empirical studies were not re-analysed but taken as the starting point for compiling and, more importantly, for categorising these issues. The important contribution to the AI ethics discourse that this paper makes is in the conceptualisation and proposed narrative which is based on the overall findings. It is therefore only possible to provide a high-level overview of the empirical work, all of which is published elsewhere. 4. Findings and Discussion Interestingly, our analysis of human rights concerns, undertaken in parallel with the case studies, found that the human rights issues that can be found in the literature are closely related to and overlap with the The purpose of this paper is to contribute to the discussion about how 379 nable opment CS05 Science CS06 Insurance CS07 Energy and utilities CS08 Communications, media and cybersecurity CS09 Retail and wholesale tr ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● B.C. Stahl et al. Journal of Business Research 124 (2021) 374–388 Table 3 Table 3 Selection of Rights and Freedoms (EU) with potential relevance to AI. Table 3 Selection of Rights and Freedoms (EU) with potential relevance to AI. Rights ECHR EUCFR Right to human dignity Article 1 Right to the integrity of the person Article 3 Right to liberty and security Article 5 Article 6 Right to respect for private and family life Article 8 Article 7 Protection of personal data Article 8 Freedom of thought, conscience and religion Article 9 Article 10 Freedom of expression and information Article 10 Article 11 Prohibition of discrimination Article 14; Article 1, Protocol 12 Article 21 Right of property Article 1, Protocol 1 Article 17 Right to education Article 1, Protocol 2 Article 14 Right to free election Article 3, Protocol 1 Articles 39–40 Freedom of movement Article 2, Protocol 4 Article 45 Freedom to choose an occupation and right to engage in work Article 15 Freedom to conduct a business Article 16 Rights of the child Article 24 Rights of the elderly Article 25 Integration of persons with disabilities Article 26 Right to health care Article 35 Consumer protection Article 38 Right to good administration Article 41 Right of access to documents Article 42 Freedom of movement and residence Article 45 n of Rights and Freedoms (EU) with potential relevance to AI. In recognition that the relationship between ethics and human rights is complex (van Est & Gerritsen, 2017) and that human rights have an ethical core that must be safeguarded, the human right considerations were analysed on a scale of concepts and notions: (See Fig. 6) Formalising human rights at different levels is based on the recog nition that humans have ethical rights that need to be explicitly safe guarded. Human rights declarations such as the Universal Declaration are political statements, but they have long found their way into positive law. Europe, for example, has the European Convention on Human Rights (ECHR) at the international level, which originates from the Council of Europe but is authoritative in the EU legal order. The EU has its own legally binding human rights instrument, the EU Charter of Fundamental Rights (EUCFR), whose scope can be said to be wider and deeper than the ECHR, as evidenced in the table below. Table 3 This is relevant because it means that many of the ethical issues identified by our research are not just ethical issues, but, where they relate to human rights questions, may well be open to adjudication in a court of law. The following overview of human rights that may be affected by AI is therefore interesting, as it shows where and how these human rights are enshrined in existing human rights instruments, namely the ECHR and the EUCFR (See Table 3). All of these rights and freedoms were raised as a concern in at least one of our case studies or scenarios, thus further demonstrating the broad range of concerns. The reason for including this table in the text was that for readers not familiar with the detail of our case studies, it shows a list of human rights that are easy to associate with AI use and demonstrates that there are legal instruments that could deal with these. As several overlaps were identified in terms of, for example, relevant legal instruments and types of solution proposed, these were intertwined in the establishment of a ‘blueprint’ of current and future practice in relation to the interrelationship of human rights, law, ethics and smart information systems, those systems that have AI and big data analytics at their core (Stahl & Wright, 2018). application of human rights to AI as a key way of addressing these issues (Access Now Policy Team, 2018; BSR, 2018; Committee on Bioethics (DH-BIO), 2019; Council of Europe, 2019; Latonero, 2018; World Eco nomic Forum, 2019). (See Fig. 5) (See Fig. 5) Fig. 4. Delphi survey responses covering the most important issues of AI and big data (Santiago, 2020). Fig. 4. Delphi survey responses covering the most important issues of AI and big data (Santiago, 2020). ethical issues described above. The human rights gap analysis was based on a combination of methodological approaches emanating from the interim results of case studies and scenarios, preliminary in-depth in terviews on cyberthreats and ethics as well as desktop research. Initial findings were cross-checked and combined to identify the most burning challenges to human rights in the digital world and start formulating solutions. The findings were mindful of the diversity and breadth of data collected and expected results. A list of commonly perceived human rights issues and challenges in the digital world was derived from the process, encompassing general and specific considerations as follows: Fig. 5. Key human rights issues in AI. Fig. 5. Key human rights issues in AI. 381 Journal of Business Research 124 (2021) 374–388 B.C. Stahl et al. (See Fig. 5) 4.1.1. Specific issues of machine learning i Machine learning and the various techniques used to achieve it have some characteristics at the core of particular ethical issues. Two of these characteristics seem most likely to raise ethical issues: First, many of the current machine-learning techniques are opaque, which means that even experts with relevant equipment cannot determine why and how inputs are transformed into outputs, e.g. how exactly a personal profile leads to a classification in terms of a mortgage application or parole decision. Second, these systems require access to big amounts of data for training and validation purposes. Other larger-scale societal concerns have to do with the question about what technology can and should do. A high-profile example of this is the use of AI-driven autonomous weaponry that has the potential to change the face of modern warfare (Defense Innovation Board, 2019; Sparrow, 2009). But there are many other examples where it is not clear- cut what machines can and should do, e.g., with regard to autonomous vehicles or care robots (Decker, 2008; Sharkey & Sharkey, 2010; Bernd Carsten Stahl & Coeckelbergh, 2016). i Resulting ethical issues are, for example, those having to do with bias and discrimination (Johnson et al., 2019; Macnish, 2012), which can arise on the basis of undetected biases included in the training data. Further specific issues are linked to the use of data, which may be per sonal data and which may allow additional insights into individual personal behaviour through AI and big data analysis. Questions around privacy and data protection therefore arise on this level as do concerns about security and integrity of systems, algorithms and data (Stahl & Wright, 2018). i Our empirical findings support the relevance of these issues. In fact, and somewhat surprisingly, ethical issues in this category constitute the majority of issues. Economic consequences, employment, fairness, freedom, the ability of having human contact, individual autonomy, inequality, integrity, justice, ownership, military use, power asymmetry, responsibility and sustainability all fall into the category. These issues are common to most specific applications of AI. Algo rithmic biases, discrimination, security and transparency are issues that are directly linked to the characteristics of machine learning. Others, such as quality and accuracy of data are closely related. Our case study analysis suggests that these issues are prominent among the AI users in organisations. They materialise and present themselves in specific and context-dependent ways. 4.1.2. General questions about living in a digital world Our second category of ethical issues includes those that have less to do with the specific capabilities of AI and more with the way societies use technologies many of which incorporate elements of machine learning or other AI techniques. These issues play to a general feeling of unease with the way in which industrialised societies develop and the role that technology plays in promoting certain developments and inhibiting others. These issues are currently discussed in the context of AI because the expectation is that AI will greatly influence them, but they are better understood as questions that relate to how modern so cieties organise themselves using technologies such as AI. A key char acteristic of these technologies seems to be autonomy, i.e., the ability to act without direct human input. This, combined with higher levels ability to detect patterns and act accordingly, can lead to the replace ment of humans by machines. Another key feature of digital technolo gies with high ethical relevance is that they increasingly constitute the environment in which humans live. The malleability of digital tech nologies means that our realities can easily be changed. Maybe even more importantly, the constitutive element of digital technologies in modern social reality means that the owners and controllers of these technologies become immensely powerful in many different ways. l The following figure aims to summarise these three types of ethical issues of AI (See Fig. 7). ii The classification suggested in the above figure serves as a starting point to explore how governance structures are positioned to address ethical issues. 4.1.1. Specific issues of machine learning From the perspective of this paper, an inter esting feature is that these issues can become visible and are – at least to some degree – capable of being addressed on the project or organisa tional level. While these ethical issues represent some of those most frequently mentioned in our empirical research, it is important to see that many of the ethical issues do not seem to be linked to the technical properties of AI as machine learning but point to the broader socio- economic context in which these technologies are used. 4.1.3. Metaphysical questions i The final set of questions concerns what machines should be allowed to do and points to some of the deeper philosophical and metaphysical questions about the future of AI and autonomous machines and their relationship with humans. There has been a long-standing discussion about the change of human nature due to machines, the emergence of cyborgs (Latimer, 2017) and transhumans (Livingstone, 2015). In par allel, there has been discussion whether machines can ever become sentient or conscious (Carter et al., 2018; Dehaene, Lau, & Kouider, 2017), whether there will be a singularity (Kurzweil, 2006) at which point machines will develop superintelligence (Bostrom, 2016). These developments are controversially discussed and highly contentious. They are based on the idea of general AI (Baum, 2017). In this paper, we do not take a position on whether these developments are likely or even possible. We also refrain from taking a position on whether current narrow AI can lead to general AI or whether a funda mentally different approach would be needed. In our case studies, we found no evidence of their being considered a current priority, but in our scenarios, there are examples of technologies that come close to this category. In the Delphi study, there was reference to ’awakening of AI’, which falls into this category. The reason for including them here is thus less their current practical relevance and more the fact that they are prominent and highly visible in science fiction, the media and increas ingly in policy discussions – which, of course, in no way reduces the legitimacy of the concerns they raise. i B.C. Stahl et al. the case of Cambridge Analytica (Isaak & Hanna, 2018) is the most visible case in point. But while Cambridge Analytica represents a rela tively clear-cut case of misuse of AI and big data, there are broader questions about the economic and resulting political power amassed by the tech industry (Macnish & Galliott, 2020). The market capitalisation and therefore economic power of the big tech companies is now such that many observers are increasingly worried about the mere possibility of oversight of these actors. One resulting question is that of justice of distribution of costs and benefits. Big tech companies have the technical infrastructure and know-how to create ever-larger data sets and benefit from these whereas smaller competitors lack the means to catch up. The role of consumers and end users at present is predominantly passive; they produce data and consume services, but have little control over the use of their data. In the course of our research and engagement with the ethics of AI, it emerged that there are types of issues that can be clustered in a way conducive to finding appropriate responses. We propose three types of issues that have significant specificities to allow them to be clustered: specific issues of machine learning, general questions about living in a digital world and metaphysical questions. 4.1. Classification of ethical issues This paper has so far confirmed that there are many potential ethical issues related to AI. We have furthermore shown that there are numerous governance approaches, including human rights legislation, that can address many of these issues. An ongoing problem, however, is the complexity of the landscape, the fact that there are too many ethical issues and ways of addressing them to allow scholars or practitioners to keep an overview. Human rights touch on or are directly part of many of the issues related to AI. Our analysis shows the breadth of human rights concerns using the European human rights framework. It seems plausible that a more direct application of human rights legislation to AI can provide some clarity on related issues and point the way to possible solutions. It is therefore not surprising that there are many voices that point to the Fig. 6. Concepts and notions employed for human rights analysis of AI. Fig. 6. Concepts and notions employed for human rights analysis of AI. 382 Journal of Business Research 124 (2021) 374–388 B.C. Stahl et al. 4.2. Governing the three types of issues The purpose of classifying ethical issues in the previous section was to render the broad array of ethical issues more manageable and impose some order on the chaos of AI ethics. In this section, we now look at how this can help identify suitable governance structures. Before we start Key examples of these issues are the influence of technology on economic and political power, the future of warfare or distribution of costs and benefits of AI. The high-profile example of the misuse of social media data for purposes of the manipulation of democratic elections in 383 B.C. Stahl et al. Journal of Business Research 124 (2021) 374–388 Journal of Business Research 124 (20 Fig. 7. Classification of ethical issues of AI. tahl et al. Fig. 7. Classification of ethical issues of AI. issues, general questions, metaphysical questions). this, we should make it clear that we understand that the above classi fication is analytic in nature and represents one possible way of thinking about AI ethics among many. We hope that the distinction makes sense and is plausible, but we are happy to concede that it is not exclusive. Privacy, to take an example, materialises on the local and project level, but is seen as a societal question (Roessler & Mokrosinska, 2015), sub ject to legislation and regulation and arguably based on human needs arising from human nature (Locke, 2010). With this contingent nature of the classification in mind, we now look at how this translates to governance mechanisms. , g q , p y q ) In many cases, the issues that we classified as specific arising from machine learning appear easiest to address. They can be subject to the application of existing governance mechanisms. Data protection or se curity issues provide a good example. Data protection is furthermore governed by legislation and regulation, even though the details of these legislative requirements differ significantly between jurisdictions. Not only is there ample regulation, there are also structures such as data protection impact assessments (CNIL, 2017a, 2017b) or standards such as the ISO 27000 family that allow structured approaches. Similar ob servations apply to other examples of the specific issues arising from machine learning, such as algorithmic biases and subsequent discrimi nation. Unlike data protection, these are less comprehensively defined and regulated. A key problem here is that it may be difficult to under stand what exactly the issue is. 4.2. Governing the three types of issues There are several high-profile examples of these problems, e.g., where a system evaluating job applications leads to discrimination on the basis of gender or where predictive policing or parole decisions lead to discrimination on the basis of race. There is broad consensus that discrimination on the basis of gender or race is immoral and to be avoided. However, it is not clear whether other systems discriminate on the basis of other properties that are less high- profile and of which individuals are unaware. It is conceivable that a medical diagnostic system might lead to discrimination on the basis of blood type or a dating system would prefer individuals on the basis of their height. This raises questions about how we would even know how to check for relevant characteristics and, even if confirmed, whether and why this would constitute an ethical issue. In our discussion of governance, we take the position that the desirable purpose of AI should be to support human flourishing. As discussed earlier, this does not preclude uses of AI for efficiency max imisation or control but implies that the overall aim of development and deployment of the technology should be to improve human lives. This position has the disadvantage of muddying the waters, raising difficult questions of what counts as flourishing and who determines this. It is also likely to require the balancing of competing goods, values and in terests. But it is arguably the purpose of AI research, development, funding and application on which most citizens in democratic states, and maybe most human beings, can agree. It is also aligned with the EU’s AI policies. Based on an agreement that AI is to support human flourishing, the question then is how ethical issues can be addressed and which governance structures can help us deal with them. i To return to the suggested classification of ethical issues (Fig. 6) and compare this with the earlier description of governance proposals (Fig. 2), it is easy to see that there is no simple and/or linear relation ship. The two sets of classification do not align, but can be better un derstood as a matrix, where all levels of governance (individual, organisational, policy) can refer to all types of ethical issues (specific These issues can be described in terms of transparency and explainability. 4.2. Governing the three types of issues At the heart of the problem is the fact that humans expect explanations that they can understand, whereas machine learning 384 B.C. Stahl et al. Journal of Business Research 124 (2021) 374–388 algorithms classify individual cases on the basis of complex statistical calculations that defy simple translations into forms that are accessible to humans (Mittelstadt et al., 2019). religious characteristics [overcoming the body, the distinction between (mortal) body and (immortal) essence of the human], they may be most suitably dealt with by theology. While it may be difficult to deal these issues, they did not receive much attention in our empirical findings. But that does not mean that proactive attention to these issues would be misplaced or that it might not be advisable to think about early warning signs or trigger points where more attention should be paid to such issues. This tension between human reasoning and machine-learning data processing is a reason why transparency is among the most visible ethical issue linked to AI. The prominence of transparency as a perceived issue may explain why the same term is used to denote an ethical principle that pervades the AI ethics guidelines (Jobin, Ienca, & Vayena, 2019). The ethical principle of transparency is described as a way to minimise harm, improve AI, foster trust and improve AI (ibid). It is also linked to dialogue, participation and democracy. Elsewhere we have tried to unpack the normative implications of ethical principles, including the principle of transparency (Ryan & Stahl, 2020). Of course, while transparency may be perceived as obligatory, it does not in itself resolve the ethical or privacy risks at issue. Transparency only gets us part of the way to solutions. Insights from our case studies suggest that this way of looking at ethical issues and governance clarifies current activities. This paper does not provide the space for a comprehensive analysis, so suffice it to say that the organisations in our case studies focused on the issues arising from machine learning and on established and practical ways of addressing them. The dominant topics were security and data protec tion, which companies dealt with in ways to ensure they met their legal responsibilities. They focused on technical approaches to achieve these. In addition, companies developed oversight mechanisms and reflective capabilities, e.g., by instituting ethics boards or engaging with stake holders. 5. Conclusion In this paper, we have reported some findings and their implications from a multi-dimensional research study into the ethics of AI. Our starting point was the highly complex and often overwhelming AI ethics discourse that motivated our attempt to bring better empirical insights but also conceptual clarity to the discussion. i The second set of issues, those relating to general questions about living in a digital world, are not capable of being dealt with on the micro or meso level of the individual or organisation. This does not mean that there are no existing governance mechanisms that could be applied to AI. Nation states can make use of policy, legislative and regulatory op tions and this is happening at large scale with regard to AI. At the same time, there are many existing structures that may well be suited to dealing with at least some of the challenges related to AI, even if such structures can be criticised as having failed to deal adequately with the ethical, privacy and societal issues. Consumer protection legislation, competition law, anti-trust law, data protection law as well as human rights legislation may well be capable of regulating AI and its conse quences, at least in part. Similarly, existing regulators may make a claim that they can address the challenges of AI. This suggests a close affinity between this type of ethical issue and the policy level of governance options. The key to the resolution of these issues does indeed seem to be on a policy level, as they touch on constitutive questions of modern societies (e.g., who can own what, how do we distribute wealth and risks, the asymmetries in the power wielded by the big tech companies through their algorithms). Ethical issues of AI may be subject to existing regulation, but it is entirely possible that additional regulation and legislation will be required to facilitate human flourishing. Nevertheless, the policy level needs to be supplemented by actions on the organisa tional and individual levels. i One outcome of this work was the classification of ethical issues into specific issues arising from machine learning, general questions about living in a digital world and metaphysical questions. We hope that this classification is helpful in identifying issues and finding or developing appropriate governance mechanisms. 4.2. Governing the three types of issues Respondents were aware of the policy issues but not actively engaged with them. Similarly, as noted, the metaphysical issues did not play a role in the social reality of the case studies. The example of transparency indicates that the ethical issues arising from AI are not clearly defined. The perception that something consti tutes an ethical issue does not by itself clarify whether this perception is accurate or on which grounds such an evaluation would be possible. It says little about the exact nature of the issue and its status from a theoretical ethical perspective. One interesting point to observe is that in our case study research, there was little reference even to most of the governance mechanisms that we categorised as aimed at the organisational level. Activities like risk management, impact assessments or human rights integration into company policies were not mentioned by our respondents. This does not mean that the companies did not engage in them or that they were unsuccessful, but that in the responses to our questions, the respondents did not associate them with ethics of AI or big data. It will therefore be important for future studies to evaluate the effectiveness of these governance approaches. In the case of transparency, there is now a quickly growing research community that focuses on such questions of transparency and explainability of AI (USACM, 2017). While these questions remain open, there is at least reason to hope that they can be addressed on the local level by designing, testing and reviewing systems using appropriate methods, relying on professional expertise of developers and users and organisational capacity to develop the relevant expertise. The ISO is developing guidance for developers (e.g., TR 24368). Discussion within ISO has recognised that AI is not neutral, that values embedded in al gorithms are intentionally or inadvertently shaped by the pro grammers’, operators’, and third-parties’ own worldviews and cognitive biases and that ethical violations could also result from AI deployed or developed prematurely, applied without proper consideration of the ways it could negatively impact individuals or society. References Access Now Policy Team. (2018). The Toronto Declaration: Protecting the right to equality and non-discrimination in machine learning systems. Access No. https:// www.accessnow.org/cms/assets/uploads/2018/08/The-Toronto-Declaration_ENG_ 08-2018.pdf. Access Now Policy Team. (2018). The Toronto Declaration: Protecting the right to equality and non-discrimination in machine learning systems. Access No. https:// www.accessnow.org/cms/assets/uploads/2018/08/The-Toronto-Declaration_ENG_ 08-2018.pdf. Adam, A. (2001). Computer ethics in a different voice. Information and Organization, 11 (4), 235–261. The nature of integrating human rights into organisations and the creation of policy provides another pointer to lessons learned from our work. The way we deal with the ethics of AI will need to be sensitive to the conceptually challenging and changing nature of the technologies in question and the social perceptions they engender. We should simply not assume that we can provide a permanently stable definition of AI or of the ethical issues related to it. Instead, we need to embrace a world where concepts are changing and contested, where moral preferences change over time, where scientific, media and political discourses dynamically interact and where impacts of new technologies such as AI need to be adequately assessed. Ethical positions based on process and exchange, such as discourse ethics (Mingers & Walsham, 2010; Rehg, 2014), are well suited to reflect the need for the ongoing negotiations of facts and values needed to make new technologies work in society. ALLEA & Royal Society. (2019). Flourishing in a Data-enabled Society (ALLEA Discussion Paper No. 4). https://www.allea.org/wp-content/uploads/2019/06/ DiscussionPaper_DataGov_Digital.pdf. Anabo, I. F., Elexpuru-Albizuri, I., & Villard´on-Gallego, L. (2019). Revisiting the Belmont Report’s ethical principles in internet-mediated research: Perspectives from disciplinary associations in the social sciences. 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Future of Life Institute. https:// futureoflife.org/ai-principles/. i Our work immediately suggests further areas of study. 5. Conclusion This new perspective is based on a novel catego risation of ethical issues that point towards next steps and the bigger question about how the different types of issues can be addressed, which can be used to generate recommendations for organisations. Many companies are interested in exploiting the advantages of AI but are unsure about how to deal with societal and ethical issues. Our research shows that there are numerous existing activities, many of which are related and interlinked, that will go a long way towards showing that a company is serious about engaging with these questions. The integration of CSR structures into organisations in a way that takes into account the organisation’s research and development is one such step. An explicit commitment to human rights and the adoption of processes designed to integrate human rights into corporate processes would be another such step. These suggestions are, of course, no guarantees that nothing will go wrong, but they are serious steps in ensuring that the corporate culture supports human flourishing, through AI and otherwise. most suitable ways of moving on. It also shows that AI ethics is not a problem to be ‘solved’ in the sense that there are clear solutions that will make problems go away. Instead, uncertainties around definitions, ethics and values can form the basis for a creative but unpredictable journey towards a desirable future where new technologies including AI are conducive to human flourishing. l This paper provides a clearer, more structured and inclusive narra tive of ethics of AI. It draws on a set of different research activities to develop a way of thinking about AI and ethics that covers the broad range of technologies that fall under the heading of AI, that covers the manifold ethical issues associated with AI and that provides a theoretical ethical position that can be used to reflect on all of these. It is clear, however, that this is only the basis for further work. The difficult questions about what stakeholders need to do, about which laws and regulations need to be updated or developed, about definitions of pro fessional or organisational responsibilities etc. still need to be addressed. Undertaking these next steps is important and urgent. It will also be greatly helped by an empirically based conceptual view of ethics and AI as we offer in this paper. 5. Conclusion Our discussion and mapping of existing proposals for AI governance to the three classes of ethical issues give rise to suggestions for further research as well as organisational practice. One important observation refers to the concept of AI and the implications of the use of a particular concept for subsequent insights. In this study, we started with an in clusive view of AI that covers not only narrow AI and machine learning, but also broader socio-technical systems incorporating AI techniques and artificial general intelligence. Our empirical findings show that the focus of attention in current use of AI is on machine learning and, to some degree, on broader socio-technical systems. This raises the ques tion whether it would be desirable to limit the scope of reflection, e.g., by focusing exclusively on machine learning. We suggest that the term AI is broader than machine learning and a rich conceptualisation of AI and its ethical consequences needs to take this into consideration. This paper has shown that a key next step in the AI ethics discourse needs to be a more detailed and thorough mapping exercise that not only lists and clearly defines ethical issues but explores to what degree these are novel or manifest inadequate regulatory structures and in need of novel solutions. As we have suggested above, the broad array of extant governance structures may be able to cover many of the issues raised by AI. Whether they do so adequately is another question. Where no rem edies currently exist, or existing ones are insufficient, further and novel The most difficult area to govern is the metaphysical. Questions of general AI and its consequences are partly technical and empirical in terms of what technology can achieve in the hands of the big tech companies, intelligence agencies and cyber attackers (for example). They are also partly philosophical insofar as they refer to basic concepts and arguments. To some extent, e.g., where transhumanism displays 385 Journal of Business Research 124 (2021) 374–388 B.C. Stahl et al. solutions may well be required. The work presented here suggests that the AI ethics debate can benefit from a different perspective, which we have offered above. References The case studies, for example, while covering a breadth of activities, could be expanded to be even more comprehensive. Our approach was open and exploratory. Future empirical work could also test the adequacy of existing governance mechanisms as well as proposals for new ones, their impacts and possible side effects (e.g., a loss of social trust in existing mechanisms). A direct mapping of ethical issues and governance pro posals would be helpful in assessing the effectiveness of the governance proposals, an important factor in developing these further. Further research should also be undertaken to broaden the geographical scope of the study. Our European focus means that we simply took for granted certain aspects of the AI ecosystem that may not be applicable else where. This includes the strong legal data protection regime of the Eu ropean Union or the existing regulatory, industrial and professional settings that shape the way in which AI is developed, deployed and used. The international nature of AI renders necessary such further work. l Baum, S. (2017). A Survey of Artificial General Intelligence Projects for Ethics, Risk, and Policy (SSRN Scholarly Paper ID 3070741). Social Science Research Network. https:// papers.ssrn.com/abstract=3070741. Beard, M., & Longstaff, S. (2018). Ethical By Design: Principles For Good Technology. THE ETHICS CENTRE. Beauchamp, T. L., & Childress, J. F. (2009). Principles of Biomedical Ethics (6th ed.). OUP USA. Becker, H. A., & Vanclay, F. (2003). The International Handbook of Social Impact Assessment: Conceptual and Methodological Advances. Edward Elgar Publishing. Bevir, M. (2012). Governance: A Very Short Introduction. OUP Oxford. Boden, M., Bryson, J., Caldwell, D., Dautenhahn, K., Edwards, L., Kember, S., … Winfield, A. (2017). Principles of robotics: Regulating robots in the real world. Connection Science, 29(2), 124–129. https://doi.org/10.1080/ 09540091.2016.1271400. Boenink, M., Swierstra, T., & Stemerding, D. (2010). Anticipating the Interaction between Technology and Morality: A Scenario Study of Experimenting with Humans in Bionanotechnology. Studies in Ethics, Law, and Technology, 4(2). https://doi.org/ 10.2202/1941-6008.1098. Bostrom, N. (2016). Superintelligence: Paths. Dangers, Strategies (Reprint edition): OUP Oxford. Braithwaite, J., & Drahos, P. (2000). Global Business Regulation. Cambridge University Press. The high-level view of the AI ethics debate presented in this paper should make an important contribution to theory and practice. We believe that our proposed categorisation is helpful for both scholars and practitioners. 5. Conclusion l A similar conclusion offers itself to policymakers and decision- makers. Just as companies can look through a portfolio of existing governance mechanisms, policymakers should take stock of the ade quacy of existing policy and regulatory options while developing spe cific steps to address AI. Some of the issues discussed in the context of AI and the general questions arising in our increasingly digital world have little to do with AI in the narrow sense. Questions of justice, distribution and power may be exacerbated by particular technologies but exist in dependent of them. As a consequence, it may well be that existing legislation or regulatory bodies are in a good position to deal with at least some of these questions and the task for policymakers is to ensure that existing policy and the adequacy of its application are taken into account in the rush to develop new policy. 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Ethics and Information Technology. https://doi.org/10.1007/s10676-010- 9234-6. Alexey Kirichenko is research collaboration manager at F-Secure, coordinating F-Secure’s participation in European and Finnish national research projects. He also represents the company in the Steering Board of WG6 (SRIA) of European Cyber Security Organisation (ECSO). His interests are mainly in applications of Machine Learning to cybersecurity. Solove, D. J. (2002). Conceptualizing Privacy. California Law Review, 90(4), 1087–1156. Sparrow, R. (2009). Predators or plowshares?: Arms control of robotic weapons. IEEE Technology and Society Magazine, 28(1). https://doi.org/10.1109/MTS.2009.931862. Kevin Macnish is Assistant Professor in Ethics and Technology at the University of Twente. His interests include ethical questions regarding privacy, surveillance, security, and AI. Stahl, B. C., & Wright, D. (2018). Ethics and Privacy in AI and Big Data: Implementing Responsible Research and Innovation. IEEE Security & Privacy, 16(3), 26–33. https:// doi.org/10.1109/MSP.2018.2701164. l g Stahl, Bernd Carsten (2012). Morality, Ethics, and Reflection: A Categorization of Normative IS Research. Journal of the Association for Information Systems, 13(8), 636–656. https://doi.org/10.17705/1jais.00304. Andrew Patel is a researcher in F-Secure Corporation’s Artificial Intelligence Center of Excellence. His main research areas include natural language processing, disinformation hunting and analysis, graph analysis and reinforcement learning. References His interests cover philosophical issues arising from the intersections of business, tech nology, and information. This includes ethical questions of current and emerging of ICTs, critical approaches to information systems and issues related to responsible research and innovation. Morley, J., Floridi, L., Kinsey, L., & Elhalal, A. (2019). From What to How- An Overview of AI Ethics Tools. ArXive: Methods and Research to Translate Principles into Practices. https://arxiv.org/abs/1905.06876. Nerurkar, M., Wadephul, C., & Wiegerling, K. (2016). Ethics of Big Data: Introduction. International Review of Information. Ethics, 24. Andreas Andreou works as a Researcher and Project Manager at the human rights non governmental organisation Aequitas which is based in Cyprus. He holds a BA Humanities from the University of Essex and an LLM Master of Laws from the University of Central Lancashire. He has a research interest and experience in artificial intelligence and human rights, LGBTQI rights and hate speech/hate crime. He published academic articles and a research book. OECD. (2017). Recommendation of the OECD Council on Health Data Governance. http://www.oecd.org/health/health-systems/Recommendation-of-OECD-Council- on-Health-Data-Governance-Booklet.pdf. Patel, A., Hatzakis, T., Macnish, K., Ryan, M., & Kirichenko, A. (2019). SHERPA Deliverable D1.3: Security Issues, Dangers and Implications of Smart Information Systems. SHERPA project. https://dmu.figshare.com/articles/D1_3_Cyberthreats_ and_countermeasures/7951292. Ramirez, R., Mukherjee, M., Vezzoli, S., & Kramer, A. M. (2015). Scenarios as a scholarly methodology to produce “interesting research”. Futures, 71, 70–87. https://doi.org/ 10.1016/j.futures.2015.06.006. Philip Brey (PhD, University of California, San Diego, 1995) is professor of philosophy of technology at the Department of Philosophy, University of Twente, the Netherlands. He is on the editorial board of eleven leading journals and book series in his field, including Ethics and Information Technology, Nanoethics, Philosophy and Technology, Techn´e, Studies in Ethics, Law and Technology and Theoria. He is also former president of the International Society for Ethics and Information Technology (INSEIT), and former presi dent of the Society for Philosophy and Technology (SPT). His research focuses on ethics and philosophy of emerging technologies, in particular digital technologies, AI, robotics, biomedical technology and sustainable technology. He currently coordinates the EU H2020 SIENNA project on the ethical and human rights dimensions of emerging tech nologies, and the NWO-funded 10-year research programme Ethics of Socially Disruptive Technologies. j Rehg, W. (2014). Discourse ethics for computer ethics: A heuristic for engaged dialogical reflection. Ethics and Information Technology, 17(1), 27–39. https://doi.org/10.1007/ s10676-014-9359-0. Roessler, B., & Mokrosinska, D. (Eds.). (2015). Social Dimensions of Privacy: Interdisciplinary Perspectives. Cambridge University Press. References He is also a frequent contributor to F-Secure’s blog. Stahl, Bernd Carsten, & Coeckelbergh, M. (2016). Ethics of healthcare robotics: Towards responsible research and innovation. Robotics and Autonomous Systems. https://doi. org/10.1016/j.robot.2016.08.018. Mark Ryan works as a Digital Ethics Researcher at Wageningen Economic Research, Wageningen University & Research. His interests are around topics in the ethics of tech nology, with a particular focus on ethical issues pertaining to the use of artificial intelli gence and Big Data. He has published on topics, such as the ethics of smart cities, self- driving vehicles, agricultural data analytics, social robotics, and trusting AI. g j Tavani, H. (2008). Informational Privacy: Concepts, Theorie and Controversies. In J. V. D. Hoven, & J. Weckert (Eds.), Information Technology and Moral Philosophy (pp. 131–164). Cambridge University Press. g y Taylor, L. (2016). The ethics of big data as a public good: Which public? Whose good? Phil. Trans. R. Soc. A, 374(2083), 20160126. https://doi.org/10.1098/ rsta.2016.0126. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. (1979). The Belmont Report—Ethical Principles and Guidelines for the Protection of Human Subjects of Research. Department of Health, Education, and Welfare. http://videocast.nih.gov/pdf/ohrp_belmont_report.pdf. David Wright is Director of Trilateral Research (London and Waterford), a company he founded in 2004. The company has partnered in more than 60 EU-funded projects. He has published four books on ambient intelligence, privacy and surveillance and more than 60 articles in peer-reviewed journals. He coined the term ethical impact assessment and published the first article on EIA. The ISO privacy impact assessment standard is based on the methodology he developed as is the CEN Workshop Agreement on ethical impact assessment. His interests include scenario construction, horizon scanning, impact assess ments, cybersecurity, artificial intelligence, privacy, ethics and surveillance. USACM. (2017). Statement on Algorithmic Transparency and Accountability. ACM US Public Policy Council. van Est, R., & Gerritsen, J. (2017). Human rights in the robot age—Challenges arising from the use of robotics, artificial intelligence, and virtual and augmented reality [Report to the Parliamentary Assembly of the Council of Europe (PACE)]. Rathenau Instituut. https://www.rathenau.nl/en/file/9605/download?token=OQgFllZS. 388
https://openalex.org/W2244979610	https://journals.openedition.org/belgeo/pdf/8184	English	null	J. Robinson, Ordinary cities : Between Modernity and Development.	BELGEO	2,009	cc-by	1,631	J. Robinson, Ordinary cities : Between Modernity and Development. London, Routledge, 2006, xiv + 204 p. J. Robinson, Ordinary cities : Between Modernity and Development. London, Routledge, 2006, xiv + 204 p. J. Robinson, Ordinary cities : Between Modernity and Development. London, Routledge, 2006, xiv + 204 p. Nick Schuermans Publisher: Publisher: National Committee of Geography of Belgium, Société Royale Belge de Géographie Nick Schuermans Nick Schuermans J. Robinson, Ordinary cities : Between Modernity and Development. J. Robinson, Ordinary cities : Between Modernity and Development. London, Routledge, 2006, xiv + 204 p. Nick Schuermans J. Robinson, Ordinary cities : Between Modernity and Development. 1 1 Electronic reference Nick Schuermans, « J. Robinson, Ordinary cities : Between Modernity and Development. », Belgeo [Online], 1 \| 2009, Online since 19 May 2013, connection on 22 September 2020. URL : http:// journals.openedition.org/belgeo/8184 ; DOI : https://doi.org/10.4000/belgeo.8184 This text was automatically generated on 22 September 2020. Belgeo est mis à disposition selon les termes de la licence Creative Commons Attribution 4.0 International. REFERENCES The second chapter deepens this critique by juxtaposing the accounts of the Chicago School with mid-twentieth-century studies on comparative urbanism in the Zambian Copper Belt. While Park and Wirth described the big cities of America as sites of alienation, individualism and indifference, members of the Manchester School considered the industrialised and multicultural cities of the Copper Belt to be places full of interaction associated with urban modernity through cultural practices that had previously been considered outside the realms of urban life. In the third chapter, Robinson further seeks to shatter the conventional illusions of modernity by arguing that so-called Western modernities are almost always hybridisations and that urban innovation is generally a result of cosmopolitan interdependence. She illustrates this tension between discourse and reality with the fact that critics look to New York as the trendsetting city in the field of urban architecture, while many so-called modern innovations actually originated in Renaissance Italy, Aztec Mexico or Rio de Janeiro. been perceived as primitivity is an essential part of urban life all over the world. The second chapter deepens this critique by juxtaposing the accounts of the Chicago School with mid-twentieth-century studies on comparative urbanism in the Zambian Copper Belt. While Park and Wirth described the big cities of America as sites of alienation, individualism and indifference, members of the Manchester School considered the industrialised and multicultural cities of the Copper Belt to be places full of interaction associated with urban modernity through cultural practices that had previously been considered outside the realms of urban life. In the third chapter, Robinson further seeks to shatter the conventional illusions of modernity by arguing that so-called Western modernities are almost always hybridisations and that urban innovation is generally a result of cosmopolitan interdependence. She illustrates this tension between discourse and reality with the fact that critics look to New York as the trendsetting city in the field of urban architecture, while many so-called modern innovations actually originated in Renaissance Italy, Aztec Mexico or Rio de Janeiro. In the remainder of her book, Robinson examines the implications of her critical rethinking of the idea of modernity for academic theorization and policy development. First, Robinson criticises the world cities literature for putting the emphasis on a relative small sector of the global economy and for dropping most cities in the world from its vision. REFERENCES J. Robinson, Ordinary cities : Between Modernity and Development, London, Routledge, 2006, xiv + 204 p. J. Robinson, Ordinary cities : Between Modernity and Development, London, Routledge, 2006, xiv + 204 p. 1 Jennifer Robinson’s “Ordinary Cities” delivers a powerful critique of the spatial division of academic theorization. Her central thesis is that urban theory development has been hampered for too long by the assumed dichotomy between innovative “global cities” in rich countries and imitative “third world” cities in poor countries. It is Robinson’s contention that theoretical insights cannot be based on the experiences of a few wealthy cities only, and that a post-colonial field of urban studies should assume the potential for learning in a broad range of different settings. For this reason, she envisages an urban theory that does not rest on pre-given categories of cities but on a cosmopolitan comparativism that places all cities within the same analytical field. Within this field, the differences across and within cities must be thought of as diversity rather than exemplars of a hierarchical division. In order to learn from different contexts, Robinson argues, it is not global cities or third world cities that should be central to academic analysis and policy recommendations, but what she calls “ordinary” cities, in all their complexity, diversity and peculiarity. 2 To substantiate this claim, Robinson begins with a critical rethinking of the concepts of modernity and development in urban studies. In the first chapter, she challenges Park’s and Wirth’s parochial and ethnocentric understandings of the Western city as the cradle of civilization and modernity as opposed to the primitivity and traditionality of the countryside and cities in other countries. Drawing on Walter Benjamin’s work, she shows that modernity and tradition are mutually interdependent and that what has 2 To substantiate this claim, Robinson begins with a critical rethinking of the concepts of modernity and development in urban studies. In the first chapter, she challenges Park’s and Wirth’s parochial and ethnocentric understandings of the Western city as the cradle of civilization and modernity as opposed to the primitivity and traditionality of the countryside and cities in other countries. Drawing on Walter Benjamin’s work, she shows that modernity and tradition are mutually interdependent and that what has Belgeo, 1 \| 2009 J. Robinson, Ordinary cities : Between Modernity and Development. 2 been perceived as primitivity is an essential part of urban life all over the world. REFERENCES Because of the focus on advanced business and producer services, cities like Lusaka or Kuala Lumpur largely fall off the world cities map, despite the fact that they are tied to the rest of the world through a wide range of economic activities such as the trade in second hand clothing or Islamic forms of global activity. As the world cities literature reproduces hierarchical relations amongst cities where some urban places are defined as modern and others as in need of development, it is not only a problematic framework for theorization on cities, however, but also for policy development. Robinson elaborates this argument in the fifth chapter through a detailed analysis of the Johannesburg 2030 vision for the city. In order to secure economic growth and improved service delivery in Johannesburg, Robinson claims that a “city development strategy” has to start from a city-wide view of urban features that takes the diversity of needs and activities in poor and wealthy parts of the city seriously. For this reason, Robinson calls, in the last chapter, for theoretical repertoires that are appreciative of the diversity of cities. These have to focus on the close intertwining of social welfare and economic activities in both poor and rich cities by acknowledging that all cities are assembling and inventing diverse ways of being modern. In the post-colonial urbanism Robinson sets out in Ordinary Cities, a cosmopolitan and comparative theoretical endeavour will enrich the divided form of urban studies. This means that policy makers and academics in Western cities have to question their understandings of cultural and economic aspects of city life by revisiting them through the lens of poor cities, and vice versa. This idea does not imply, however, that well- resourced scholars should start globe-trotting to study cities around the world. Robinson pleads, on the contrary, for the kind of armchair comparativism that forces scholars to think comparatively. In her own words (p. 168), she suggests “that any research on cities needs to be undertaken in a spirit of attentiveness to the possibility that cities elsewhere might perhaps be different and shed stronger light on the processes being studied. The potential to learn from other contexts, other cities, would need to always be kept open and hopefully acted upon”. Belgeo, 1 \| 2009 J. Robinson, Ordinary cities : Between Modernity and Development. REFERENCES 3 While Robinson’s provocative thesis definitely breaks down the binary thinking that has shaped the way in which cities have been classified and studied, it is surprising that all her attention goes to the deconstruction of the dichotomy between the West and the rest. By focusing her effort on the hierarchical categorisation of all cities as developed or undeveloped, Robinson implicitly reproduces the marginal position that non-English and non-American Western geographers take up in the international production of urban theory. Belgian geographers, for example, clearly belong to the side of the West in Robinson’s analysis. Nevertheless, it is one of their frustrations that theorizations about Belgian cities will never be taken seriously by the urban studies academics in the United States or Britain. Very rarely, a scholar from the Anglo-Saxon heartland would be expected to cite a Belgian case study for the sake of the originality of the theory, and not just to embellish his list of references with a publication from an exotic country imitating and confirming the theories produced in London, Los Angeles or New York. In addition, it must be noted that the examples Robinson elaborates, originate largely in big cities. The silencing of smaller cities, towns, villages and other settlement forms is problematic because it seems to reinforce the modernist notion criticised in the first chapter of the book that innovations take place in cities and that other areas are, by definition, traditional, primitive and undynamic. By breaking down the binary between urban and rural geographies, and by bridging the divide between Anglo-Saxon and Continental urban theories, I believe it is possible to fully envisage the scope of Jennifer Robinson’s powerful and inspiring arguments. Belgeo, 1 \| 2009
https://openalex.org/W2887950972	https://www.mdpi.com/1099-4300/20/8/574/pdf	English	null	Investigating Information Geometry in Classical and Quantum Systems through Information Length	Entropy	2,018	cc-by	8,173	Received: 19 July 2018; Accepted: 1 August 2018; Published: 3 August 2018 Abstract: Stochastic processes are ubiquitous in nature and laboratories, and play a major role across traditional disciplinary boundaries. These stochastic processes are described by different variables and are thus very system-speciﬁc. In order to elucidate underlying principles governing different phenomena, it is extremely valuable to utilise a mathematical tool that is not speciﬁc to a particular system. We provide such a tool based on information geometry by quantifying the similarity and disparity between Probability Density Functions (PDFs) by a metric such that the distance between two PDFs increases with the disparity between them. Speciﬁcally, we invoke the information length L(t) to quantify information change associated with a time-dependent PDF that depends on time. L(t) is uniquely deﬁned as a function of time for a given initial condition. We demonstrate the utility of L(t) in understanding information change and attractor structure in classical and quantum systems. Keywords: stochastic processes; Langevin equation; Fokker–Planck equation; information length; Fisher information; relaxation; chaos; attractor; probability density function Entropy 2018, 20, 574; doi:10.3390/e20080574 Article Investigating Information Geometry in Classical and Quantum Systems through Information Length Eun-jin Kim ID School of Mathematics and Statistics, University of Shefﬁeld, Shefﬁeld S3 7RH, UK; e.kim@shefﬁeld.ac.uk or e.kim@shef.ac.uk; Tel.: +44-114-222-3876 Received: 19 July 2018; Accepted: 1 August 2018; Published: 3 August 2018 entropy entropy 2. Information Length Intuitively, we deﬁne the information length L by computing how quickly information changes in time and then measuring the clock time based on that time scale. Speciﬁcally, the time-scale of information change τ can be computed by the correlation time of a time-dependent PDF, say p(x, t), as follows. 1 τ2 = Z dx 1 p(x, t) ∂p(x, t) ∂t 2 . (1) (1) From Equation (1), we can see that the dimension of τ = τ(t) is time and serves as a dynamical time unit for information change. L(t) is the total information change between time 0 and t: From Equation (1), we can see that the dimension of τ = τ(t) is time and serves as a dynamical time unit for information change. L(t) is the total information change between time 0 and t: L(t) = Z t 0 dt1 τ(t1) = Z t 0 dt1 sZ dx 1 p(x, t1) ∂p(x, t1) ∂t1 2 . (2) (2) In principle, τ(t) in Equation (1) can depend on time, so we need the integral for L in Equation (2). To make an analogy, we can consider an oscillator with a period τ = 2 s. Then, within the clock time 10 s, there are ﬁve oscillations. When the period τ is changing with time, we need an integration of dt/τ over the time interval. In principle, τ(t) in Equation (1) can depend on time, so we need the integral for L in Equation (2). To make an analogy, we can consider an oscillator with a period τ = 2 s. Then, within the clock time 10 s, there are ﬁve oscillations. When the period τ is changing with time, we need an integration of dt/τ over the time interval. We now recall how τ(t) and L(t) in Equations (1) and (2) are related to the relative entropy (Kullback–Leibler divergence) [15,16]. 1. Introduction Stochastic processes are ubiquitous in nature and laboratories, and play a major role across traditional disciplinary boundaries. Due to the randomness associated with stochasticity, the evolution of these systems is not deterministic but instead probabilistic. Furthermore, these stochastic processes are described by different variables and are thus very system-speciﬁc. This system-speciﬁcity makes it impossible to make comparison among different processes. In order to understand universality or underlying principles governing different phenomena, it is extremely valuable to utilise a mathematical tool that is not speciﬁc to a particular system. This is especially indispensable given the diversity of stochastic processes and the growing amount of data. p g g Information geometry provides a powerful methodology to achieve this goal. Speciﬁcally, the similarity and disparity between Probability Density Functions (PDFs) is quantiﬁed by a metric [1] such that the distance between two PDFs increases with the disparity between them. This was the very idea behind a statistical distance [2] based on the Fisher (or Fisher–Rao) metric [3] which represents the total number of statistically different states between two PDFs in Hilbert space for quantum systems. The analysis in [2] was extended to impure (mixed-state) quantum systems using a density operator by [4]. Other related work includes [5–12]. For Gaussian PDFs, a statistically different state is attained when the physical distance exceeds the resolution set by the uncertainty (PDF width). This paper presents a method to deﬁne such distance for a PDF which changes continuously in time, as is often the case of non-equilibrium systems. Speciﬁcally, we invoke the information length L(t) according to the total number of statistically different states that a system evolves through in time. L(t) is uniquely deﬁned as a function of time for a given initial condition. We demonstrate Entropy 2018, 20, 574; doi:10.3390/e20080574 www.mdpi.com/journal/entropy Entropy 2018, 20, 574 2 of 11 the utility of L(t) in understanding information change and attractor structure in classical and quantum systems [13–21]. the utility of L(t) in understanding information change and attractor structure in classical and quantum systems [13–21]. This paper is structured as follows: Section 2 discusses information length and Section 3 investigates attractor structure. Sections 4 and 5 present the analysis of classical music and quantum systems, respectively. Conclusions are found in Section 6. 2. Information Length (9) (9) By summing dt(ti) for i = 0, 1, 2, ..., n −1 (where n = t/dt) in the limit dt →0, we have By summing dt(ti) for i = 0, 1, 2, ..., n −1 (where n = t/dt) in the limit dt →0, we have lim dt→0 n−1 ∑ i=0 dl(idt) = lim dt→0 n−1 ∑ i=0 q D[p(x, idt), p(x, (i + 1)] dt ∝ Z t 0 dt1 sZ dx(∂t1 p(x, t1))2 p(x, t1) = L(t), (10) where L(t) is the information length. Thus, L is related to the sum of inﬁnitesimal relative entropy. It cannot be overemphasised that L is a Lagrangian distance between PDFs at time 0 and t and sensitively depends on the particular path that a system passed through reaching the ﬁnal state. In contrast, the relative entropy D[p(x, 0), p(x, t)] depends only on PDFs at time 0 and t and thus does not tell us about intermediate states between initial and ﬁnal states. 3. Attractor Structure Since L(t) represents the accumulated change in information (due to the change in PDF) at time t, L(t) settles to a constant value L∞when a PDF reaches its ﬁnal equilibrium PDF. The smaller L∞, the smaller number of states that the initial PDF passes through to reach the ﬁnal equilibrium. Therefore, L∞provides us with a unique representation of a path-dependent, Lagrangian measure of the distance between a given initial and ﬁnal PDF. We will utilise this property to map out the attractor structure by considering a narrow initial PDF at a different peak position y0 and by measuring L∞ against y0. We are particularly interested in how the behaviour of L∞against y0 depends on whether a system has a stable equilibrium point or is chaotic. 2. Information Length We consider two nearby PDFs p1 = p(x, t1) and p2 = p(x, t2) at time t = t1 and t2 and the limit of a very small δt = t2 −t1 to do Taylor expansion of D[p1, p2] = R dx p2 ln (p2/p1) by using ∂ ∂t1 D[p1, p2] = − Z dx p2 ∂t1 p1 p1 , (3) ∂2 ∂t2 1 D[p1, p2] = Z dx p2 ( (∂t1 p1)2 p2 1 − ∂2 t1 p1 p1 ) , (4) ∂ ∂t2 D[p1, p2] = Z dx ∂t2 p2 + ∂t2 p2 ln p2 −ln p1 , (5) ∂2 ∂t2 2 D[p1, p2] = Z dx ∂2 t2 p2 + (∂t2 p2)2 p2 + ∂2 t2 p2 ln p2 −ln p1 . (6) (3) (4) (5) ∂t2 [p p ] Z 2 p 2 p p p ∂2 ∂t2 2 D[p1, p2] = Z dx ∂2 t2 p2 + (∂t2 p2)2 p2 + ∂2 t2 p2 ln p2 −ln p1 . (6) (6) it t2 →t1 = t (p2 →p1 = p), Equations (3)–(6) give us In the limit t2 →t1 = t (p2 →p1 = p), Equations (3)–(6) give us lim t2→t1 ∂ ∂t1 D[p1, p2] = lim t2→t1 ∂ ∂t2 D[p1, p2] = Z dx∂tp = 0, lim t2→t1 ∂2 ∂t2 1 D[p1, p2] = lim t2→t1 ∂2 ∂t2 2 D[p1, p2] = Z dx(∂tp)2 p = 1 τ2 . (7) (7) Up to O((dt)2) (dt = t2 −t1), Equation (7) and D(p1, p1) = 0 lead to Up to O((dt)2) (dt = t2 −t1), Equation (7) and D(p1, p1) = 0 lead to D[p1, p2] = 1 2 Z dx(∂tp(x, t))2 p(x, t) (dt)2, (8) D[p1, p2] = 1 2 Z dx(∂tp(x, t))2 p(x, t) (dt)2, (8) (8) 3 of 11 Entropy 2018, 20, 574 Entropy 2018, 20, 574 and thus the inﬁnitesimal distance dl(t1) between t1 and t1 + dt as and thus the inﬁnitesimal distance dl(t1) between t1 and t1 + dt as and thus the inﬁnitesimal distance dl(t1) between t1 and t1 + dt as dl(t1) = q D[p1, p2] = 1 √ 2 sZ dx(∂t1 p(x, t1))2 p(x, t1) dt. 3.1. Linear vs. Cubic Forces We ﬁrst consider the case where a system has a stable equilibrium point when there is no stochastic noise and investigate how L∞is affected by different deterministic forces [15,16]. We consider the following Langevin equation [22] for a variable x: dx dt = F(x) + ξ. (11) (11) Here, ξ is a short (delta) correlated stochastic noise with the strength D as Here, ξ is a short (delta) correlated stochastic noise with the strength D as ⟨ξ(t)ξ(t′)⟩= 2Dδ(t −t′), (12) (12) where the angular brackets denote the average over ξ and ⟨ξ⟩= 0. We consider two types of F, which both have a stable equilibrium point x = 0; the ﬁrst one is the linear force F = −γx (γ > 0 is the frictional constant) which is the familiar Ornstein–Uhlenbeck (O-U) process, a popular model for a noisy relaxation system (e.g., [23]). The second is the cubic force F = −µx3 where µ represents the frictional constant. Note that, in these models, the dimensions of γ (s−1) and µ (s−1m−2) are different. Equivalent to the Langevin equation governed by Equations (11) and (12) is the Fokker–Planck equation [22] ∂ ∂t p(x, t) = ∂ ∂x −F(x) + D ∂ ∂x p(x, t). (13) (13) As an initial PDF, we consider a Gaussian PDF As an initial PDF, we consider a Gaussian PDF p(x0, 0) = r β0 π e−β0(x0−y0)2. (14) (14) Entropy 2018, 20, 574 4 of 11 Then, for the O-U process, the PDF remains Gaussian for all time with the following form [15,16]: Then, for the O-U process, the PDF remains Gaussian for all time with the following form [15,16]: p(x, t) = r β(t) π e−β(t)(x−⟨x⟩)2. (15) (15) In Equations (14) and (15), ⟨x⟩= y0e−γt is the mean position and y0 is its initial value; β0 is the inverse temperature at t = 0, which is related to the variance at t = 0 as ⟨(x0 −y0)2⟩= 1 2β0 = D0 γ . The ﬂuctuations level (variance) changes with time, with time-dependent β(t) given by ⟨(x −⟨x⟩)2⟩= 1 2β(t) = D(1 −e−2γt) γ + e−2γt 2β0 . (16) (16) Note that, when D = D0, β(t) = β0 = γ 2D for all t, PDF maintains the same width for all t. 3.1. Linear vs. Cubic Forces Note that, when D = D0, β(t) = β0 = γ 2D for all t, PDF maintains the same width for all t. For this Gaussian process, β and ⟨x⟩constitute a parameter space on which the distance is deﬁned with the Fisher metric tensor [3] gij (i, j = 1, 2) as [16] Note that, when D D0, β(t) β0 2D for all t, PDF maintains the same width for all t. For this Gaussian process, β and ⟨x⟩constitute a parameter space on which the distance is deﬁned with the Fisher metric tensor [3] gij (i, j = 1, 2) as [16] gij = R dx 1 p(x,t) ∂p ∂zi ∂p ∂zj = 1 2β2 0 0 2β ! , (17) (17) where i, j = 1, 2, z1 = β, z2 = ⟨x⟩. This enables us to recast 1 τ2 in Equation (1) in terms of gij as = ⟨x⟩. This enables us to recast 1 τ2 in Equation (1) in terms of gij as 1 τ2 = 1 2β2 dβ dt 2 + 2β d⟨x⟩ dt 2 = gij dzi dt dzj dt . (18) (18) The derivation of the ﬁrst relation in Equation (18) is provided in Appendix A (see Equation (A2)). Using Equations (2) and (18), we can calculate L analytically for this O-U process (see also Appendix A). The derivation of the ﬁrst relation in Equation (18) is provided in Appendix A (see Equation (A2)). Using Equations (2) and (18), we can calculate L analytically for this O-U process (see also Appendix A). The derivation of the ﬁrst relation in Equation (18) is provided in Appendix A (see Equation (A2)). Using Equations (2) and (18), we can calculate L analytically for this O-U process (see also Appendix A). In comparison, theoretical analysis can be done only in limiting cases such as small and large times for the cubic process [17,24]. In particular, the stationary PDF for large time is readily obtained as In comparison, theoretical analysis can be done only in limiting cases such as small and large times for the cubic process [17,24]. In particular, the stationary PDF for large time is readily obtained as p(x) = 2β 1 4c Γ 1 4 e−βcx4, (19) (19) where βc = µ 4D. For the exact calculation of L(t), Equation (13) is to be solved numerically. where βc = µ 4D. 3.1. Linear vs. Cubic Forces For the exact calculation of L(t), Equation (13) is to be solved numerically. To summarise, due to the restoring forcing F, the equilibrium is given by a PDF around x = 0, Gaussian for linear force and quartic exponential for cubic force. If we were to pick any point in x, say y0, we are curious about how close y0 is to the equilibrium and how F(x) affects it. To determine this, we make a narrow PDF around x = y0 (see Figure 1) at t = 0 and measure L∞. The question is how this L∞depends on y0. We repeat the same procedure for the cubic process, as shown in Figure 1, and examine how L∞depends on y0. L∞as a function of y0 is shown for both linear (in red dotted line) and cubic (in blue solid line) processes in Figure 2. In the linear case we can see a clear linear relation between y0 and L∞, meaning that the information length preserves the linearity of the system. This linear relationship holds for all D and D0. In particular, when D = D0, we can show that L∞= 1 √ D/γy0 by taking the limit of t →∞ (y →0) in Equation (A10). In contrast, for the cubic process, the relation is not linear, and the log-log plot on the right in Figure 2 shows a power-law dependence with the power-law index p. This power-law index p varies between 1.52 and 1.91 and depends on the width (∝D1/2 0 ) of initial PDF and stochastic forcing amplitude D, as shown in [16]. This indicates that nonlinear force breaks the linear scaling of geometric structure and changes it to power-law scalings. In either cases here, L∞has a smooth variation with y0 with its minimum value at y0 = 0 since the equilibrium point 0 is stable. This will be compared with the behaviour in chaotic systems in Section 3.2. 5 of 11 Entropy 2018, 20, 574 -0.2 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 (a) -0.2 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 (b) t = t = 0 Figure 1. Initial (red) and ﬁnal (blue) Probability Density Functions (PDFs) for the O-U process in (a) and the cubic process in (b). 3.1. Linear vs. Cubic Forces 0.75 0 0.25 0.5 0.75 0 400 800 y0 0 −2 −1.5 −1 −0.5 0 0.5 1.5 2.5 log(y0) −2 −1.5 −1 −0.5 0 0 1 2 3 log(y0) -0.2 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 (b) t = t = 0 −2 −1 5 −1 −0 5 0 0 1 2 3 Figure 1. Initial (red) and ﬁnal (blue) Probability Density Functions (PDFs) for the O-U process in (a) and the cubic process in (b). y0 log(y0) log(y0) 0.75 0 0.25 0.5 0.75 0 1000 2000 3000 y0 0 −2 −1.5 −1 −0.5 0 0.5 1.5 2.5 log(y0) −2 −1.5 −1 −0.5 0 0.5 1.5 2.5 3.5 log(y0) L∞∝y0 p L∞∝y0 -1 -0.5 -1 -0.5 0 0 0.25 0.5 0.75 y0 log(y0) 3000 2000 1000 3.5 2.5 1.5 0.5 (a) (b) Figure 2. (a): L∞against ⟨x(t = 0)⟩= y0 for the linear process in red dashed line and for the cubic process in blue solid line; (b): L∞against ⟨x(t = 0)⟩= y0 for the cubic process on log-log scale (data from [17]). −2 −1.5 −1 −0.5 0 0.5 1.5 2.5 3.5 L∞∝y0 p -1 -0.5 -1 -0.5 0 log(y0) 3.5 2.5 1.5 0.5 (b) 5 0 0.25 0.5 0.75 0 1000 2000 3000 −2 −1.5 −1 −0.5 0 0.5 1.5 2.5 L∞∝y0 0 0.25 0.5 0.75 y0 3000 2000 1000 (a) y0 log(y0) log(y0) Figure 2. (a): L∞against ⟨x(t = 0)⟩= y0 for the linear process in red dashed line and for the cubic process in blue solid line; (b): L∞against ⟨x(t = 0)⟩= y0 for the cubic process on log-log scale (data from [17]). number so as to make each leak out of th quent time st 4. Music: Can We See the Music? p p( , ) represents the probability of ﬁnding an orbit in bin x at time t. Using the random initial distribution of M = 9 × 107 points, centred at x0 = −0.553, we ﬁrst check the validity of approximat- ing Eq. (5) with Eq. (10). Interestingly, Fig. 1 shows that L given in a slope of transition the system resulting i occupying Q w. This results in the slope of !L(t) < 1. Yet since the rate in which orbits leave the bin that includes x = −1 is constant, the reduced slope is also constant. The constant slope is equivalent to the system taking the path of minimum available work through Eq. (10). To understand how the initial position x0 and the unsta- ble ﬁxed points are related to L, we plot in Fig. 4 the total change in L starting from different initial delta functions uni- formly spread over the domain. The total change in L between t = 0 and the ﬁnal time when the evolution reaches its in- In this paper we have in merically the information le We have shown that dL dt > equilibrium as long as the s operator. On of the sets that contr of probability not being used servation of probability, whe PDF at the next time step o meaning the system has no Our methodology is not system-speciﬁc and applicable to any stochastic processes. In particular, given any time-dependent PDFs that are computed from a theory, simulations or from data, we can compute L(t) to understand information change. As an example, we apply our theory to music data and discuss information change associated with different pieces of classical music. In particular, we are interested in understanding differences among famous classical music in view of information change. To gain an insight, we used the MIDI ﬁle [26], computed time-dependent PDFs and the information length as a function of time [14]. Eq. (5) plotted in the solid black line with solid dots agrees very well for most of the evolution with L given by Eq. (10) shown by the line with circles, respectively. It is seen from Fig. 1 that initially, the PDFs never overlap at the two consecutive times, oc- The ﬁn artiﬁcially and increa orbits for variant density varies with the initial position x0. 3.2. Chaotic Attractor Section 3.1 demonstrates that the minimum value of L∞occurs at a stable equilibrium point [15,16]. We now show that in contrast, in the case of a chaotic attractor, the minimum value of L∞ occurs at an unstable point [13]. To this end, we consider a chaotic attractor using a logistic map [13]. The latter is simply given by a rule as to how to update the value x at t + 1 from its previous value at t as follows [25] (20) xt+1 = 1 −ax2 t , (20) where x = [−1, 1] and a is a parameter, which controls the stability of the system. As we are interested in a chaotic attractor, we chose the value a = 2 so that any initial value x0 evolves to a chaotic attractor given by an invariant density (shown in the right panel of Figure 3). A key question is then whether all values of x0 are similar as they all evolve to the same invariant density in the long time limit. To address how close a particular point x0 is to equilibrium, we (i) consider a narrow initial PDF around x0 at t = 0, (ii) evolve it until it reaches the equilibrium distribution, (iii) measure the L∞between initial and ﬁnal PDF, and (iv) repeat steps (i)–(iii) for many different values x0. For example, for x0 = 0.7, the initial PDF is shown on the left and ﬁnal PDF on the right in Figure 3. We show L∞against x0 in Figure 4. A striking feature of Figure 4 is an abrupt change in L∞for a small change in x0. This means that the distance between x0 and the ﬁnal chaotic attractor depends sensitively on x0. This sensitive dependence of L∞on x(t = 0) means that a small change in the initial condition x0 causes a large difference in a path that a system evolves through and thus L∞. This is a good illustration of a chaotic equilibrium and is quite similar to the sensitive dependence of the Lyapunov exponent on the initial condition [25]. That is, our L∞provides a new methodology to 6 of 11 Entropy 2018, 20, 574 our set represe test chaos. Another interesting feature of Figure 4 are several points with small values of L∞, shown by red circles. 3.2. Chaotic Attractor Fo ferent initial x0, the domain, [−1, 1] with the width of each bin 2 The n ken into M bins, with the width of each bin M . The number bins used is a free parameter after all “There is no law of na- e that deﬁnes the coarse grains” [17] Here we have ﬁxed the where lows t do not Fig. 4. The evolution of L as a function of time for many initial conditions spread over the domain. Most initial conditions travel a distance of between 13 and 16 be- fore reaching p0(x). The points x0 = [−1, −0.96, −0.708, −0.5, 0, 0.5, 0.708, 0.96, 1] whose initial conditions are marked with circles, start at or quickly occupy the bin of a ﬁxed point and thus reach p0(x) in a far shorter distance. Fig. 5. The evolution of L into four main phases. 0 < holds the x = −1 ﬁxed poi orbits is reducible. 7 < t ≤ changes. t > 16 the system Figure 4. L∞against the peak position x = x0 of an initial PDF in the chaotic regime of a logistic map (Reprinted from Physics Letters A, 379, S.B. Nicholson & E. Kim, Investigation of the statistical distance to reach stationary distributions, 83-88, Copyright (2015), with permission from Elsevier). 3.2. Chaotic Attractor In particular, x0 = 0.5 has the smallest value of L∞, indicating that the unstable point is closest to the chaotic attractor. That is, an unstable point is most similar to the chaotic attractor and thus minimises L∞. inﬁnite lengths. We recall that the logistic map is governed by the following mapping xt 1 = 1 ax2 (17) x -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Equilibrium PDF 0 20 40 60 80 100 120 140 160 x -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Initial PDF around x0=0.7 0 10 20 30 40 50 60 +1 1 axt , (17) hich describes the position of an orbit xt+1 at time t + 1 as a nction of its position xt at the earlier time t. a is the control rameter, which is taken to be 2 for simulating a chaotic region. he stationary density for a = 2 is given by p0 = 1/π(1 −x2)1/2. this chaotic region, the map has the two unstable ﬁxed points = −1 and x = 1/2, which turn out to play an interesting role in L(t) as shown later. A key question of our interest is how an initial state far from Fig. 3. P with the 15 < t x0=0.7 (a) (b) Figure 3. (a): an initial narrow PDF at the peak x0 = 0.7; (b): the invariant density of a logistic map. S.B. Nicholson, E.-j. Kim / Physics Letters A 379 (2015) 83–88 x -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Initial PDF around x0=0.7 0 10 20 30 40 50 60 axt , escribes the position of an orb of its position xt at the earlie er, which is taken to be 2 for s onary density for a = 2 is give haotic region, the map has the nd x = 1/2, which turn out to shown later. x0=0.7 (a) x -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Equilibrium PDF 0 20 40 60 80 100 120 140 160 (17) it xt+1 at time t + 1 as a r time t. a is the control mulating a chaotic region. n by p0 = 1/π(1 −x2)1/2. 3.2. Chaotic Attractor two unstable ﬁxed points play an interesting role in Fi w (b) A key question of our interest is how an initial state far from 15 < t Figure 3. (a): an initial narrow PDF at the peak x0 = 0.7; (b): the invariant density of a logistic map. S.B. Nicholson, E.-j. Kim / Physics Letters A 379 (2015) 83–88 quilibrium approaches p0(x) in probability space in terms of L(t). For instance, is there any unique property of "L(t) that n be identiﬁed for all evolutions starting from different initial nditions? To answer this question, we perform numerical sim- ation of Eq. (17) starting from an initial PDF which is strongly calised at x = x0, approximated by a delta function. For each mulation using different initial x0, the domain, [−1, 1] will be oken into M bins, with the width of each bin 2 M . The number bins used is a free parameter after all “There is no law of na- re that deﬁnes the coarse grains” [17]. Here we have ﬁxed the not in PDF on and Eq tionary We hav other in The where lows th do not Fig. 4. The evolution of L as a function of time for many initial conditions spread over the domain. Most initial conditions travel a distance of between 13 and 16 be- fore reaching p0(x). The points x0 = [−1, −0.96, −0.708, −0.5, 0, 0.5, 0.708, 0.96, 1] whose initial conditions are marked with circles, start at or quickly occupy the bin of a ﬁxed point and thus reach p0(x) in a far shorter distance. Fig. 5. The evolution of L st into four main phases. 0 < t holds the x = −1 ﬁxed poin orbits is reducible. 7 < t ≤16 changes. t > 16 the system se Figure 4. L∞against the peak position x = x0 of an initial PDF in the chaotic regime of a logistic map (Reprinted from Physics Letters A, 379, S.B. Nicholson & E. Kim, Investigation of the statistical distance to reach stationary distributions, 83-88, Copyright (2015), with permission from Elsevier). hes p0(x) in probability space in te is there any unique property of "L( r all evolutions starting from different er this question, we perform numeric tarting from an initial PDF which is s approximated by a delta function. number so as to make each leak out of th quent time st 4. Music: Can We See the Music? 1 τ = dL dt for different composers shown in Figure 5 (from [14]). number so as to make each leak out of th quent time st 4. Music: Can We See the Music? Interestingly, the total change in L takes the minimum value for the initial ensembles starting from or quickly entering the two unstable ﬁxed points x = −1, 1/2. Some of these initial conditions x0 = [−1, −0.96, −0.708, −0.5, 0, 0.5, 0.708, 0.96, 1] are marked with the circles in Fig. 4. All of these initial conditions reach ﬁxed points in 5 iterations or less. Since L represents the statistical dis- tance between the initial PDFs and the ﬁnal, invariant density, this the system’s PDFs start inte we have non-zero Q w and time. This is because the av (measured with DS) is redu able probability in Q p. The our results. An interesting re tem almost always follows appears to deviate from thi from a non-stationary distrib Speciﬁcally, the midi ﬁle stores a music by the MIDI number according to 12 different music notes (C, C#, D, D#, E, F, F#, G, G#, A, A#, B) and 11 different octaves, with the typical time ∆t between the two adjacent notes of order ∆t ∼10−3 s. In order to construct a PDF, we specify 129 statistically different states according to the MIDI number and one extra rest state (see Table 1 in [14]) and calculate an instantaneous PDF (see Figure S1 in [14]) from an orchestra music by measuring the frequency (the total number of times) that a particular state is played by all instruments at a given time. Thus, 7 of 11 Entropy 2018, 20, 574 the time-dependent PDFs are deﬁned in discrete time steps with ∆t ∼10−3, and the discrete version of L (Equation (7) in [14]) is used in numerical computation. Figure 5 shows L(t) against time for Vivaldi’s Summer, Mozart, Tchaikovsky’s 1812 Overture, and Beethoven’s Ninth Symphony 2nd movement. We observe the difference among different composers, in particular, more classical, more subtle in information change. We then look at the rate of information change against time for different music by calculating the gradient of L ( dL dt = 1/τ) in Figure 6, which also manifests the most subtle change in information length for Vivaldi and Mozart. Figure 5. L(t) against time T for different composers (from [14]). Figure 5. L(t) against time T for different composers (from [14]). Figure 6. 1 τ = dL dt for different composers shown in Figure 5 (from [14]). Figure 6. 5. Quantum Systems (23) (23) Equation (22) clearly shows that the PDF is Gaussian, with the mean ⟨x⟩= ¯hk0t m + Ft2 2m and the variance Equation (22) clearly shows that the PDF is Gaussian, with the mean ⟨x⟩= ¯hk0t m + Ft2 2m and the variance Var(t) = ⟨(x −⟨x⟩)2⟩= 1 4β = 1 4β0 + β0¯h2t2 m2 = Var(0) + ¯h2t2 4Var(0)m2 . (24) (24) In Equation (24), Var(0) = ⟨(x(0) −⟨x(0)⟩)2⟩= 1 4β0 = Dx 2 is the initial variance. We note that the last term in Equation (24) increases quadratically with time t due to the quantum effect, the width of wave function becoming larger over time. Obviously, this effect vanishes as ¯h →0. In Equation (24), Var(0) = ⟨(x(0) −⟨x(0)⟩)2⟩= 1 4β0 = Dx 2 is the initial variance. We note that the last term in Equation (24) increases quadratically with time t due to the quantum effect, the width of wave function becoming larger over time. Obviously, this effect vanishes as ¯h →0. g g y Since the PDF in Equation (22) is Gaussian, we can use Equation (18) to ﬁnd (e.g., see [16]) 1 τ2 = 2t2 1 (T2 + t2)2 + 2β0 T2 T2 + t2 v2 0 1 + Ft ¯hk0 2 , (25) (25) where T = m 2¯hβ0 , the time scale of the broadening of the initial wave function [21]. It is interesting to note that when there is no external constant force F, the two terms in Equation (25) decrease for large time t, making τ large. The situation changes dramatically in the presence of F in Equation (25) as the second term approaches a constant value for large time. The region with the same value of τ signiﬁes that the rate of change in information is constant in time, and was argued to be an optimal path to minimise the irreversible dissipation (e.g., [16]). Physically, this geodesic arises when when the broadening of a PDF is compensated by momentum Ft which increases with time. Mathematically, the limit t →∞reduces Equation (25) and thus L to 1 τ ∼FDx ¯h , L ∼(Ft)Dx ¯h . (26) (26) Since Ft = P and Dx = (2β0)−1/2 is the width of the wave function at t = 0, FtDx in Equation (26) represents the volume in the P −x phase space spanned by this wave function. 5. Quantum Systems Finally, we examine quantum effects on information length [21]. In Quantum Mechanics (QM), the uncertainty relation ∆x∆P ≥¯h 2 between position x and momentum P gives us an effect quite similar to a stochastic noise. We note here that we are using P to denote the momentum to distinguish it from a PDF (p(x, t)). For instance, the trajectory of a particle in the x −P phase space is random and not smooth. Furthermore, the phase volume h plays the role of resolution in the phase space, one unit of information given by the phase volume h. Thus, the total number of states is given by the total Entropy 2018, 20, 574 8 of 11 phase volume divided by h. This observation points out a potentially different role of the width of PDF in QM in comparison with the classical system since a wider PDF in QM occupies a larger region of x in the phase space, with the possibility of increasing the information. phase volume divided by h. This observation points out a potentially different role of the width of PDF in QM in comparison with the classical system since a wider PDF in QM occupies a larger region of x in the phase space, with the possibility of increasing the information. To investigate this, for simplicity, we consider a particle of mass m under a constant force F and assume an initial Gaussian wave function around x′ = 0 [21] ψ(x′, 0) = 2β0 π 1 4 e−β0x′2+ik0x′, (21) (21) where k0 = P0/¯h is the wave number at t = 0, Dx = (2β0)−1/2 is the width of the initial wave function, and P0 is the initial momentum. A time-dependent PDF p(x, t) is then found as (e.g., see [21,27]): where k0 = P0/¯h is the wave number at t = 0, Dx = (2β0)−1/2 is the width of the initial wave function, and P0 is the initial momentum. A time-dependent PDF p(x, t) is then found as (e.g., see [21,27]): p(x, t) = \|ψ(x, t)\|2 = r β(t) π e−β(t)(x−⟨x⟩)2. (22) (22) Here, β(t) = 2β0m2 m2 + (2¯hβ0t)2 , ⟨x⟩= ¯hk0t m + Ft2 2m . 5. Quantum Systems This reﬂects the information changes associated with the coverage of a phase volume ¯h. Interestingly, similar results are also obtained in the momentum representation where L is computed from the PDF p(P, t) in the momentum space: Since Ft = P and Dx = (2β0)−1/2 is the width of the wave function at t = 0, FtDx in Equation (26) represents the volume in the P −x phase space spanned by this wave function. This reﬂects the information changes associated with the coverage of a phase volume ¯h. Interestingly, similar results are also obtained in the momentum representation where L is computed from the PDF p(P, t) in the momentum space: p(P, t) = r λ π e−λ(p−(mv0+Ft))2, 1 τ2 = 2λF2, L = √ 2λFt, (27) (27) 9 of 11 Entropy 2018, 20, 574 where λ = 1 2¯h2β0 . In Equation (27), τ is obviously constant, and L linearly increases with time t. We can see even a strong similarity between Equation (27) and Equation (26) as t →∞once using L ∝ √ 2λFt ∼(Ft)Dx/¯h. In view of the complementary relation between position and momentum in quantum systems, the similar result for L in momentum and position space highlights the robustness of the geodesic. where λ = 1 2¯h2β0 . In Equation (27), τ is obviously constant, and L linearly increases with time t. We can see even a strong similarity between Equation (27) and Equation (26) as t →∞once using L ∝ √ 2λFt ∼(Ft)Dx/¯h. In view of the complementary relation between position and momentum in quantum systems, the similar result for L in momentum and position space highlights the robustness of the geodesic. where λ = 1 2¯h2β0 . In Equation (27), τ is obviously constant, and L linearly increases with time t. We can see even a strong similarity between Equation (27) and Equation (26) as t →∞once using L ∝ √ 2λFt ∼(Ft)Dx/¯h. In view of the complementary relation between position and momentum in quantum systems, the similar result for L in momentum and position space highlights the robustness of the geodesic. 6. Conclusions We investigated information geometry associated with stochastic processes in classical and quantum systems. Speciﬁcally, we introduced τ(t) as a dynamical time scale quantifying information change and calculated L(t) by measuring the total clock time t by τ. As a unique Lagrangian measure of the information change, L∞was demonstrated to be a novel diagnostic for mapping out an attractor structure. In particular, L∞was shown to capture the effect of different deterministic forces through the scaling of L∞again the peak position of a narrow initial PDF. For a stable equilibrium, the minimum value of L∞occurs at the equilibrium point. In comparison, in the case of a chaotic attractor, L∞ exhibits a sensitive dependence on initial conditions like a Lyapunov exponent. We then showed the application of our method to characterize the information change associated with classical music (e.g., see [14]). Finally, we elucidated the effect of the width of a PDF on information length in quantum systems. Extension of this work to impure (mixed-state) quantum systems and investigation of Riemannian geometry on the space of density operators would be of particular interest for future work. Funding: This research received no external funding. Acknowledgments: This paper is a review and summary of work carried out over several years with numerous co-authors. Among these, I am particularly grateful to Rainer Hollerbach, Schuyler Nicholson and James Heseltine for their contributions and valuable discussions regarding different aspects of information length. Conﬂicts of Interest: The author declares no conﬂict of interest. Conﬂicts of Interest: The author declares no conﬂict of interest. Appendix A. L for the O-U Process To make this paper self-contained, we provide here the main steps for the derivation of L for the O-U process [15,16]. We use y = ⟨x⟩= y0e−γt in p(x, t) in Equation (12) and differentiate it to ﬁnd ∂p ∂t = ˙β 1 2β −(x −y)2 + 2β(x −y) ˙y p. (A1) (A1) Equations (A1) and (1) and using the properties of a Gaussian PDF [⟨(x −y)2⟩= 1 2β, ⟨(x −y)4⟩= 3⟨(x −y)2⟩2] lead to Equations (A1) and (1) and using the properties of a Gaussian PDF [⟨(x −y)2⟩= 1 2β, ⟨(x −y)4⟩= 3⟨(x −y)2⟩2] lead to 1 τ2 = 1 2β(t)2 dβ dt 2 + 2β dy dt 2 . (A2) (A2) We express β in Equation (16) in terms of T = 2β0D(e2γt −1) + γ as β = γβ0e2γt T . Differentiating this and using r = 2β0D −γ then give We express β in Equation (16) in terms of T = 2β0D(e2γt −1) + γ as β = γβ0e2γt T . Differentiating this and using r = 2β0D −γ then give ˙β2 2β2 = 2γ2r2 1 T2 . (A3) Similarly, using dy dt = −γy0e−γt, T = 2β0D(e2γt −1) + γ and q = β0γy02, we obtain 2β ˙y2 = 2qγ2 1 T . (A4) ˙β2 2β2 = 2γ2r2 1 T2 . (A3) ˙β2 2β2 = 2γ2r2 1 T2 . (A3) Similarly, using dy dt = −γy0e−γt, T = 2β0D(e2γt −1) + γ and q = β0γy02, we obtain 2β ˙y2 = 2qγ2 1 T . 2β ˙y2 = 2qγ2 1 T . (A4) (A4) Using these results, Equations (A3) and (A4) in (A2) gives us Using these results, Equations (A3) and (A4) in (A2) gives us Using these results, Equations (A3) and (A4) in (A2) gives us Using these results, Equations (A3) and (A4) in (A2) gives us Entropy 2018, 20, 574 Entropy 2018, 20, 574 Entropy 2018, 20, 574 10 of 11 1 τ2 = 1 2β2 dβ dt 2 + 2β dy dt 2 = 2γ2 T2 (r2 + qT). (A5) (A5) Again, in Equation (A5), q = β0γy02, r = 2β0D −γ, andT = 2β0D(e2γt −1) + γ [15–17]. Appendix A. L for the O-U Process Thus, Equation (2) can easily be calculated directly from Equation (A5) with the result L = 1 √ 2 Z Tf Ti √q T 3 2 dT = − p 2q 1 √ T Tf Ti = 1 p D/γ [y0 −y] , (A10) (A10) where again y = ⟨x⟩= y0e−γt. References Appendix A. L for the O-U Process It is worth noting that q and r, respectively, arise from the difference in mean position at t = 0 and t →∞ (i.e., y0 ̸= y(t →∞)) and in PDF width at t = 0 and t →∞(i.e., D0 ̸= D). Thus, the ﬁrst and second terms in Equation (A5) represent the information change due to the change in PDF width and the movement of the PDF, respectively. Using D0 = γ 2β0 , we express r, q and T in Equation (A5) as β0 q = γ2y2 0 2D0 , r = γ D D0 −1 , T = γ h D D0 (e2γt −1) + 1 i . Equations (A5) and (2) then give us L = 1 √ 2 Z Tf Ti 1 T 1 T + r q r2 + qT dT, (A6) (A6) where Ti = T(t = 0) and Tf = T(t). To compute Equation (A6) for r ̸= 0, we use Y = p r2 + qT and integrate L = 1 √ 2 ln Y −r Y + r Yf Yi + √ 2 r H, H = Z Yf Yi qr −r2 Y2 + qr −r2 dY, (A7) (A7) where Yi = Y(t = 0) and Yf = Y(t). To calculate H in Equation (A7), we need to consider the two cases where q ≥r or q < r. First, when q ≥r, we use the change of the variable Y = p qr −r2 tan θ to ﬁnd Y H = q qr −r2 " tan−1 Y p qr −r2 !#Yf Yi . (A8) (A8) When q < r, we let Y = p r2 −qr sec θ and ﬁnd When q < r, we let Y = p r2 −qr sec θ and ﬁnd When q < r, we let Y = p r2 −qr sec θ and ﬁnd When q < r, we let Y = p r2 −qr sec θ and ﬁnd H = − p r2 −qr 2 " ln Y − p r2 −qr Y + p r2 −qr !#Yf Yi . (A9) (A9) When D = D0 (r = 0), β(t) = β0 for all t. Thus, Equation (2) can easily be calculated directly from Equation (A5) with the result When D = D0 (r = 0), β(t) = β0 for all t. References 1. Gibbs, A.L.; Su, F.E. On choosing and bounding probability metrics. Int. Stat. Rev. 2002, 70, 419–435. [CrossRef] 2. Wootters, W.K. Statistical distance and Hilbert space. Phys. Rev. D 1981, 23, 357. [CrossRef] 3. Frieden, B.R. Science from Fisher Information; Cambridge University Press: Cambridge, UK, 2000. 4. Braunstein, S.L.; Caves, C.M. Statistical distance and the geometry of quantum states. Phys. Rev. Lett. 1994, 72, 3439. [CrossRef] [PubMed] 4. Braunstein, S.L.; Caves, C.M. Statistical distance and the geometry of quantum states. Phys. 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https://openalex.org/W4392701647	https://revistas.pucp.edu.pe/index.php/themis/article/download/28363/26255	es		Responsabilidad de directores por ganancias ilícitas	Thēmis	2,023	cc-by	17,324	10.18800/themis.202302.009 RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS DIRECTORS’ LIABILITY FOR UNLAWFUL PROFITS Sergio García Long* Pontificia Universidad Católica del Perú Directors’ liability has certain aspects that demand a special legal treatment different from the general rules of civil liability. One such aspect is the commission of profitable wrongs, which are situations where a damage is caused intentionally to obtain a benefit. This makes compensation insufficient and requires for restitution and unjustified enrichment, and even figures of deterrence and punitive nature such as disgorgement of profits and punitive damages, which is a lively debate in comparative law. This paper analyzes the legal consequences derived from wrongs that generate illicit profits and shows the defectiveness of the current corporate regulation in this regard. Keywords: Directors; companies; unlawful profits; tort law; restitution; unjustified enrichment; profit stripping; punitive damages. * La responsabilidad de los directores tiene ciertos aspectos que demandan un tratamiento jurídico especial y distinto a las reglas generales de la responsabilidad civil. Uno de tales aspectos es la comisión de daños rentables, que son situaciones donde se comete una infracción de manera intencional para conseguir un beneficio. Lo anterior hace que la compensación sea insuficiente y que se necesite de la restitución y el enriquecimiento injustificado, e incluso de conceptos de naturaleza disuasiva y punitiva como el disgorgement of profits y los punitive damages, lo cual es un debate constante en el derecho comparado. Este trabajo analiza las consecuencias jurídicas derivadas de los ilícitos que generan beneficios ilegales y muestra los defectos de la actual regulación societaria al respecto. Palabras clave: Directores; sociedades; beneficios ilícitos; responsabilidad civil; restitución; enriquecimiento injustificado; despojo de ganancias; daños punitivos. Abogado. Candidato al LLM en International Business and Commercial Law por la University of Manchester. Especialización en Law & Economics of Mergers and Acquisitions por London School of Economics and Political Science. Miembro de la Comisión Consultiva en Derecho Civil del Ilustre Colegio de Abogados de Lima. Miembro del European Law Institute (ELI). Contacto: sergio.garcialong@postgrad.manchester.ac.uk Nota del Editor: El presente artículo fue recibido por el Consejo Ejecutivo de THĒMIS-Revista de Derecho el 13 de julio de 2023, y aceptado por el mismo el 5 de septiembre de 2023. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 149 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS I. INTRODUCCIÓN: EL ESTADO DE LA CUESTIÓN La responsabilidad de los directores de sociedades es uno de los temas más sensibles debido al impacto que puede tener en la gestión de los negocios. Un inadecuado sistema de responsabilidad incrementaría los costos y desincentivaría la toma de decisiones comerciales. No obstante, también puede ocurrir lo contrario, esto es, que el derecho sea deficiente al respecto, generando inadecuados incentivos y fallas en el mercado. Aquello es lo que ocurre con los daños rentables (profitable wrongs)1 o culpa lucrativa (faute lucrative)2 en el derecho societario y bursátil, el cual requiere de normas especiales sobre responsabilidad civil y enriquecimiento injustificado. En principio, todo tipo de daño puede ser compensado a través de las reglas generales de la responsabilidad civil, conforme al Código Civil. Sin embargo, existen supuestos identificados que pueden requerir una regulación particular, adicional a las reglas generales. Este es el caso de la responsabilidad de directores. La experiencia ha identificado que un director puede ser negligente en su actuar y ocasionar un daño a la sociedad y sus dueños. No obstante, en un gran número de casos, el director no se tropieza y comete el daño, sino que realiza un actuar consciente y premeditado con el fin de obtener una ganancia ilícita derivada. Esta situación particular requiere un tratamiento diferenciado que vaya más allá de las reglas de la responsabilidad civil, ya que es usual que el beneficio ilícito obtenido sea mayor al monto de la indemnización y que no tenga relación con el daño. Como tal, inclusive si el director es obligado a pagar una compensación, seguirá enriquecido por su actuar ilícito. 1 Por ello, se requiere de una tutela adicional, la restitutoria, para lograr la devolución de la ganancia ilícita. En este contexto, se debe estudiar la interacción entre la indemnización y la restitución. Pero, de manera adicional, existe una discusión en torno a la naturaleza de esta restitución, ya que en muchos casos no hay una conexión entre el beneficio y un respectivo empobrecimiento, lo cual hace que esta acción vaya más allá de la restauración o reversión patrimonial (la función típica de la restitución) para acercarse hacia el campo de la disuasión y punición. Por este motivo, se diferencia entre restitución en sentido estricto (give back) y despojo (give up o disgorgement) de ganancias ilícitas3. Esta distinción es particularmente importante en torno a la responsabilidad de los directores, en tanto se trata de un supuesto donde el derecho debe actuar para disuadir y castigar una conducta ilícita y ello se materializa con la liquidación y el pago de un monto dinerario extra-compensatorio. Todas estas discusiones nos harán evidenciar la deficiencia del derecho peruano en materia societaria y bursátil, y qué es lo que se debe mejorar. Para ello, se tomará como referencia el derecho comparado, en especial el derecho inglés (Common Law) en relación con la law of restitution y el derecho chileno (Civil Law Latinoamericano) por su cercanía geográfica y lo mucho que se puede aprender de nuestro país vecino en torno a su regulación sobre acciones restitutorias (sin necesidad de ir muy lejos para mirar a un país europeo, usualmente con una cultura y realidad jurídica distinta). En el presente artículo no me enfocaré a fondo en la responsabilidad civil por deberes fiduciarios en el ámbito corporativo ni sobre la aplicación de doctrinas angloamericanas u otros aspectos vinculados, como la pretensión individual versus la En el derecho inglés, el término ‘profitable wrongs’ es uno referencial, ya que podría ser denominado de diferente manera. En principio, pertenece a la restitution for wrongs, como opuesto a los casos de restitution by subtraction, para referirse a la restitución que surge de la comisión de un ilícito (wrong), como distinto a los casos donde la transferencia patrimonial se da por error, por ejemplo. En este caso, se usa el término ‘daños rentables’ para llamar la atención sobre su denominación. Véase la discusión en Birks (1999), Giglio (2007) y Burrows (2011). También véase el recuento de Helms (2012), comparando Civil Law y Common Law para subrayar que el disgorgement of profits (degüelle o despojo de ganancias ilícitas) como tal puede tener distintas manifestaciones, algunas limitadas que puedan explicarse bajo el principio general contra el enriquecimiento injustificado en jurisdicciones civilistas y otras que no (más anglosajonas). Para estas últimas es que guarda la denominación disgorgement of profits. 150 2 En Francia se usa el término ‘faute lucrative’ para referirse a los casos donde se comete un ilícito con la intención de obtener un beneficio derivado. Este término es usado de manera expresa por la doctrina y diversos proyectos de reforma del Código Civil francés, los cuales intentaron incorporar daños punitivos para corregir tales situaciones lucrativas. Véase a García Rubio (2017) y Vingiano (2017). 3 A veces, la diferencia no es totalmente clara, ya que se considera, por un lado, que give back incluye give up, mientras que otros consideran importante la distinción para diferenciar algunos casos particulares de give up que cumplan funciones distintas a la simple restauración patrimonial. Véase la discusión en Birks (1985), McCamus (2011), Grantham y Rickett (2003). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 pretensión social, la responsabilidad del directorio versus la responsabilidad de directores, los tipos de daños resarcibles, entre otros, porque ya varios profesores de derecho se han encargado de ello4. Por el contrario, lo que me interesa es centrarme en lo que es un vacío en el derecho peruano, así como en otras jurisdicciones civilistas: el despojo de ganancias ilícitas contra los directores de entes corporativos. nes que van más allá de una simple devolución de un beneficio obtenido que genera un respectivo empobrecimiento, lo cual no califica como una restitución en sentido restricto (devolver algo previamente entregado). Por tal motivo, se emplea ‘despojo de ganancias ilícitas’ para subrayar la diferencia de tales casos que se alejan de la restauración y compensación, pero que se acercan a la disuasión y sanción. Este vacío se debe a varios aspectos. Por un lado, a veces se trata al derecho societario como uno distinto al derecho civil, cuando en realidad el segundo es la base y regla supletoria del primero. A pesar de ello, usualmente en la academia, los civilistas se desentienden del derecho societario y, a su vez, los corporativistas del derecho civil. Bajo este escenario, parecería responsabilidad de los corporativistas la deficiencia de la legislación societaria peruana en torno al tratamiento de las ganancias ilícitas de los directores. Sin embargo, la misma restitución y enriquecimiento injustificado son poco conocidos por los propios civilistas. Tal materia no tiene un curso propio en las facultades de Derecho en el Perú y, como la acción general por enriquecimiento injustificado es una acción subsidiaria y residual, es poco usada y estudiada5. Por ello, se exige un esfuerzo conjunto entre expertos en derecho civil y societario para que el despojo de las ganancias ilícitas sea correctamente regulado por el derecho. De este modo, la sección II explica el supuesto de responsabilidad de directores y pone en evidencia sus particulares, así como la atención que debe tener la legislación al respecto. La sección III desarrolla conceptos preliminares para entender la distinción entre restitución en sentido estricto (restitution) y despojo en torno a las ganancias ilícitas obtenidas por el infractor (disgorgement). La sección IV aterriza en el caso del despojo de ganancias ilícitas en el derecho societario y bursátil, para lo cual se toma como referencia el derecho chileno y el peruano. La sección V presenta la acción de provecho del dolo ajeno, ausente en el Código Civil peruano, pero regulado en el chileno, a efectos de ser tomado como referencia. La sección VI concluye con las ideas centrales del artículo. LA RESPONSABILIDAD DE DIRECTORES Y LOS PROBLEMAS SIN SOLUCIÓN Un tema que es claro en el derecho societario es que la responsabilidad de los directores deriva del incumplimiento de deberes fiduciarios. Una relación fiduciaria es aquella donde una parte denominada ‘agente’ subordina su interés para la satisfacción del interés de la otra parte, denominada ‘principal’. Este contexto es distinto al de una relación contractual en la cual, si bien las partes colaboran entre sí para lograr la satisfacción de una meta conjunta, ambas partes –en principio– son egoístas en la ejecución de sus prestaciones. Se encuentran en una situación adversarial. Por ejemplo, en un contrato de compraventa, el comprador quiere el bien al menor precio posible, mientras que el vendedor solicitará el mayor precio que el comprador esté dispuesto a pagar. Si las partes llegan a un punto medio en relación con sus precios de reserva, se logrará un acuerdo y se ejecutará el contrato, pero siempre cada 4 La doctrina peruana ya ha escrito sobre varios aspectos relacionados a la responsabilidad de directores, pero aún no sobre las ganancias ilícitas, tal vez por ser un tema que se aleja de la tutela resarcitoria para acercarse a la restitutoria e, incluso, que puede ir más allá para entrar al ámbito de la disuasión y punición. En general, sobre el derecho peruano, véase a Espinoza (1998), Beaumont (2005), Escobar (2012), Navarrete (2021), Hernández Peña (2022), Salazar (2023), Jiménez Vargas-Machuca (2023) y también compárese con Hernández Gazzo y Gotuzzo (2004). Sobre el derecho chileno, véase a Alcalde (2019). Para el derecho colombiano, véase a Sabogal (2012). Para el derecho argentino, véase a Abdala (2014). 5 Una crítica similar, sobre el estado del enriquecimiento injustificado en el Perú y sus razones, puede encontrarse en León (2021). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long Fíjese que acá se usa el término ‘responsabilidad’ en sentido amplio y no como sinónimo de tutela resarcitoria. Lo que quiero graficar es que el actuar ilícito de un director puede generar diversas consecuencias jurídicas, entre ellas, el pago de una indemnización y del beneficio ilícito obtenido. Uno podría diferenciar estas dos acciones, la primera como ‘compensatoria’ y la segunda como ‘restitutoria’. Sin embargo, existen diversos matices y discusiones a tener presente. Por ello, este trabajo hace referencia a una ‘responsabilidad por ganancias ilícitas’ sin intención de limitarse a la tutela resarcitoria, porque el pago de las ganancias ilícitas corresponde a la tutela restitutoria. Sin embargo, tampoco se hace una referencia directa a una ‘restitución por ganancias ilícitas’, porque quiero resaltar aquellas situacio- II. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 151 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS uno en la búsqueda de su interés personal. Por el contrario, en una relación fiduciaria, el agente somete voluntariamente su propio interés al interés ajeno del principal, usualmente a cambio de una contraprestación, y esto sirve como guía de la ejecución contractual. En caso el agente priorice su propio interés o el de otra persona distinta, incurrirá en un conflicto de interés que lo expondrá a responsabilidad. En el caso de los directores, estos se vinculan frente a un ente corporativo y sus dueños en una relación fiduciaria con el fin de administrar al ente legal con dinero ajeno (el de los dueños), en interés de aquellos. Dentro de esta relación jurídica se debe cumplir con los conocidos deberes fiduciarios de lealtad (loyalty) y cuidado (care), e incluso con un deber fiduciario de buena fe (good faith), conforme las cortes de Delaware en Estados Unidos6. Con relación al derecho peruano, aunque en teoría se entiende que la responsabilidad de los directores se deriva por el incumplimiento de deberes fiduciarios, ello no es claro conforme a los artículos 171-184 de la Ley General de Sociedades (en adelante, LGS), lo cual es reconfirmado por el Anteproyecto de Reforma de la Ley General de Sociedades de 2018 (arts. 147-154) que propuso una modificación y regulación más amplia para aclarar lo anterior. Sin perjuicio de las mejoras, algo que aún no se ha tratado correctamente en la LGS ni en el Anteproyecto son las consecuencias jurídicas o remedios aplicables en el caso de responsabilidad de directores por ganancias ilícitas. En principio, si un director es responsable, deberá pagar daños. Esto podría servir para el caso de daños ocasionados por culpa. Sin embargo, la situación es distinta frente a los daños rentables. Si el director comete un daño de manera intencional y premeditada –entiéndase, con dolo– con el fin de obtener un beneficio ilícito, por ejemplo, con el uso de información confidencial de la sociedad para realizar una inversión, entonces el pago de una indemnización será insuficiente, en tanto el monto del beneficio es mayor a la cuantía de los daños. En estos casos es necesario que se complemente la tutela resarcitoria con la tutela res- 152 titutoria y el enriquecimiento injustificado. Sobre todo, habrá complicaciones adicionales sobre la cuantificación y pago del beneficio si el mismo no tiene conexión con un respectivo empobrecimiento y si el beneficio tiene como origen el esfuerzo y astucia del director, lo cual genera problemas de causalidad. Ante tal escenario, puede ser más importante la aplicación de remedios que permitan disuadir y castigar conductas ilícitas. En relación con estos aspectos, el derecho corporativo peruano tiene mucho por mejorar, ya que la tutela restitutoria y punitiva frente a las ganancias ilícitas en el ámbito corporativo es poco conocida. III. RESTITUCIÓN Y ENRIQUECIMIENTO INJUSTIFICADO A. Restitución en sentido estricto La tutela resarcitoria y restitutoria tienen grandes diferencias. La primera se enfoca en el daño, requiere de un criterio de imputación (principalmente la culpa, pero también el riesgo), y tiene fines compensatorios. La segunda se enfoca en el beneficio o enriquecimiento, no requiere de un criterio de imputación (incluso un inocente puede verse obligado a desprenderse de un enriquecimiento si no tiene justificación), tiene fines restaurativos (muy similares a la compensación) y su fundamento es la falta de justificación en una transferencia patrimonial7. Por ejemplo, si alguien sufre un accidente automovilístico puede cobrar una indemnización igual a los daños sufridos. Como los fines son compensatorios, la tutela resarcitoria mira a la víctima y su daño para efectos de mantenerlo indemne. Por ello, aplica la regla de la réparation intégrale, por el cual, se debe reparar todo el daño, pero solo el daño y nada más que el daño. No se puede pagar una suma mayor que coloque a la víctima en una mejor posición en comparación a la situación anterior al perjuicio, el cual debe probarse en su ocurrencia fáctica y cuantía. Además, para que proceda el pago de una indemnización, debe existir una razón que justifique trasladar el costo del daño de la víctima al demandado (los criterios de imputación). 6 Los clásicos deberes fiduciarios son el de cuidado y lealtad. Sin embargo, se discute sobre la presencia de un deber fiduciario de buena fe, distinto al deber contractual, y al respecto, se habla sobre cuál es su relación con los clásicos deberes fiduciarios. A veces, se considera que sería parte del deber de lealtad. Sobre la discusión de los deberes fiduciarios en USA, véase a Eisenberg (2006), Strine, Hammermesh, Balotti y Gorris (2009), Gold (2009), Velasco (2010) y Bruner (2013). 7 Sobre las diferencias entre la responsabilidad civil y el enriquecimiento injustificado, véase a Díez Picazo (2011) y Gallo (2018). Es importante tener en cuenta estas diferencias como premisa porque se tratan de tutelas distintas, sobre todo a efectos de las técnicas legislativas que se emplean en los código civiles, ya que es usual que se legisle al enriquecimiento injustificado como si fuera una especie de indemnización, cuando en realidad se trata de un remedio restitutorio. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 B. Daños rentables Los daños rentables se refieren a situaciones donde una persona comete un daño intencional con el fin de lograr un beneficio ilícito. La restitución y el enriquecimiento injustificado no se limitan a los casos de enriquecimiento por sustracción, sino que también incluye supuestos donde la restitución se desencadena por un ilícito (wrong). Por ejemplo, si alguien infringe la propiedad intelectual de otro, se está beneficiando en perjuicio del propietario, porque usa la propiedad ajena sin autorización (lo que se conoce como un enriquecimiento por intromisión o Eingriff). Si la reacción del derecho es a través de la tutela resarcitoria, deberá pagar el monto de los daños ocurridos. Sin embargo, dado que en tales casos puede ser complicado demostrar los daños a la propiedad intangible, se toman en cuenta otros criterios definidos por ley, entre ellos, la regalía hipotética (royalty damages) y el beneficio ilícito obtenido (restitutionary damages8). En la regalía hipotética, el infractor debe pagar al propietario el monto que hubiera acordado al haber negociado previamente con él por usar la propiedad o sino, se podría pagar una indemnización basada en el beneficio ilícito obtenido (restitutionary damages). 8 Como alternativa, se podría salir de la responsabilidad civil para reconocer una específica acción restitutoria enfocada en el beneficio ilícito y desprendida del daño, de manera que su procedencia esté sujeta a reglas distintas de las que regula a la responsabilidad civil, como las referidas a la probanza y cuantificación del daño. Esta solución sería más adecuada en lugar de ampliar el ámbito de aplicación de la indemnización a criterios que no le corresponden (esto pasa con los restitutionary damages, que es una indemnización basada en el beneficio del demandado y no en el daño del demandante). La opción de una u otra vía legal dependerá del derecho aplicable y la política legislativa. En cierta manera, aunque la restitución del pago indebido sea diferente a la restitución por infracción de la propiedad intelectual, se considera que en ambos casos se da la restauración o reversión de un patrimonio por la conexión que existe entre el empobrecimiento y enriquecimiento, de manera que se sigue aplicando la regla del enriquecimiento indebido. Quien paga por error 100, solo tiene derecho a pedir la devolución de 100, ello porque 100 es el monto en el que el demandante se empobrece y en el que el demandado se enriquece. De manera similar, se considera que hay una restitución patrimonial cuando alguien paga el beneficio ilícito obtenido por la infracción del derecho ajeno, ya que sería un monto que le corresponde al propietario por tener tal calidad, lo cual lo hace titular de todos los provechos y beneficios que puedan derivarse de su bien, de manera que se corrige una especie de expropiación privada9. Sin embargo, existen otras situaciones más complejas donde no se puede apreciar una conexión entre enriquecimiento y empobrecimiento, lo cual impide que se pueda estar ante una verdadera restitución. Incluso el beneficio puede tener diferentes fuentes distintas al patrimonio del demandante, entre ellas el propio trabajo del demandado. Por ello, se trata de casos donde el derecho debe actuar para desincentivar malas conductas lucrativas y sancionarlas10. El uso de la propiedad patrimonial ajena podría no generar tantos dolores de cabeza, a diferencia de la comercialización forzada de los derechos de la personalidad. La diferencia estaría en el bien ju- También denominados como disgorgement damages, aunque acá se reserva el término disgorgement para una situación diferente, conforme se explica más adelante en este ensayo. 9 Estas y otras circunstancias se desarrollan en Ripstein (2016). 10 Véase a Friedman (1980), Edelman (2001) y Edelman (2002). Este sector doctrinario hace énfasis en la necesidad de diferenciar este tipo de casos de otras situaciones –digamos, simples– de restitución. La mejor manera de distinguirlos es a través de las funciones que cumplen. El disgorgement of profits puede cumplir funciones de restauración y compensación, pero también de disuasión y punición, los cuales son, por definición, ajenos a la restitución y el enriquecimiento injustificado. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long Por el contrario, si alguien recibe un pago que no corresponde, el empobrecido puede solicitar la devolución de lo pagado, aunque el enriquecido no haya tenido la culpa y sea inocente. Lo que interesa es restaurar el patrimonio disminuido sin justificación o, en otras palabras, generar una reversión patrimonial. Como tal, la premisa es que exista una conexión entre el enriquecimiento y el empobrecimiento, de manera que el empobrecido pueda solicitar la devolución del mismo monto pagado, ya que representa la suma del enriquecimiento. El ejemplo más claro es el pago indebido por error (répétition de l’indu), que pertenece a los casos de enriquecimiento por sustracción. En este caso, se puede hablar de una restitución en sentido estricto, en tanto el demandante solicita la devolución de lo mismo que previamente entregó y que corresponde al mismo monto de su empobrecimiento. Esta es la noción original del enriquecimiento injustificado: la experiencia de un enriquecimiento que genera un respectivo empobrecimiento. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 153 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS rídico involucrado y en la ineficacia del derecho. En la propiedad intelectual, se afecta un bien patrimonial y la infracción se puede corregir con el pago de una suma dineraria. Por el contrario, en la comercialización forzada de un derecho de la personalidad como la imagen, intimidad, voz, y similares, alguien afecta un bien moral, cuya reparación in natura no es posible, pues el infractor puede obtener un beneficio ilícito (piénsese en las ganancias por ventas y publicidad), y, sobre todo, si el demandado se ve obligado a pagar una indemnización, quedando un remanente a su favor si el beneficio es mayor al daño. En este tipo de casos, se requiere una reacción más severa del derecho que permita disuadir y castigar la conducta ilícita. La discusión está en que la reacción legal va más allá de la simple compensación y restauración. Para lograr una correcta disuasión y punición, se debe hacer uso de las sanciones privadas. Esto puede lograrse si, además de solicitar una indemnización, se puede cobrar punitive damages o, en todo caso, si procede una acción basada en el beneficio ilícito y que no calificaría como una restitución en sentido estricto, porque no habría conexión entre el beneficio del demandado y el empobrecimiento del demandante. Esta acción es denominada como disgorgement of profits, para diferenciarlo de la restitución en sentido estricto. Reservo el término ‘disgorgement’ para esta noción, ya que es comúnmente usado de manera indistinta y como sinónimo de restitution. Una manera ilustrativa de diferenciar ambas acciones es por las funciones. La restitución y el enriquecimiento indebido no pueden explicar situaciones donde el demandante puede solicitar el pago de un monto que no tiene conexión con el supuesto empobrecimiento y que, incluso, lo colocaría en una mejor posición que la situación anterior al desequilibrio patrimonial. Por ejemplo, si un famoso sufre una invasión a su privacidad por la publicación de fotos íntimas, podrá solicitar el pago del daño moral, pero no el pago de las ganancias obtenidas por ventas y publicidad, debido a que tal monto no tendría relación con su daño ni su patrimonio. Por lo anterior, se insiste en subrayar la presencia de la función disuasiva y punitiva del disgorgement of profits, porque se trataría de una acción que se enfoca solo en el beneficio y, como tal, está desconectada del empobrecimiento, hasta el punto de que 11 154 en la práctica funcionaría de manera similar a los punitive damages. Esta acción no puede ser explicada a través de la prohibición contra el enriquecimiento injustificado, ya que si el beneficio se paga al demandante, este terminaría en una mejor posición en comparación al momento anterior al empobrecimiento. Por ello, se prefiere hablar de despojo de ganancias ilícitas como traducción de disgorgement of profits (aunque sea usual que se use el término ‘restitución’ de manera omnicomprensiva). Debo subrayar que el destino del pago del beneficio ilícito aún es un tema debatible en el derecho comparado. La disuasión y el castigo se logra imponiendo al infractor el despojo de toda la ganancia ilícita, pero ello no significa que automáticamente el monto deba pagarse al demandante. A manera de ejemplo, en Estados Unidos de América (en adelante, EE.UU.) se han creado diversos mecanismos para distribuir el pago de una condena punitiva entre la víctima, fondos de compensación, el tesoro público y otros fines sociales11. De esta manera, se logra el efecto disuasivo y punitivo deseado frente al infractor y, a su vez, se eliminan los incentivos perversos del agraviado que permitirían –curiosamente– que se enriquezca sin justificación. Todas estas discusiones desde el derecho comparado son importantes para evidenciar lo que somos en virtud de lo que no tenemos. En específico, el Common Law nos enseña que en jurisdicciones civilistas no existe una acción de disgorgement of profits en el sentido explicado en el presente artículo y ello es un tema a considerar hacia una futura reforma en favor de la mejora del derecho en torno a las ganancias ilícitas. En un interesante estudio publicado en el 2015 y dirigido por los comparatistas Ewoud Hondius y André Janssen, se convocó a diversos ponentes nacionales de Alemania, Austria, Grecia, Francia, Bélgica, Italia, España, Portugal, Romania, China, Japón, Brasil, Chile, entre otros. El objetivo era que expusieran si en su derecho nacional existe algo similar al disgorgement y que indicaran si su sistema nacional es adecuado para otorgar alguna solución frente al despojo de ganancias ilícitamente obtenidas. La conclusión de Hondius y Janssen fue que no existe una teoría coherente del disgorgement en jurisdicciones civilistas, a pesar del uso parcial de disgorgement damages para ciertas situaciones (Hondius & Janssen, 2015). En efecto, en EE.UU. se han publicado leyes que distribuyen el pago de los punitive damages entre la víctima y otros fines. Así, en promedio, solo se deja un 25% en favor de la víctima. Esta pequeña porción suele estar justificada por diferentes motivos, entre ellos, por particularidades o deficiencias del sistema de daños americano. Por ejemplo, como no hay reembolso de costas y costas, o ciertos conceptos de daños no son resarcibles bajo ciertos supuestos, se pueden pagar punitive damages para cubrir esos vacíos, de manera que tal condena punitiva en realidad cumple fines compensatorios. Para mayor información, véase a García Long (2019). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 DIRECTORES Y GANANCIAS ILÍCITAS A. La discusión sobre el disgorgement of profits La ocurrencia de un tort permite cobrar compensatory damages, la comisión de un breach of contract otorga expectation damages, un unjust enrichment concede restitution, mientras que la infracción de deberes fiduciarios y de confianza (equitable wrongs) dan lugar al disgorgement of profits12. Por ello, es importante diferenciar las distintas circunstancias, porque cada una de ellas justifica una reacción legal distinta. Por ejemplo, en el derecho americano está el Chapter 5 ‘Restitution for wrongs’ del Restatement (Third) of Restitution and Unjust Enrichment, que regula el incumplimiento de fiduciary duties y confidence duties, y el respectivo despojo de la ganancia obtenida vía restitution13. Si estamos ante una relación fiduciaria por la cual el agente subordina su interés en favor del interés del principal, se considera doblemente reprochable una situación de conflicto de interés si el agente opta por sobreponer su interés para obtener un beneficio en perjuicio del interés del principal. Ante tal situación, procede el despojo del beneficio obtenido y no solo el resarcimiento de algún daño. De manera similar, si se trata de un breach of confidence, se considera que su infracción lucrativa justifica el despojo de la ganancia obtenida (piénsese en deberes de confidencialidad, no competencia, exclusividad y similares). En el caso inglés Attorney-General v. Guardian Newspapers Ltd (1990), el diario Sunday Times fue encontrado responsable por account of profits14 debido a un breach of confidence frente a la Corona inglesa, por la publicación de extractos del libro Spycatcher de Peter Wright, antes de que la información fuera de dominio público (de manera anticipada). Como señaló Lord Goff: La afirmación de que no se debe permitir que un hombre se beneficie de su propio mal está en términos muy generales, y no proporciona por sí misma ninguna orientación segura para la solución de un problema en cualquier caso particular. Que hay grupos de casos en los que no se permite que un hombre se beneficie de su propio daño, es efectivamente cierto. Una parte importante del derecho de la restitución se refiere a los casos en los que el demandado debe restituir los beneficios adquiridos por su propio acto ilícito, en particular los casos de renuncia a la responsabilidad extracontractual [waiver of tort], los beneficios adquiridos por determinados actos delictivos, los beneficios adquiridos por incumplimiento de una relación fiduciaria y, por supuesto, los beneficios adquiridos por abuso de confianza [breach of confidence]. La pretensión de restitución del demandante se suele hacer valer mediante la rendición de cuentas de los beneficios [account of profits] obtenidos por el demandado a través de su ilícito a costa del demandante. Este remedio de una cuenta [account] es alternativo al remedio de los daños y perjuicios, que en los casos de violación de la confianza está ahora disponible, a pesar de la naturaleza equitativa de la infracción, a través de una interpretación benéfica de la Chancery Amendment Act 1858 (Lord Cairns’ Act), y que en razón de las dificultades que conlleva la toma de una cuenta se considera a menudo como un remedio más satisfactorio, al menos en los casos en que la información confidencial es de naturaleza comercial, y por lo tanto se puede haber sufrido un daño cuantificable (Attorney-General v. Guardian Newspapers Ltd, 1990)15. En el leading case inglés Attorney General v. Blake (2000), George Blake había sido miembro de la seguridad e inteligencia británica entre 1944 y 1961, mientras que en 1951 se convirtió en agente espía para la Unión Soviética y compartió información al gobierno soviético durante nueve años. Blake fue condenado a cuarenta y dos años de prisión por su traición, conforme al Official Secrets Act, pero escapó de prisión en 1966 hacia Berlín y luego emigró a Moscú, donde escribió una autobiografía sobre sus memorias como espía y firmó un contrato con una editorial para publicar su libro a cambio de 50 000 libras a la firma y dos cuotas adicionales del mismo monto a la entrega del manuscrito y a su publicación. El Attorney General inglés tomó conocimiento de la publicación e inició acciones legales por el incumplimiento de Blake de su contrato de trabajo con la Corona inglesa, donde se había obligado a “not to divulge any official information gained by [him] as a result of [his] employment, either in the press or in book form” (Attorney General v. Blake, 2000). 12 Véase a Worthington (1999). 13 Sobre el derecho americano, véase a Jones (1968), Jones (1970), Miller (2013) e Israel y O’Neill (2014). 14 ‘Accounts of profits’ es la terminología inglesa para referirse al disgorgement of profits. Tal denominación también es usada en jurisdicciones anglosajonas de influencia inglesa (Australia, Escocia, Nueva Zelanda, entre otros). 15 Véase también a la English Law Commission (1997). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long IV. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 155 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS Aparentemente, la Corona no había sufrido ningún daño pecuniario. Entonces, la discusión se centró en definir si Blake debía pagar solo un resarcimiento por el incumplimiento contractual (que sería irrisorio o inexistente) o debía proceder el despojo de las ganancias ilícitas obtenidas por el incumplimiento contractual, lo cual permitiría la liquidación de un monto dinerario superior al resarcimiento. Este caso hacía evidente que, si Blake se limitaba a pagar el resarcimiento, aún quedaría en su patrimonio parte de las ganancias obtenidas de manera ilícita. Ante tal situación, Lord Nicholls admitió que debía proceder el account of profits: Una account of profits será apropiada en circunstancias excepcionales. Normalmente, los remedios de daños [compensatorios], el cumplimiento específico y el injunction, junto con la caracterización de algunas obligaciones contractuales como fiduciarias, proporcionarán una respuesta adecuada a un incumplimiento de contrato. Solo en casos excepcionales, cuando estos remedios sean inadecuados, se planteará la cuestión de la contabilización de los beneficios. No se pueden prescribir reglas fijas. El tribunal tendrá en cuenta todas las circunstancias, incluido el objeto del contrato, la finalidad de la disposición contractual que se ha incumplido, las circunstancias en las que se produjo el incumplimiento, las consecuencias del mismo y las circunstancias en las que se solicita la reparación. Una guía general útil, aunque no exhaustiva, es si el demandante tiene un interés legítimo en impedir la actividad lucrativa del demandado y, por tanto, en privarle de su beneficio (Attorney General v. Blake, 2000). En el Civil Law, el derecho societario no ha sido el más sofisticado al momento de regular las acciones de resarcimiento y de despojo de ganancias ilícitas para la responsabilidad de directores, sea porque no contiene alguna regulación (como el caso peruano) o porque contiene una regulación imprecisa. Una correcta regulación societaria debe considerar la procedencia de dos acciones autónomas e independientes, una de responsabilidad civil para solicitar el pago de una indemnización por el daño ocurrido y otra de restitución o disgorgement –según corresponda– para despojar al infractor de la ganancia ilícita. La ley aplicable es la que debe permitir acumular la acción resarcitoria y el despojo de las ganancias ilícitas ante infracciones de deberes fiduciarios por parte de directores. Esto significa que el afectado podría demandar el pago de daños conforme a las reglas generales de la responsabilidad civil y, además, solicitar el despojo de ganancias ilícitas. De esta manera, aunque no hubiera un daño resarcible, procedería el despojo de la ganancia ilícita. 156 Una cuestión importante es definir qué tipo de despojo permite la ley aplicable. Da la impresión que los legisladores societarios no son expertos en restitución y enriquecimiento injustificado, pues, usualmente, la ley es redactada de tal manera que no queda claro si la acción que se puede acumular al resarcimiento es una acción restitutoria o simplemente se trata de una indemnización que puede calcularse en base al beneficio ilícito obtenido (restitutionary damages). La diferencia no es solo teórica, ya que, si se trata de una indemnización, se deberá cumplir con los elementos de la responsabilidad civil, lo cual no es necesario para la procedencia de una acción restitutoria o de disgorgement, que no gira en torno al daño, sino al beneficio. Por ello, no es recomendable regular la procedencia de una indemnización basada en el beneficio, porque ello ata la acción a las reglas de la responsabilidad civil, lo cual puede hacer inútil una acción que debe girar en torno al beneficio, independientemente del daño. Además, esta distinción es importante para definir el alcance y liquidación del pago de los beneficios ilícitos. El beneficio ilícito puede ser mayor al monto de resarcimiento e incluso estar desvinculado de cualquier perjuicio en el patrimonio del demandante. Por ello, es preferible la procedencia de una acción independiente y autónoma, separada y distinta del pago de daños y sus requisitos. En general, la lógica del régimen societario de responsabilidad de directores es que, por un lado, el infractor responda por el daño ocasionado y, por otro lado, se despoje del beneficio ilícito obtenido, sea que el beneficio se considere que pertenecía al afectado y por ello corresponda restituirse (restitución en sentido estricto) o porque no pueda considerarse que el beneficio sale del patrimonio del afectado, pero que igual deba despojarse y pagarse en favor del demandante para disuadir ese tipo de conductas o, en todo caso, pueda definirse un destino distinto para el pago. Así, el director debe desprenderse de cualquier beneficio obtenido con su actuar, sin importar la fuente del beneficio, si el mismo fue obtenido por su trabajo y esfuerzo, y si el beneficio se paga al perjudicado o no. En el derecho bursátil se observa una mejor regulación en comparación a la ley societaria e, incluso, se presentan algunas novedades, en específico, cuando se dispone que las ganancias ilícitas deben ser despojadas y pagadas al tesoro público si no puede identificarse alguna víctima concreta (véase más adelante el derecho chileno). Sobre este punto, se apertura un interesante campo de aplicación de un disgorgement of profits de naturaleza pública con fines de disuadir y castigar. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Para aterrizar la discusión en torno al disgorgement of profits en el derecho societario y bursátil, se tomará en cuenta el derecho chileno y el peruano. B. Derecho chileno: una referencia En el derecho chileno17, el artículo 42 de la Ley 18046, Ley sobre Sociedades Anónimas de Chile, regula diferentes prohibiciones frente a los directores y, ante el incumplimiento de alguna de ellas, se dispone el despojo de los beneficios obtenidos en favor de la sociedad, sin perjuicio de la procedencia de la acción por resarcimiento y cualquier otra sanción que pueda imponer la autoridad competente18. Al respecto, la norma en cuestión sostiene lo siguiente: Artículo 42.- Los directores no podrán: 1. Proponer modificaciones de estatutos y acordar emisiones de valores mobiliarios o adoptar políticas o decisiones que no tengan por fin el interés social, sino sus propios intereses o los de terceros relacionados; 2. Impedir u obstaculizar las investigaciones destinadas a establecer su propia responsabilidad o la de los ejecutivos en la gestión de la empresa; 3. Inducir a los gerentes, ejecutivos y dependientes o a los inspectores de cuenta o auditores, a rendir cuentas irregulares, presentar informaciones falsas y ocultar información; 4. Presentar a los accionistas cuentas irregulares, informaciones falsas y ocultarles informaciones esenciales; 5. Tomar en préstamo dinero o bienes de la sociedad o usar en provecho propio, de sus parientes, representados o sociedades a que se refiere el inciso segundo del artículo 44, los bienes, servicios o créditos de la sociedad, sin previa autorización del directorio otorgada en conformidad a la ley; 6. Usar en beneficio propio o de terceros relacionados, con perjuicio para la sociedad, las oportunidades comerciales de que tuvieren conocimiento en razón de su cargo, y 7. En general, practicar actos ilegales o contrarios a los estatutos o al interés social o usar de su cargo para obtener ventajas indebidas para sí o para terceros relacionados en perjuicio del interés social. Los beneficios percibidos por los infractores a lo dispuesto en los tres últimos números de este artículo pertenecerán a la sociedad, la que además deberá ser indemnizada por cualquier otro perjuicio. Lo anterior, no obsta a las sanciones que la Superintendencia pueda aplicar en el caso de sociedades sometidas a su control (1981) [el énfasis es nuestro]. Conforme a la norma citada, es clara la procedencia de dos acciones independientes, pero que puedan acumularse: una acción de resarcimiento y otra de despojo de beneficios ilícitos, las cuales, independientemente de su fuente o cómo hayan 16 Sobre la función de private enforcement de los punitive damages, véase a Vanleenhove (2017). 17 Sobre la responsabilidad de directores en Chile y el despojo de ganancias ilícitas, véase a Pino Emhart (2019). 18 Aunque ya se encuentre derogados, vale mencionar que el artículo 147, inciso 7, de la Ley 18046 señalaba que, en el caso de operaciones con partes relacionadas, la sociedad o los accionistas podrían demandar “el reembolso en beneficio de la sociedad de una suma equivalente a los beneficios que la operación hubiera reportado a la contraparte relacionada, además de la indemnización de los daños correspondientes” (1981); mientras que el artículo 148, inciso 3, contenía una regulación similar para los casos en que un director o accionista se apropiase de una oportunidad comercial que le correspondiera a la sociedad (1981). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long Aquí entramos en una discusión interesante relacionada al destino de los beneficios ilícitos. La función disuasiva puede conseguirse con el simple despojo de los beneficios ilícitos obtenidos por el infractor. No es necesario el pago de tal monto en favor del perjudicado, a quien, en principio, no le corresponde y terminaría –paradójicamente– en una mejor posición, esto es, indebidamente enriquecido. Ante ello, el pago podría dirigirse a otro destino diferente del afectado, como el tesoro público, fines sociales, fondos de compensación, entre otros. Por el contrario, si el monto es pagado en favor del perjudicado, el disgorgement funcionaría de manera similar a los punitive damages. Ello no es un problema necesariamente, en todo caso, tiene que definirse la naturaleza y fines del disgorgement. Por ejemplo, se considera que es correcto que un demandante se beneficie con el pago en su favor de punitive damages, porque ello es como una recompensa por su labor de private enforcement (lograr que el derecho se cumpla para el infractor y las demás víctimas que no pueden acceder al sistema judicial)16. Otra justificación está en las limitaciones o deficiencias que pueda tener el derecho aplicable en torno al sistema de daños, de manera que el pago del beneficio ilícito a través de una sanción cumpla funciones compensatorias complementarias. La misma lógica podría seguirse para el disgorgement of profits. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 157 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS sido obtenidas, deben ser pagadas en favor de la sociedad. Esta regulación chilena debe ser tomada como referencia para el derecho societario peruano, ya que la actual LGS no regula una acción independiente y con alcance general a todo tipo de ente corporativo frente al beneficio ilícito (a diferencia de la ley bursátil peruana que sí lo hace dentro del ámbito de su aplicación). ha puesto en el supuesto en que la infracción a las normas bursátiles pueda generar un daño general al correcto funcionamiento del mercado de valores, independientemente de que se pueda identificar alguna víctima específica. Esto queda como un tema de agenda. C. Derecho peruano Por otro lado, el artículo 172 de la Ley 18045, Ley de Mercado de Valores de Chile, señala que toda persona perjudicada por infracciones a las disposiciones sobre uso de información privilegiada tendrá una acción de resarcimiento contra el infractor y, además, el infractor deberá pagar al fisco el beneficio ilícito obtenido si no hubiera un perjudicado identificado (1981)19. El texto del artículo en cuestión es el siguiente: 1. Derecho societario: mejoras pendientes Artículo 172.‐ Toda persona perjudicada por actuaciones que impliquen infracción a las disposiciones del presente Título, tendrá derecho a demandar indemnización en contra de las personas infractoras. La acción para demandar perjuicios prescribirá en cuatro años contado a partir de la fecha en que la información privilegiada haya sido divulgada al mercado y al público inversionista. Las personas que hayan actuado en contravención a lo establecido en este Título, deberán entregar a beneficio fiscal, cuando no hubiere otro perjudicado, toda utilidad o beneficio pecuniario que hubieren obtenido a través de transacciones de valores del emisor de que se trate (1981) [el énfasis es nuestro]. La disposición citada es interesante, pues señala que el destino de los beneficios ilícitos será el fisco cuando no hubiera un afectado. Así, se logra el efecto disuasivo y punitivo deseado al realizar el despojo del beneficio ilícito, independientemente del destino del pago que, en este caso, no se paga en favor de un perjudicado como sucedería en una acción restitutoria en sentido estricto. Esta es una interesante alternativa cuando se trata de afectaciones a intereses difusos que impiden la identificación de una víctima específica. A diferencia de lo regulado en el artículo 172 chileno, el derecho bursátil peruano no contiene alguna disposición en su regulación sobre uso de información privilegiada que señale que el beneficio despojado se pague al fisco u otro destinado diferente al perjudicado. El legislador peruano no se 19 158 La actual LGS contiene una regulación deficiente en sus artículos 171-184 en torno a los deberes fiduciarios de directores y las consecuencias de su incumplimiento. Uno de los problemas que se puede identificar es la falta de referencia al despojo de beneficios ilícitos, lo cual es particularmente relevante en relaciones fiduciarias donde el fiduciario (administrador de la sociedad) incurre en un conflicto de interés al perseguir el suyo propio en perjuicio del agente (la sociedad y sus dueños). Entonces, si se presume que habrá algún beneficio derivado, es esencial que la ley disponga algún mecanismo para que el infractor pierda la ganancia. En el 2011 se intentó poner un parche a la LGS para curar la ausencia de una acción que permitiera el despojo de ganancias ilícitas. Ello se materializó con la promulgación de la Ley 29720, que promueve las emisiones de valores mobiliarios y fortalece el mercado de capitales, cuyo artículo 4 modifica el artículo 181 de la LGS sobre pretensión social de responsabilidad contra los directores, aunque no modifica directamente el texto del artículo 181, sino que añade reglas adicionales a efectos de aplicar el artículo 181 cuando se trata de sociedades con acciones inscritas en la bolsa de valores; en otras palabras, el artículo 181 debe leerse junto con el artículo 4. El artículo 4 de la Ley 29720 sostiene que: Artículo 4. Pretensión social de responsabilidad Para los fines de la aplicación del artículo 181 de la Ley 26887, Ley General de Sociedades, tratándose de emisores con acciones inscritas en las bolsas de valores, para poder ejercer directamente la pretensión social de responsabilidad se requiere ser titular de al menos el diez por ciento del capital social de dicho emisor. En tal caso, no constituye requisito previo para iniciar la pretensión social de responsabilidad la celebración de la junta general de accionistas ni la falta del acuerdo respectivo. Sobre el despojo de ganancias ilícitas conforme a la normativa bursátil chilena, véase a Pino Emhart (2018). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 La indemnización por daños y perjuicios a favor de la sociedad incluye la restitución del beneficio obtenido en dicha transacción, sin perjuicio de la responsabilidad penal a que hubiera lugar (2011) [el énfasis es nuestro]. Por su parte, el texto del artículo 181 de la LGS es el siguiente: Artículo 181.- Pretensión social de responsabilidad La pretensión social de responsabilidad contra cualquier director se promueve en virtud de acuerdo de la junta general, aun cuando la sociedad esté en liquidación. El acuerdo puede ser adoptado aunque no haya sido materia de la convocatoria. Los accionistas que representan por lo menos un tercio del capital social pueden ejercer directamente la pretensión social de responsabilidad contra los directores, siempre que se satisfaga los requisitos siguientes: 1. Que la demanda comprenda las responsabilidades a favor de la sociedad y no el interés particular de los demandantes; 2. Que, en su caso, los actores no hayan aprobado la resolución tomada por la junta general sobre no haber lugar a proceder contra los directores. Cualquier accionista puede entablar directamente pretensión social de responsabilidad contra los directores, si transcurrido tres meses desde que la junta general resolvió la iniciación de la pretensión no se hubiese interpuesto la demanda. Es aplicable a este caso lo dispuesto en los apartados 1 y 2 de este artículo. Los bienes que se obtengan en virtud de la demanda entablada por los accionistas son percibidos por la sociedad, y los accionistas tienen derecho a que se les reembolse los gastos del proceso. 20 Los acreedores de la sociedad sólo pueden dirigirse contra los directores cuando su pretensión tienda a reconstituir el patrimonio neto, no haya sido ejercitada por la sociedad o sus accionistas y, además, se trate de acto que amenace gravemente la garantía de los créditos (1997) [el énfasis es nuestro]. En este caso, el artículo 4 parece integrar las ganancias ilícitas al resarcimiento, pues señala que “la indemnización […] incluye la restitución del beneficio obtenido” (2011). Se trataría de una indemnización (restitutionary damages) y no de un verdadero disgorgement. Sin embargo, aún quedaría la duda de cómo aplicar la norma y hasta dónde puede extenderse el resarcimiento. Hay dos opciones: (i) si se trata de un daño de difícil probanza, la corte podría tomar como referencia los beneficios obtenidos como base de cálculo; o (ii) si no estamos ante un daño de difícil probanza y la corte puede determinar la cuantía del daño, se deberá añadir sobre el resarcimiento el monto de las ganancias ilícitas. De esta manera, por concepto de resarcimiento, se otorgaría un monto mayor al que correspondería por el daño real y efectivo. En este caso, el resarcimiento y el despojo de las ganancias ilícitas estarían conceptualmente separadas, pero se otorgarían como un pago único y por concepto de resarcimiento. Lo ideal hubiera sido que la norma fuera más precisa en su redacción y no señalase que el resarcimiento incluye a los beneficios ilícitos, sino que, adicional al pago de daños, procede pagar los beneficios ilícitos. De esta manera, se evitaría cualquier cuestionamiento que, por concepto de resarcimiento, se condene al pago de un monto extra-compensatorio en violación del principio de réparation intégrale y que incluso pueda ser considerado como una sanción privada similar a los punitive damages. Esto ya ha ocurrido en la jurisprudencia peruana20. Lo mejor es el reconocimiento de dos acciones autónomas y diferenciadas, cada una procedente ante requisitos distintos. Si la intención de la norma fue la de fortalecer el mercado de valores mediante el reconocimiento de acciones judiciales idóneas, entonces la norma debe leerse según su finalidad, sin perjuicio de su redacción imperfecta, para concluir que lo que en el fondo se regula es una acción restitutoria o de disgorgement y así se pueda despojar de todos los beneficios obtenidos y no solo un monto dinerario Una corte de segunda instancia alegó expresamente a la función punitiva para aumentar la cuantía de la indemnización. Ello generó varios problemas procesales, por ejemplo, en torno al derecho de defensa y la debida motivación de las resoluciones judiciales. Por tal motivo, en casación se revocó la decisión (Casación 464-2018-La Libertad). Véase a García Long (2021). Sin perjuicio de que sea deseable la función punitiva en la responsabilidad civil, la misma debe hacerse en respeto de las garantías constitucionales que rigen a todas las sanciones, sean públicas, privadas o híbridas. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long Durante el proceso judicial, los demandantes pueden solicitar al directorio de forma genérica todos los documentos relacionados al acto o decisión que se investiga y el directorio debe proporcionarlos. Dicho pedido se tramita a través del juez competente. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 159 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS que deba cumplir con las reglas de la responsabilidad civil (entiéndase, que deba ser compensatorio). Tengamos en cuenta que el artículo 4 de la Ley 29720 precisa los alcances de la pretensión social de responsabilidad en relación con las sociedades anónimas abiertas, de manera que el despojo de ganancias ilícitas estaría excluido para los demás tipos de sociedades anónimas. Por ello, con posterioridad al 2011, el Anteproyecto de Reforma de la Ley General de Sociedades (2018) propuso corregir en parte esta deficiencia y, respecto al deber de lealtad, señala con alcance general que su incumplimiento desencadena una acción resarcitoria y otra para despojar el beneficio ilícito, conforme a su artículo 148: Artículo 148.- Deber de lealtad en el ejercicio del cargo 148.1. El director desempeña el cargo con la lealtad de un representante leal, obrando de buena fe y en el mejor interés de la sociedad, procurando evitar incurrir en conflicto de intereses. 148.2. La infracción del deber de lealtad determina no solo la obligación de indemnizar el daño causado a la sociedad, sino también la de devolver a la sociedad el enriquecimiento indebido obtenido. 148.3. Cada director tiene el mismo deber de lealtad para con la sociedad. 148.4. La actuación del director no debe estar destinada a promover o defender los intereses de los accionistas que los eligieron o de las personas vinculadas a estos o al propio director (2018) [el énfasis es nuestro]. Fíjese que el artículo 148.2 del Anteproyecto es más preciso que el artículo 4 de la Ley 29720, pues no señala que la indemnización incluye los beneficios, sino que, además del resarcimiento, debe devolverse los beneficios. De esta manera, queda claro que se reconoce una acción independiente y autónoma que se acumula al resarcimiento y que no debe confundirse con este. Asimismo, complementando la regulación sobre deberes fiduciarios y su incumplimiento, el artículo 157.2 del Anteproyecto define los casos de conflictos de intereses, entre ellos, el aprovecharse de oportunidades de negocio de la sociedad, usar los activos de la sociedad para fines propios, obtener ventajas o remuneraciones de terceros, competir con la sociedad, entre otros casos donde se presume que el infractor obtendrá un beneficio ilícito 160 con su actuar (2018). Por su parte, el artículo 159 del Anteproyecto señala que, por junta general de accionistas o el directorio, según corresponda, se puede dispensar a un director para realizar ciertos actos que serían considerados de conflicto de interés conforme al artículo 157.2 y así, se evita la responsabilidad (2018). Finalmente, en relación con las sociedades anónimas abiertas y de manera similar a la redacción del artículo 4 de la Ley 29720, el artículo 239 del Anteproyecto señala que, en la pretensión social de responsabilidad, el resarcimiento incluye a los beneficios obtenidos (2018). Al respecto, el artículo 239 sostiene lo siguiente: Artículo 239.- Pretensión social de responsabilidad Son de aplicación a las sociedades anónimas abiertas, las siguientes normas especiales con respecto a la pretensión social de responsabilidad a que se refiere el artículo 162: 1. Pueden ejercer directamente la pretensión social de responsabilidad de acuerdo con el numeral 162.2 del artículo 162 accionistas que representen al menos el diez por ciento del capital. En tal caso, no constituye requisito previo para iniciar la pretensión social de responsabilidad la celebración de la junta general ni la falta del acuerdo respectivo, siempre que se hubiera solicitado notarialmente la convocatoria a junta general y el directorio no hubiera cumplido con realizarla en el plazo legalmente establecido. 2. Antes y durante el proceso judicial, los demandantes pueden solicitar al directorio, en forma genérica, todos los documentos relacionados al acto o decisión que se investiga y el directorio debe proporcionarlos. Dicho pedido se tramita a través del juez del domicilio de la sociedad. 3. La indemnización por daños y perjuicios a favor de la sociedad incluye la restitución del beneficio obtenido del acto o decisión señalado en el inciso 2, sin perjuicio de la responsabilidad penal a que hubiera lugar (2018) [el énfasis es nuestro]. Es buena la intención del Anteproyecto de mejorar la regulación sobre deberes fiduciarios y responsabilidad de directores y que, para ello, haya incluido el pago de las ganancias ilícitas. Sin embargo, aún tal regulación genera algunas dudas sobre qué remedio se ha regulado: ¿restitutionary damages, restitución de ganancias ilícitas o un verdadero disgorgement of profits? THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Si tenemos presente la intención del Anteproyecto de actualizar la regulación sobre la responsabilidad de los directores, debe concluirse que sería procedente una acción adicional y acumulativa a la resarcitoria, ello con distintas liquidaciones dinerarias. En general, frente a una nueva propuesta de reforma, es necesario ser preciso. No es deseable el reconocimiento de restitutionary damages, es insuficiente. Es necesario una acción restitutoria, pero incluso la misma podría ser limitada si se toma en cuenta que, en muchos casos de conflicto de interés, el beneficio ilícito obtenido por el director no tendrá referencia a un respectivo empobrecimiento en la sociedad o sus dueños (podría tener origen en una operación financiera con un tercero), de manera que será necesario el reconocimiento de una acción por disgorgement of profits que sea disuasiva y punitiva, similar a los punitive damages. 2. Mercado de valores: un buen camino La ley es más clara en torno a la procedencia de una acción independiente para despojar el beneficio ilícito en el derecho de mercado de valores, en relación con el uso de información privilegiada. Mientras el artículo 40 del Texto Único Ordenado de la Ley de Mercado de Valores (en adelante, TUO de la LMV), aprobado por Decreto Supremo 093-2002-EF, define qué se entiende por información privilegiada, el artículo 43 indica que las personas que incumplan las prohibiciones sobre uso de información privilegiada deben entregar, a quien corresponda según el caso, el beneficio obtenido. Al respecto, los artículos en cuestión sostienen lo siguiente: Artículo 40.- Definición Para los efectos de esta ley, se entiende por información privilegiada cualquier información referida a un emisor, a sus negocios o a uno o varios valores por ellos emitidos o garantizados, no divulgada al mercado; y cuyo conoci- miento público, por su naturaleza, sea capaz de influir en la liquidez, el precio o la cotización de los valores emitidos. Comprende, asimismo, la información reservada a que se refiere el artículo 34 de esta Ley y aquella que se tiene de las operaciones de adquisición o enajenación a realizar por un inversionista institucional en el mercado de valores, así como aquella referida a las ofertas públicas de adquisición […] Artículo 43.- Prohibiciones Las personas que posean información privilegiada, están prohibidas de: a) Revelar o confiar la información a otras personas hasta que ésta se divulgue al mercado; b) Recomendar la realización de las operaciones con valores respecto de los cuales se tiene información privilegiada; y, c) Hacer uso indebido y valerse, directa o indirectamente, en beneficio propio o de terceros, de la información privilegiada. Estas personas están obligadas a velar porque sus subordinados acaten las prohibiciones establecidas en este artículo. Las personas que incumplan las prohibiciones establecidas en el presente artículo deben hacer entrega al emisor o fondo, cuando se trate de información relativa a las operaciones de los fondos mutuos, de los fondos de inversión, de los de pensiones o de otros fondos administrados por inversionistas institucionales, de los beneficios que hayan obtenido (2022) [el énfasis es nuestro]. Fíjese que en el caso del artículo 43 del TUO de la LMV se regula una acción independiente, sin perjuicio de la acción por daños y perjuicios conforme a las normas generales (societarias o civiles). De manera complementaria, el artículo 6 del Reglamento contra el abuso de mercado y normas sobre uso indebido de información privilegiada y manipulación de mercado, aprobado por Resolución SMV 005-2012-SMV-01, señala que lo regulado por el artículo 43 del TUO de la LMV aplica independientemente de que el infractor haya obtenido una ganancia o haya evitado una pérdida. El artículo 6 señala lo siguiente: Artículo 6.- Prohibiciones Sergio García Long Visto de manera conjunta, el artículo 148.2 del Anteproyecto, para las sociedades anónimas, señala que “la infracción del deber de lealtad determina no solo la obligación de indemnizar el daño causado a la sociedad, sino también la de devolver a la sociedad el enriquecimiento indebido obtenido” (2018), con lo cual sería procedente una acción para despojar el beneficio ilícito y otra resarcitoria. Sin embargo, luego, el artículo 239.3 del Anteproyecto, para las sociedades anónimas abiertas, señala que “la indemnización por daños y perjuicios a favor de la sociedad incluye la restitución del beneficio obtenido del acto o decisión” (2018), con lo cual, el resarcimiento se calcularía teniendo en cuenta el beneficio obtenido, no siendo procedente una acción adicional y acumulativa a la acción resarcitoria. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 6.1. En el marco de lo establecido en el artículo 43 de la Ley del Mercado de Valores es- THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 161 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS tán prohibidos de hacer uso de información privilegiada, además de las personas naturales, los inversionistas institucionales que administran recursos de terceros que van a ser destinados a la inversión en valores de oferta pública, emisores, proveedores de precios, entidades valorizadoras, empresas clasificadoras de riesgos, agentes de intermediación y las demás personas jurídicas que posean información privilegiada. Se incurre en la prohibición de uso prevista en el artículo 43 inciso c) de la Ley del Mercado de Valores cuando se realizan operaciones con valores respecto de los cuales se tiene información privilegiada. 6.2. Las sanciones establecidas en virtud de lo dispuesto en el artículo 43 de la Ley del Mercado de Valores, son de aplicación, independientemente de que la persona que haya revelado, recomendado o hecho uso de información privilegiada, haya obtenido una ganancia o evitado una pérdida. Asimismo, dichas sanciones aplican a todas las personas naturales o jurídicas que hubieren revelado, recomendado o hecho uso de información privilegiada obtenida a través de cualquier medio, sea porque les ha sido voluntaria o involuntariamente transmitida o porque la hubieran obtenido de manera lícita o a través de medios ilícitos, indebidos o fraudulentos (2012) [el énfasis es nuestro]. La precisión anterior es importante por dos razones: (i) frente a una técnica legislativa sobre cómo regular el despojo de ganancias ilícitas no solo se debe tener en cuenta el enriquecimiento positivo, sino también el negativo, lo cual puede consistir en el ahorro de un costo; en consecuencia, es correcto que la norma bursátil haga referencia a que “haya obtenido una ganancia o evitado una pérdida” (Resolución SMV 005-2012-SMV-01); y (ii) le da independencia al despojo de ganancias ilícitas, pues lo que importa es que la infracción se haya realizado con la intención de lograr un objetivo, sin perjuicio que se haya tenido éxito o no en conseguir el objetivo. Asimismo, el artículo 44 del TUO de la LMV señala que, independientemente de las prohibiciones sobre el uso indebido de información privilegiada, los directores y gerentes de un emisor, así como ciertas personas que participen en la compra o venta de los valores emitidos por el emisor, deberán desprenderse de toda ganancia realizada en favor del emisor o el patrimonio, según corresponda (2012). El artículo 44 señala lo siguiente: 162 Artículo 44. Devolución de ganancias de corto plazo Toda ganancia realizada por directores y gerentes del emisor, así como los directores, gerentes, miembros del comité de inversiones y personas involucradas en el proceso de inversión de las sociedades administradoras, de las sociedades administradoras de fondos de inversión y de administradoras de fondos de pensiones, proveniente de la compra y venta o de la venta y compra, dentro de un período de tres meses, de valores emitidos por el emisor, debe ser entregada íntegramente al emisor o al patrimonio, según corresponda. Lo dispuesto en este párrafo es independiente del uso indebido de información privilegiada. Mediante disposiciones de carácter general, Conasev puede regular lo dispuesto en el presente artículo, así como los supuestos de excepción a la obligación de devolver la ganancia (2012) [el énfasis es nuestro]. El artículo 44 del TUO de la LMV regula en su primer párrafo una acción para despojar las ganancias ilícitas que operan sin perjuicio de la procedencia de la acción por daños y perjuicios. Además, conforme a la disposición del segundo párrafo del artículo 44, se promulgó la Resolución SMV 0182015-SMV-01, que aprobó las normas aplicables para la devolución de ganancias de corto plazo, las cuales, entre otros aspectos, regulan cuáles son las operaciones consideradas para el cálculo de las ganancias a corto plazo (artículo 3), supuestos de excepción (artículo 4), la metodología para determinar el monto de devolución de las ganancias de corto plazo (artículo 5), plazo para el pago (artículo 8), entre otros. Por su parte, el artículo 51 del TUO de la LMV dispone ciertas obligaciones sobre el emisor de valores inscritos, entre las cuales, se encuentran prohibiciones que exigen al infractor el despojo de los beneficios obtenidos en favor del emisor, sin perjuicio –señala la norma– de la acción por daños y perjuicios (2012). El artículo 51 señala lo siguiente: Artículo 51.- Obligación del Emisor Los emisores con valores inscritos en el Registro se sujetan a las siguientes normas: a) Los directores y gerentes están prohibidos de recibir en préstamo dinero o bienes de la sociedad, o usar en provecho propio, o de quienes tengan con ellos vinculación, los bienes, servicios o créditos de la sociedad, sin contar con autorización del Directorio; THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 b) Los directores y gerentes están prohibidos de valerse del cargo para, por cualquier otro medio y con perjuicio del interés social, obtener ventajas indebidas para sí o para personas con las que tengan vinculación; y, c) Para la celebración de cada acto o contrato que involucre al menos el cinco por ciento de los activos de la sociedad emisora con personas naturales o jurídicas vinculadas a sus directores, gerentes o accionistas que directa o indirectamente representen más del diez por ciento del capital de la sociedad, se requiere la aprobación previa del directorio, sin la participación del director que tenga vinculación. Para los fines de la determinación del cinco por ciento, deben tenerse en cuenta los últimos estados financieros que correspondan. En las transacciones en las cuales el accionista de control de la sociedad emisora también ejerza control de la persona jurídica que participa como contraparte en el respectivo acto o contrato sujeto a aprobación previa por el directorio, se requiere adicionalmente la revisión de los términos de dicha transacción por parte de una entidad externa a la sociedad emisora. Se considerará como entidad externa a dicha sociedad a las sociedades auditoras u otras personas jurídicas que mediante disposiciones de carácter general determine Conasev. La entidad que revise la transacción no debe estar vinculada a las partes involucradas en ella, ni a los directores, gerentes o accionistas titulares de cuando menos el diez por ciento del capital social de dichas personas jurídicas. Se considera, entre otros, que se encuentra vinculada a la sociedad emisora la entidad que hubiese auditado sus estados financieros en los últimos dos años. Por otro lado, el artículo 72 del TUO de la LMV, sobre las Ofertas Públicas de Adquisición (en adelante, OPA), señala que el incumplimiento de las normas bursátiles, en relación con el procedimiento de realizar una OPA para obtener participación significativa, desencadena una obligación de devolver la ganancia de capital derivada de la venta de las acciones adquiridas en favor de los accionistas transferentes, ello en primer lugar. El artículo 72 sostiene lo siguiente: Artículo 72. Incumplimiento En caso de incumplimiento de las normas precedentes, se aplican las siguientes reglas: a) Quien adquiera o incremente su participación significativa, sea que se trate de una adquisición directa o indirecta, actuando de manera individual o conjunta, sea que actúen o no de manera concertada sin observar el procedimiento respectivo, queda suspendido por mérito del pronunciamiento de Conasev en el ejercicio de los derechos políticos inherentes a las acciones adquiridas y obligado a su venta en los términos y plazos que determine Conasev mediante norma de carácter general. Mientras dure la suspensión de los valores indicados, los mismos no se computan para el quórum. Sin perjuicio de lo anterior, Conasev podrá excepcionalmente determinar, adicionalmente, la suspensión de los derechos inherentes a las acciones que tuviese de manera previa a la adquisición o incremento de participación significativa, así como determinar que dicha tenencia previa no se computa para efectos del quórum. En caso de obtener una ganancia de capital como producto de la venta de las acciones adquiridas, debe entregarla a los accionistas que le transfirieron tales valores. Es nulo todo acuerdo adoptado por los órganos de la sociedad cuando para la adopción de los mismos o para la elección de los miembros de dichos órganos se ha ejercido la representación de acciones adquiridas con prescindencia de la obligación de realizar una oferta pública de adquisición, así como todo acto de disposición que se efectúe con tales valores. La nulidad a la que se hace referencia opera de pleno derecho por el mérito de la resolución del órgano competente de Conasev que se pronuncie sobre la infracción de las normas que regulan las ofertas públicas de adquisición. Sergio García Long Corresponde a Conasev definir los alcances de los términos control y vinculación y regular la participación de la entidad externa a la sociedad y los demás aspectos del presente artículo. se reconocen dos acciones independientes que pueden acumularse. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 163 Los beneficios que se perciban con infracción de lo establecido en este Artículo deben ser derivados a la sociedad, sin que ello obste a la correspondiente reclamación de daños y perjuicios, ni a la interposición de las acciones penales a que hubiere lugar (2012) [el énfasis es nuestro]. Respecto al artículo 51 del TUO de la LMV, también es preciso al señalar que el despojo de los beneficios ilícitos se realiza “sin que ello obste a la correspondiente reclamación de daños y perjuicios” (2012), con lo cual, queda claro que THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS Conasev puede sustituir, a solicitud de parte y siempre que sea más beneficioso para el mercado, la obligación de venta por la de una OPA dirigida al cien por ciento del capital social. En este caso, la OPA debe formularse por el mayor precio pagado por las acciones adquiridas indebidamente o el que determine una entidad valorizadora de acuerdo con lo que establezca Conasev mediante norma de carácter general, el que resulte mayor, y siempre que cumpla de manera previa con el pago del íntegro de la multa por infringir las normas de OPA. Asimismo, de existir un diferencial positivo entre el precio de la OPA y el precio al que transfirieron las acciones que se adquirieron de manera indebida, el oferente debe realizar el pago de dicho diferencial. Lo dispuesto en el presente inciso es de aplicación en lo pertinente a los casos de incumplimiento de la obligación de efectuar una oferta pública de adquisición cuando se trate de supuestos distintos de la adquisición de acciones a que se refiere el primer párrafo. c) Conasev no excluye el valor del registro si previamente no se ha efectuado la oferta pública de compra a que se refiere el artículo 69. La interposición de una acción contenciosoadministrativa no impide la ejecución de lo dispuesto por Conasev salvo que, mediante una medida cautelar judicial motivada se disponga lo contrario y siempre que, en tal caso, se hubiera otorgado como contracautela una garantía suficiente en efectivo, póliza de caución o carta fianza bancaria por el equivalente al cincuenta por ciento del monto de la OPA que debió realizarse, de acuerdo con los criterios que Conasev determine mediante norma de carácter general. No es admisible la caución juratoria como contracautela ni ninguna otra distinta a las enunciadas anteriormente (2012) [el énfasis es nuestro]. En este caso, el artículo 72 del TUO de la LMV regula una acción independiente como supuesto de sanción y, como tal, acumulativa a alguna acción de resarcimiento, pues la intención de la norma es disuadir el incumplimiento del procedimiento para realizar una OPA y, en consecuencia, su objetivo es despojar al adquirente de la ganancia de capital que pueda obtener de una venta de las acciones adquiridas. Es importante mencionar que las citadas disposiciones bursátiles sobre despojo de ganancias ilí- 164 citas fueron todas agregadas o modificadas en el 2011 por la Ley 29720, la misma que, a través de su artículo 4, aclaró los alcances del artículo 181 sobre la pretensión social de responsabilidad para las sociedades anónimas abiertas. En efecto, fue el artículo 6 de la Ley 29720 la que agregó el último párrafo del artículo 43 y modificó el artículo 44 del TUO de la LMV para reconocer acciones para el despojo de ganancias ilícitas, independientes y cumulativas a la acción por daños y perjuicios. El último párrafo 51 del TUO de la LMV ya disponía de manera previa al 2011 la respectiva acción sobre ganancias ilícitas, al igual que el artículo 72, el cual fue modificado por la Ley 29720, pero no en relación con la acción de despojo, la cual ya estaba reconocida. Finalmente, la Ley 29720 también modificó el Código Penal peruano para reforzar la sanción de ilícitos lucrativos, al añadir: (i) el artículo 251-A, que sanciona penalmente el uso de información privilegiada; y (ii) el artículo 213-A, que sanciona al fiduciario que, en beneficio propio o de un tercero, realiza un acto que contraviene el fin para el cual fue constituido el patrimonio. Los artículos mencionados son los siguientes: Artículo 213-A.El factor fiduciario o quien ejerza el dominio fiduciario sobre un patrimonio fideicometido, o el director, gerente o quien ejerza la administración de una sociedad de propósito especial que, en beneficio propio o de terceros, efectúe actos de enajenación, gravamen, adquisición u otros en contravención del fin para el que fue constituido el patrimonio de propósito exclusivo, será reprimido con pena privativa de libertad no menor de dos (2), ni mayor de cuatro (4) años e inhabilitación de uno a dos (2) años conforme al Artículo 36, incisos 2 y 4. […] Artículo 251-A.El que obtiene un beneficio o se evita un perjuicio de carácter económico en forma directa o a través de terceros, mediante el uso de información privilegiada, será reprimido con pena privativa de la libertad no menor de uno (1) ni mayor de cinco (5) años. Si el delito a que se refiere el párrafo anterior es cometido por un director, funcionario o empleado de una Bolsa de Valores, de un agente de intermediación, de las entidades supervisoras de los emisores, de las clasificadoras de riesgo, de las administradoras de fondos mutuos de inversión en valores, de las administradoras de THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 fondos de inversión, de las administradoras de fondos de pensiones, así como de las empresas bancarias, financieras o de seguros, la pena no será menor de cinco (5) ni mayor de siete (7) años (1991) [el énfasis es nuestro]. usada y quede relegada, sobre todo si procede una acción por responsabilidad civil. Sin embargo, este no debería ser un inconveniente si no fuera por la mala redacción de la norma peruana, sin perjuicio del reconocimiento legal de la subsidiariedad. Como vemos, a diferencia del derecho societario peruano, la ley bursátil ha prestado mayor atención al problema de la comisión de infracciones para obtener un beneficio ilícito y, ante ello, ha reconocido diferentes acciones para lograr el despojo de las ganancias ilícitas. Fíjese que el artículo 1955 está redactado en términos funcionales. Otras normas como el equivalente 1303.3 del Código Civil francés señala que: “El empobrecido no tiene acción sobre este fundamento cuando dispone de otra acción” (1804) [traducción propia]22, ello significa que basta que la ley haya dispuesto la aplicación de otra acción sin importar cuál fuese tal acción. Si la ley francesa hace aplicable una acción menos beneficiosa, la misma deberá aplicarse y el perjudicado no podrá acudir al enriquecimiento injustificado, aunque esta fuera más beneficiosa. Por el contrario, la norma peruana señala que “no es procedente cuando la persona que ha sufrido el perjuicio puede ejercitar otra acción para obtener la respectiva indemnización” (Código Civil, art. 1955, 1984), lo cual significa que solo está precluida la acción por enriquecimiento injustificado cuando existe una acción igual de beneficiosa, entiéndase, que permita obtener el mismo resultado. Derecho civil: ¿la solución? Uno podría preguntarse si la norma supletoria podría ser de alguna ayuda. El Código Civil regula la acción general por enriquecimiento injustificado, la cual podría ser invocada en un caso de responsabilidad de directores, ante la deficiencia de la norma especial. Tal acción restitutoria se encuentra regulada en los artículos 1954 y 1955 del Código Civil, los cuales sostienen lo siguiente: Artículo 1954.- Aquel que se enriquece indebidamente a expensas de otro está obligado a indemnizarlo. Artículo 1955.- La acción a que se refiere el artículo 1954 no es procedente cuando la persona que ha sufrido el perjuicio puede ejercitar otra acción para obtener la respectiva indemnización (1984). Así, la regulación peruana adoptó el clásico modelo subsidiario de influencia francesa y, además, tiene algunas deficiencias en su redacción. Todo esto hace complicado invocar a la acción restitutoria21. Para empezar, la acción por enriquecimiento injustificado debe ser subsidiaria. Esto significa, como indica el artículo 1955, que no debe ser procedente otra acción para obtener la respectiva indemnización. Si es procedente otra acción de tal característica, el perjudicado no tiene la opción de elegir una acción más beneficiosa, sino que debe proceder por la que tiene a su disposición. Solo si no existe otra acción para obtener la respectiva indemnización, podría accionar por enriquecimiento injustificado. Este presupuesto de la subsidiariedad hace que la acción por enriquecimiento injustificado no sea El problema con los artículos 1954 y 1955 del Código Civil peruano es que hacen referencia a una ‘indemnización’ –como lo hacen jurisdicciones de influencia francesa– en lugar de mencionar ‘restitución’, como el Código Civil de Alemania (§ 812), Suiza (artículo 62), Japón (artículo 703) y Cuba (artículos 101 y 103). Esta no es una exquisitez terminológica; la acción por enriquecimiento injustificado es una acción restitutoria, no resarcitoria. Por ello, es incorrecto hablar de ‘indemnización’. Tal vez se podría aceptar el uso coloquial, polisémico y amplio del término23. Sin embargo, tal opción genera un problema práctico al momento de superar la subsidiariedad. Si la norma peruana dijera ‘para obtener la respectiva restitución’, la procedencia de una acción indemnizatoria no sería obstáculo, ya que, por un lado, se trata de una acción de distinta naturaleza y, por el otro, la liquidación del monto del beneficio ilícito podría ser superior a lo que correspondería por indemnización de daños y perjuicios. Bajo este escenario, podría acumularse una acción resarcitoria con una acción restitutoria, sin que la 21 Estas cuestiones están desarrolladas en García Long (2022, 2023a y 2023b) y León (2021 y 2023). 22 Traducción propia de: «L’appauvri n’a pas d’action sur ce fondement lorsqu’une autre action lui est ouverte ou se heurte à un obstacle de droit, tel que la prescription” (1804). 23 Así lo propone entre nosotros Fernández (2015). Sin embargo, a su vez, Fernández propone en el Anteproyecto de Reforma del Código Civil (2019) que se modifique el artículo 1954 para reemplazar ‘indemnización’ por ‘restitución’. Curiosamente, la misma propuesta no se hace para el artículo 1955. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long 3. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 165 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS subsidiariedad sea un obstáculo para ello. Por el contrario, si la norma es leída en su sentido literal como ‘para obtener la respectiva indemnización’, se podría pensar –equivocadamente– que la existencia de una acción indemnizatoria impediría a su vez accionar por enriquecimiento injustificado. Entonces, no podría darse el cúmulo de acciones por la simple razón que la acción por enriquecimiento injustificado estaría siendo confundida y equiparada a una acción indemnizatoria. Existe jurisprudencia que confunde al enriquecimiento injustificado con la responsabilidad civil24. Por lo anterior, podría intentarse invocar a la acción por enriquecimiento injustificado para lograr el despojo del beneficio ilícito, independiente del resarcimiento. Sin embargo, por la mala redacción de las normas peruanas y por haberse adoptado el presupuesto de la subsidiariedad, tal alternativa podría ser más viable en teoría que en la práctica. Finalmente, el enriquecimiento injustificado podría ser insuficiente si lo que se busca es despojar al infractor de un beneficio ilícito que no tiene conexión con un empobrecimiento o si, de existir, es menor al beneficio; sobre todo, piénsese en los beneficios que se obtienen por el mismo trabajo o astucia del infractor. El modelo tradicional de influencia francesa requiere la presencia de cinco presupuestos para la procedencia de la acción general por enriquecimiento injustificado, esto es el enriquecimiento, empobrecimiento, conexión entre enriquecimiento y empobrecimiento, falta de justificación o causa y la subsidiariedad. Ante este escenario, una visión tradicional del enriquecimiento injustificado impediría una liquidación dineraria superior al monto del empobrecimiento o daño y que pueda aumentarse de manera irrestricta por el monto de cualquier beneficio obtenido por el infractor. Aparentemente, se considera que el derecho peruano ha seguido tal modelo tradicional. Sin embargo, como he explicado previamente25, este es un falso consenso, porque el artículo 1954 no requiere la presencia de los cinco presupuestos, a diferencia de sus equivalentes europeos que sí siguen de manera expresa este modelo, como el artículo 1303 del Código Civil francés o el artículo 2041 del Código Civil italiano. La norma peruana está redactada de manera tan amplia que –en realidad– reconoce al principio general contra el 166 enriquecimiento injustificado cuando señala que: “Aquel que se enriquece indebidamente a expensas de otro está obligado a indemnizarlo” (Código Civil, art. 1954, 1984). Este tipo de redacción permite una interpretación extensiva de la figura para que no esté limitada al modelo tradicional, sino más bien enfocada en el presupuesto del ‘enriquecimiento a expensas de otro’, sin estar limitado a un respectivo empobrecimiento. De esta manera, el derecho peruano está más cerca a jurisdicciones anglosajonas que civilistas en torno al enriquecimiento injustificado. El problema sería su aplicación práctica, ya que se conoce poco del enriquecimiento injustificado o lo que se conoce es equivocado. Ello haría difícil explicar a un juez que el modelo peruano es uno amplio, esto sin perjuicio de los cuestionamientos que se podrían hacer a las liquidaciones extra-compensatorias (como ya he advertido) si se estira la figura del enriquecimiento injustificado hasta el punto de que funcione como punitive damages. V. LA ACCIÓN DEL PROVECHO DE DOLO AJENO Una acción restitutoria que no está regulada en el Perú, pero sí en Chile, es la acción del provecho por dolo ajeno, según los artículos 1458 y 2316 del Código Civil de Chile, como muestra de la expresión del principio contra el enriquecimiento injustificado26. Dichos artículos sostienen lo siguiente: Art. 1458. El dolo no vicia el consentimiento sino cuando es obra de una de las partes, y cuando además aparece claramente que sin él no hubieran contratado. En los demás casos el dolo da lugar solamente a la acción de perjuicios contra la persona o personas que lo han fraguado o que se han aprovechado de él; contra las primeras por el total valor de los perjuicios, y contra las segundas hasta concurrencia del provecho que han reportado del dolo. […] Art. 2316. Es obligado a la indemnización el que hizo el daño, y sus herederos. El que recibe provecho del dolo ajeno, sin ser cómplice en él, sólo es obligado hasta concu- 24 Véase a la Casación 4689-2017-LIMA. 25 Sobre todo, véase a mi comentario al artículo 1954 del Código Civil peruano en García Long (2023a). 26 Sobre la acción de provecho del dolo ajeno en Chile –altamente debatido– véase a Pizarro Wilson (2009), Domínguez Águila (2009), Pino Emhart (2016), García Mekis (2016), González Castillo (2020), Momberg (2022), San Martín Neira (2022) y González González (2022). Compárese con Prado (2018). THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Esta es una interesante acción restitutoria que debe tomarse como referencia para una futura reforma peruana. Entonces, queda como tema en agenda para el legislador peruano el estudio de la acción de provecho del dolo ajeno y su incorporación en el derecho peruano. La acción de provecho del dolo ajeno, como su nombre lo indica, está pensada para aquellas situaciones donde alguien termina beneficiado del ilícito de otro. Como tal, la norma dispone diferentes consecuencias según los intervinientes en el ilícito. Frente al infractor, es claro que será procedente una acción resarcitoria y otra frente al beneficio ilícito27, pero también será aplicable alguna acción frente a las personas que no hayan cometido el ilícito de manera directa pero igual se beneficien de este. Por ello, el artículo 1458 señala que los beneficiados que no han cometido el ilícito responden “hasta concurrencia del provecho que han reportado del dolo” (Código Civil [Chile], 2000), o “hasta concurrencia de lo que valga el provecho” (Código Civil [Chile], 2000) como señala el artículo 2316. Por la forma en cómo está redactada la norma, la doctrina chilena debate si se trata de una acción indemnizatoria o restitutoria. Sin perjuicio de ello, entiéndase como una acción restitutoria enfocada en el beneficio ilícito. Pizarro Wilson la considera una institución maravillosa y de muchas problemáticas (2020). El caso chileno emblemático fue Inverlink. Enzo Bertinelli, gerente general de Inverlink Corredora de Bolsa, representante legal y socio de Holding Inverlink, mantenía un romance con Pamela Andrada, quien fue secretaria de Carlos Massad, presidente del Banco Central de Chile, cuando un 29 de enero de 2003 se enteró que su secretaria filtraba información confidencial. Durante la investigación, también se averiguó que Inverlink había sobornado a Javier Moya Cucurella, tesorero de la Corporación de Fomento de la Producción (en adelante, Corfo), una agencia estatal chilena. Esto fue con el fin de que le ‘prestara’ certificados de depósito a plazo para ser usados como garantía, poder seguir operando y sacar provecho de información confidencial. Dentro de este contexto, se plantearon acciones penales y civiles, entre las últimas, acciones de provecho del dolo ajeno contra terceros (bancos, corredores de bolsas, entre otros), por las ganancias obtenidas derivadas del uso de información confidencial por parte de Inverlink, aunque tales terceros no hayan cometido los ilícitos. A la fecha, Corfo ha recuperado 55 mil millones de pesos chilenos de los 85 mil millones robados28. En uno de los casos, la Corte Suprema de Chile explicó los requisitos de la acción de provecho del dolo ajeno: “a. Que exista una actuación dolosa; b. que un tercero reciba provecho del dolo ajeno; c. que el que recibe el provecho no sea cómplice del dolo” (Corte Suprema de Chile, 2012, como se citó en Prado, 2018, p. 356). Además, explica su diferencia con la responsabilidad civil: […] aun cuando se está en presencia de una acción derivada de la responsabilidad extracontractual, ella no tiene la naturaleza de una acción indemnizatoria propiamente tal, ya que la obligación de restituir no está determinada por el monto de los perjuicios sufridos; tampoco tiene una relación directa con el hecho ilícito cometido, puesto que el tercero que debe restituir lo percibido no es el autor del daño. Esta obligación se genera sólo por percibirse un beneficio producto del dolo que ha sido cometido por otra persona. Así, es este provecho o utilidad el que debe ser objeto de restitución, sin perjuicio de que igualmente es necesario que se produzca un daño a la víctima, pero éste sólo opera como factor de limitación del monto máximo a restituir (Prado, 2018, pp. 356-357). Así, la acción del provecho del dolo ajeno muestra que una acción restitutoria que se basa en el principio contra el enriquecimiento injustificado tiene una lógica distinta a la responsabilidad civil. Una diferencia fundamental es que no necesita de un criterio de imputación. A diferencia de la responsabilidad civil, donde la culpa u otro criterio de justificación es necesario para proceder con una reasignación patrimonial (el costo de la indemnización); en la restitución, la reasignación se produce porque, en primer lugar, se realizó una asignación sin justificación, es decir, sin importar la presencia de culpa u otro criterio. Por ello, si los inversionistas de un fondo de inversión obtienen beneficios por la administración del fondo sin tener conocimiento que las operaciones fueron realizadas con información confidencial o mediante la comisión de otros ilícitos, los inversionistas, aunque sean inocentes o no hayan cometido algún ilícito, responderán por la devolución del beneficio obtenido en virtud de la prohibición del enriquecimiento injustificado. 27 Aunque debo advertir que en el derecho chileno es discutible la procedencia de una acción contra los beneficios ilícitos frente al infractor del daño. La acción de provecho del dolo ajeno no está regulada para los autores sino frente a beneficiarios inocentes. Su aplicación extensiva hacia los autores es lo discutible. 28 Véase el reportaje de Vega (2023) por los 20 años del caso. THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. e-ISSN: 2410-9592 Sergio García Long rrencia de lo que valga el provecho (2000) [el énfasis es nuestro]. THEMIS 84 \| Revista de Derecho THĒMIS-Revista de Derecho 84. julio-diciembre 2023. pp. 149-171. ISSN: 1810-9934 167 THEMIS 84 \| Revista de Derecho RESPONSABILIDAD DE DIRECTORES POR GANANCIAS ILÍCITAS Bajo el derecho peruano, siempre será posible invocar, contra beneficiarios inocentes, la acción general por enriquecimiento injustificado, conforme a los artículos 1954 y 1955 del Código Civil. Sin embargo, el presupuesto de la subsidiariedad y la mala redacción de las normas peruanas harían nula su aplicación allí donde sea necesaria. Ante tal escenario, la acción de provecho del dolo ajeno debe ser tomado como referencia para una futura reforma del derecho peruano. Si los civilistas no están preparados para incorporarlo en el Código Civil con alcance general, se podría proceder con una reforma de la Ley General de Sociedades para regular de manera más completa las consecuencias negativas de actuaciones ilícitas en el ámbito empresarial, entre ellas, las derivadas de la responsabilidad de directores por ganancias ilícitas, incluso frente a inocentes beneficiarios. VI. REFLEXIONES FINALES El estudio de la responsabilidad de directores es particularmente interesante por la interacción de diversas áreas del derecho como lo es el civil, societario y bursátil. Este tema llega a ser tan complejo que, por ello mismo, da la impresión de haber sido una de las razones por las cuales ha sido omitido incluso por los propios especialistas. En el caso del derecho peruano, hubo un vacío en torno al tratamiento del beneficio ilícito obtenido por el director con su actuar ilícito, pero los expertos en derecho societario no le dieron el tratamiento necesario. En cierta manera, podría entenderse por la complejidad de la materia, que es parte del derecho civil y no societario. No obstante, a su vez, los especialistas en civil tampoco fueron los más interesados en estudiar este supuesto especial de responsabilidad civil que por sus particularidades requiere, además, el estudio de la restitución y el enriquecimiento injustificado, las cuales son materias usualmente poco perfeccionadas entre los mismos expertos en derecho civil. 168 ilícitos de directores que generan beneficios ilícitos sean correctamente reguladas. En este sentido, el derecho peruano aún tiene mucho que aprender en relación con la restitución, disuasión y punición en el derecho corporativo, teniendo en cuenta que la responsabilidad de directores es una buena oportunidad para ponerlo en práctica. REFERENCIAS Abdala, M. (2014). La aplicación de la denominada business judgment rule en el derecho argentino. Revista de Derecho Universidad del Norte, (42), 264-284. Alcalde, E. 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https://openalex.org/W4237103508	https://www.qeios.com/read/YXLYXV/pdf	English	null	Gastric Adenomatous Polyp	Definitions	2,020	cc-by	103	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Gastric Adenomatous Polyp National Cancer Institute Qeios ID: YXLYXV · https://doi.org/10.32388/YXLYXV Source National Cancer Institute. Gastric Adenomatous Polyp. NCI Thesaurus. Code C95773. National Cancer Institute. Gastric Adenomatous Polyp. NCI Thesaurus. Code C95773. National Cancer Institute. Gastric Adenomatous Polyp. NCI Thesaurus. Code C95773 A neoplastic polyp that arises from the stomach. It is usually associated with intestinal- type differentiation. The presence of high grade dysplasia and a size of more than 2 cm are factors that increase the risk of malignant transformation. Qeios ID: YXLYXV · https://doi.org/10.32388/YXLYXV 1/1
https://openalex.org/W1531761146	https://bmcresnotes.biomedcentral.com/counter/pdf/10.1186/s13104-015-1237-2	English	null	Baseline characteristics in stroke patients with atrial fibrillation: clinical trials versus clinical practice	BMC research notes	2,015	cc-by	4,896	© 2015 Tanislav et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tanislav et al. BMC Res Notes (2015) 8:262 DOI 10.1186/s13104-015-1237-2 Tanislav et al. BMC Res Notes (2015) 8:262 DOI 10.1186/s13104-015-1237-2 Abstract Background: When applying information gathered from medical research to the clinical setting, it is imperative that the sample of the investigated patients be representative of the clinical population. Because of this fact, it is necessary to closely examine the sample’s baseline characteristics in clinical trials. Methods: We analysed baseline data of relevant trials investigating considerable proportions of patients with atrial fibrillation (AF) in the secondary stroke prevention: EAFT, SIFA, Active-W, BAFTA, RE-LY, AVERROES, ARISTOTLE and ROCKET AF. For comparing baseline data stroke patients with AF documented in a statutory stroke registry were con- sidered. In a subgroup of patients (members of a large insurance) data on subsequent prescription for oral anticoagu- lants (oAK) were available. Results: In the stroke registry (n = 15,886) the mean age was higher than in the selected clinical trials (mean 77.7 versus 70–72 years). Among insurance members (n = 1,828), those with a prescription for oAK (n = 827) were older than patients recruited in clinical trials (mean 75.1 versus 70–72 years). Results also showed that the male sex was overrepresented in clinical trials (59–63% versus 46%). The distribution of vascular risk factors in recent clinical trials was comparable to proportions in the registry (hypertension: 77–85% versus 80%; diabetes mellitus: 20–26% versus 27%). Conclusions: The majority of stroke patients with AF in the clinical setting are considerably older than those included in clinical trials. While the distribution of vascular risk factors in clinical trials corresponds to proportions observed in clinical practice, an overrepresentation of the male sex in clinical trials is evident. Keywords: Stroke, Oral anticoagulation, Atrial fibrillation, Care delivery, New anticoagulants Keywords: Stroke, Oral anticoagulation, Atrial fibrillation, Care delivery, New anticoagulants the most important and most often discussed confound- ing variables is selection bias, which occurs when con- sidering patients to participate in a study [1]. Applying information derived from medical research into the clini- cal setting depends on the generalizability of the investi- gated patients to the population of interest. Christian Tanislav1, Sonja Milde2, Sabine Schwartzkopff3, Björn Misselwitz4, Nicole Sieweke1 and Manfred Kaps1 Christian Tanislav1, Sonja Milde2, Sabine Schwartzkopff3, Björn Misselwitz4, Nicole Sieweke and Manfred Kaps1 Christian Tanislav1, Sonja Milde2, Sabine Schwartzkopff3, Björn Misselwitz4, Nicole Sieweke1 and Manfred Kaps1 Abstract Background: When applying information gathered from medical research to the clinical setting, it is imperative tha the sample of the investigated patients be representative of the clinical population. Because of this fact, it is necessar to closely examine the sample’s baseline characteristics in clinical trials. Methods: We analysed baseline data of relevant trials investigating considerable proportions of patients with atrial fibrillation (AF) in the secondary stroke prevention: EAFT, SIFA, Active-W, BAFTA, RE-LY, AVERROES, ARISTOTLE and ROCKET AF. For comparing baseline data stroke patients with AF documented in a statutory stroke registry were con sidered. In a subgroup of patients (members of a large insurance) data on subsequent prescription for oral anticoagu lants (oAK) were available. Results: In the stroke registry (n = 15,886) the mean age was higher than in the selected clinical trials (mean 77.7 versus 70–72 years). Among insurance members (n = 1,828), those with a prescription for oAK (n = 827) were older than patients recruited in clinical trials (mean 75.1 versus 70–72 years). Results also showed that the male sex was overrepresented in clinical trials (59–63% versus 46%). The distribution of vascular risk factors in recent clinical trials was comparable to proportions in the registry (hypertension: 77–85% versus 80%; diabetes mellitus: 20–26% versus 27%). Conclusions: The majority of stroke patients with AF in the clinical setting are considerably older than those included in clinical trials. While the distribution of vascular risk factors in clinical trials corresponds to proportions observed in clinical practice, an overrepresentation of the male sex in clinical trials is evident. Keywords: Stroke, Oral anticoagulation, Atrial fibrillation, Care delivery, New anticoagulants Correspondence: christian.tanislav@neuro.med.uni‑giessen.de 1 Department of Neurology, Justus Liebig University, Klinikstrasse 33, 35392 Giessen, Germany Full list of author information is available at the end of the article Methods We evaluated the baseline data of previous clinical trials with substantial proportions of stroke patients with atrial fibrillation, including: EAFT, SIFA, Active-W, BAFTA, RE-LY, AVERROES, ARISTOTLE and ROCKET AF [2–10]. Characteristics such as age, sex, vascular risk fac- tors including hypertension and diabetes mellitus were investigated. For RE-LY, ARISTOTLE and ROCKET AF baseline data and frequencies calculated in the stroke subgroups were collected for analysis. Among this sample, we selected a subgroup of patients (n = 827) from a large health insurance consortium with evidence of a prescription for oral anticoagulants (oAK). For this subgroup insurance data between 2005 and 2007 was used. In order to identify pertinent members of this insurance, we examined patients matching a set of crite- ria including date of birth, date of hospital admission and the admitting hospital. This data was linked in a pseu- donymous manner. For our analysis, we selected patients without further hospitalisation in a 30-day period within 90 days after discharge (n = 1,828). We defined evidence of a prescription for oral anticoagulants (including phen- procoumaron, warfarin and coumadin) as a marker of anticoagulation. This information was gathered from the insurance claims; 827 patients were identified. For the categorical variables, data was presented in propor- tions. A Chi squared test was used to compare propor- tions between the entire sample identified in the Hessian stroke registry, the EAFT and SIFA’s samples, and the subgroups of patients with a previous stroke or TIA in RE-LY, ARISTOTLE and ROCKET AF. For comparison, we gathered baseline data from ischaemic stroke or TIA patients with AF documented (2004–2010) in the stroke registry (n = 15,886) of the Institute of Quality Assurance Hesse (Geschäftsstelle für Qualitätssicherung, GQH) (Figure 1) [11]. Patients with a haemorrhagic stroke or intracranial bleeding 151.158 cases of ischemic stroke or TIA, bleedings and other aetiologies documented in the hessian registry (2004-2010) 18.154 bleedings or other aetiologies (e.g. Background Evidence-based medicine, identified in clinical trials, is crucial to facilitate appropriate decision-making in patient care. However, patients in clinical trials inevitably represent a selected sample of the population. As a result, physicians might challenge the external validity especially in specific subgroups such as elderly patients. Addition- ally, different sources of bias might distort results. One of A large number of studies have shown the efficacy of oral anticoagulation (OA) in atrial fibrillation (AF) [2–10]. These studies evaluated primary and second- ary stroke prevention with results demonstrating the substantial benefit of OA [2–10]. Therefore, it is crucial to determine how the baseline characteristics of study Correspondence: christian.tanislav@neuro.med.uni‑giessen.de 1 Department of Neurology, Justus Liebig University, Klinikstrasse 33, 35392 Giessen, Germany Full list of author information is available at the end of the article Correspondence: christian.tanislav@neuro.med.uni‑giessen.de 1 Department of Neurology, Justus Liebig University, Klinikstrasse 33, 35392 Giessen, Germany Full list of author information is available at the end of the article Tanislav et al. BMC Res Notes (2015) 8:262 Page 2 of 6 samples compare to those of patients encountered in clinical practice and nation-wide populations. In the presented investigation, we aimed to analyse baseline characteristics in relevant clinical trials of atrial fibrillation when compared to stroke victims documented in a larger, more representative stroke registry sample. samples compare to those of patients encountered in clinical practice and nation-wide populations. were excluded from this study sample. Patients with moderate disability (modified Rankin scale ≤3 as assessed on discharge) were selected because these patients might most meet the criteria for measures in the secondary stroke prevention such as intake of oral anticoagulants. This selection might further provide certain structural equality regarding the disability sta- tus when comparing patients in clinical trials versus daily practice. In the presented investigation, we aimed to analyse baseline characteristics in relevant clinical trials of atrial fibrillation when compared to stroke victims documented in a larger, more representative stroke registry sample. Methods CVT) 133.004 cases of ischemic stroke or TIA 28.971 documented cases of atrial fibrillation 10.851 cases with documented mRs>3 on discharge 18.120 cases with documented mRs≤3 on discharge 15.886 with documented discharge at home (n=11.336) or to an department for rehabilitation (n=4.550) 1828 patients without obvious contraindications for anticoagulants 2101 patients affiliated to the insurance treated 2005 - 2007 119 cases of recurrent stroke 103 deceased within the 90 day follow-up 51 cases without a 30 days interval at home or incomplete 90 days follow up Figure 1 Patient selection within the stroke registry data set. TIA refers to transient ischaemic attack, CVT refers to cerebral vein throm- bosis, mRs refers to modified Rankin scale. The protocol of the present study was reviewed and approved by the ethical committee of the medical faculty of the Justus Liebig University Giessen. Results The burden of leukoaraiosis, microbleeds and silent brain infarctions, which correlates with the presence of vascu- lar risk factors, substantially increases beyond 75 years of age; this results in a higher risk for intracerebral haem- orrhage [15–20]. In the BAFTA selection process there was a preference for elderly individuals without consid- erable vascular burden. Therefore, those patients with supposedly higher risk for bleeding were less preferred for inclusion. Furthermore, regarding secondary stroke prevention, the BAFTA trial is of limited benefit, as only a small proportion of patients who have had a previous stroke were investigated (13%) [8]. The proportion of hypertension and diabetes mellitus in the registry population, in the selected trials, and in the subgroups of patients with a previous stroke or TIA were similar (hypertension 80, 79 and 79.8%); diabetes: 27, 29 and 29%) (Table 1). Earlier investigations (EAFT, SIFA and BAFTA) focused on patients with a moder- ate vascular burden and had lower proportions of these risk factors (hypertension: 44–55% of the patients; dia- betes mellitus: 13–16% of the patients). In recent tri- als (Active-W, RE-LY, AVERROES, ARISTOTLE and ROCKET AF) these factors are of similar proportions when compared to the registry population (hyperten- sion: 77–86% of the patients; diabetes mellitus: 20–26% of the patients). Using a Chi squared test, we compared the propor- tions of these characteristics among the entire study sam- ple, which was composed of the Hessian stroke registry and the EAFT and SIFA populations and the subgroups of patients with stroke or TIA in RE-LY, ARISTORLE and ROCKET AF. Results showed that in the majority of the cases p values were <0.001 (Table 1). However, no relevant differences were identified in the comparison between proportions of patients with diabetes mellitus in the registry study group and the stroke or TIA sub- group in ARISTOTLE (27 versus 26%, P = 0.1317) and the gender comparison with SIFA (males: 46 versus 47%, P = 0.5889). ROCKET AF claims to address more elderly patients (median 73 years) [10]. However, in the subgroup of patients with a previous stroke, the median age decreases (median 71 years) corresponding with the subgroups in RE-LY (mean 70.5 years) and ARISTOTLE (mean 70.1 years) [7]. In this case the additional burden of a previous cerebrovascular event a further bias to younger patients. In many clinical trials males represent the majority of the participants [2–4, 21, 22]. Results In the GQH registry sample (n = 15,886) the mean age was 77.7 years. Apart from the BAFTA trial (mean age 81.5 years), the mean age in the selected trials and sub- groups of patients with a previous stroke or TIA ranged between 70 and 72 years. In the insurance subgroup the mean age was comparable to the registry sample (77.6 versus 77.7 years) (Table 1). In the insurance subgroup of patients with a prescription for oAK, the mean age was 75.1 years and in those without evidence of a prescription the mean age was 79.8.h It is also important to note that factors such as age might impact a patient’s inclusion in clinical trials on OA. In these clinical trials, there are strict inclusion criteria that determine whether or not a patient is included. The factor age may bias the inclusion in studies due to the increased risk of bleeding in the elderly and as a result elderly patients may be less selected for inclusion in clini- cal trials for OA. In our GQH registry sample the mean age within the subgroup of patients with prescriptions for oAK was lower than in the total registry sample (75.1 vs. 77.7 years respectively); however, it still ranged above the mean age observed in clinical trials (75.1 vs. 70–72 years).h The proportion of males in the registry sample was 46%, while in the insurance subgroup only 41% of patients were male. The proportion of males increased in the insurance subgroup of patients with an oAK pre- scription to 48.1%. Apart from SIFA (proportion of male patients 47%) there was a majority of males reaching 55–66% in the selected clinical trials (Table 1).h g ( y ) The BAFTA trial, which included oAK for individu- als with AF over 75 years of age, reported a 2.4% abso- lute risk reduction per year and a similar risk for major bleeding when compared to aspirin (1.9 versus 2.0%) [8]. While distributions of vascular risk factors in the recent trials are comparable to proportions calculated in the registry population, participants in the BAFTA-trial were less affected by vascular risk factors (hypertension: 80 versus 54%; diabetes mellitus 27 versus 13%) [8]. Results In contrast, in nation- wide data collections, males do not exceed 50% [23–25]. In line with this finding, the proportion of male partici- pants in our nationwide registry was also lower than in clinical trials (46% versus 55–66%). This might also be a result of the increased age of the participants as females suffer from stroke later in life than males [18]. Consistent with this assumption, the slight decrease in age within the insurance subgroups (mean 77.6 versus 75.1 years) GQHh The GQH database is a mandatory nationwide hospital- based registry spanning more than 95% of all ischaemic strokes, transient ischaemic attacks (TIA), and intracer- ebral haemorrhages in more than 6 million residents of Hesse, Germany. The GQH includes data of acute inpa- tient treatment, as well as factors proven to be relevant for the course and the prognosis of a stroke. For quality assurance purposes, the acquisition of this data is regu- lated by law and implemented as a guideline, which is elaborated by the Federal Joint Committee for hospital quality assurance in accordance with Volume V of the Social Insurance Code (§137 SGB V and §135a SGB V). Based on this regulation, the Hesse State Hospital Law contains a provision that allows the GQH to record such data legally. The publication of aggregate quality assur- ance data has been approved by the Hesse Data Protec- tion Commissioner, so no data protection problem arises here [12–14]. Tanislav et al. BMC Res Notes (2015) 8:262 Page 3 of 6 comparable in recent clinical trials with those seen in clinical practice. Discussion In the registry population patients were on average 7 years older than those included in the selected studies [2–10]. Apart from age, the comparison of baseline char- acteristics revealed an overrepresentation of male indi- viduals in clinical trials. The proportions of vascular risk factors such as hypertension and diabetes mellitus were Page 4 of 6 Tanislav et al. BMC Res Notes (2015) 8:262 Table 1 Comparison of baseline characteristics between the registry cohorts versus relevant studies providing evidence on secondary stroke prevention in atrial fibrillation a Data refers to the subgroup of patients with a previous stroke or TIA. b Indobufen is a reversible inhibitor of platelet cyclooxygenase activity. † p value <0.001 calculated with a Chi square test (comparison between the entire cohort determined in the hessian stroke registry (n = 15,886) and the EAFT cohort, the SIFA cohort and the stroke subgroups in RE-LY, ARISTOTLE and ROCKET AF). Hessian stroke registry (2004–2010) n = 15,886 Insurance (total Cohort) n = 1,828 Insurance (subgroup of patients with prescrip- tion for AK) n = 827 EAFT (1993) n = 669 SIFA (1997) n = 916 Active-W (2006) n = 6,706 BAFTA (2007) n = 973 AVERROES (2011) n = 5,599 RE-LY (2010) n = 18,113 ARISTOTLE (2011) n = 18,201 ROCKET AF (2012) n = 14,264 Age (years) mean, ±SD 77.7 (±9) 77.6 (±8) 75.1(± 8) 71 (±7) 72 (± 8) 70.2 81.5 (±8) 70 ± 9 70 (±9)a 70.1 (±9.7)a 71 (median)a (IQR 64–77) Sex Male 7,312 (46%) 764 (41.8%) 398 (48.1%) 395 (59%) 430 (47%) 4,430 (66%) 531 (55%) 3,277 (59%) 2,279 (63%)a,† 2,152 (63%)a,† 4,538(61%)a,† Female 8,574 (53%) 1,064 (58%) 429 (51.9%) 274 (41%) 486 (53%) 2,276 (34%) 442 (45%) 2,322 (41%) 1,344 (37%)a,† 1,284 (37%)a,† 2,930(39%)a,† Risk factors Hypertension 12,679 (80%) 1,444 (79%) 660 (79.8%) 294 (44%) 506 (55%) 5,522 (82%) 528 (54%) 4,837 (86%) 2,783 (77%)a,† 2,858 (83%)a,† 6,343 (85%)a,† Diabetes mellitus 4,371 (27%) 530 (29%) 240 (29%) 87 (13%) 144 (16%) 1,429 (21%) 129 (13%) 1,096 (20%) 816 (22%)a,† 902 (26%)a 5,695 (24%)a,† Proportion of Strokes or TIAs within the study population 100% 100% 100% 100% 100% 15% (n = 1,006) 13% (n = 124) 14% (n = 764) 20% (n = 3,623) 19% (n = 3,436) 52% (n = 7,468) Comparison of agents – – – Warfarin vs. placebo and aspirin vs. placebo Indobufenb vs. warfarin Aspi- rin + clopi- dogrel vs. References 1. Miller KD, Rahman ZU, Sledge GW Jr (2001) Selection bias in clinical trials. Breast Dis 14:31–40 1. Miller KD, Rahman ZU, Sledge GW Jr (2001) Selection bias in clinical trials. Breast Dis 14:31–40 2. EAFT (European Atrial Fibrillation Trial) Study Group (1993) Secondary prevention in non-rheumatic atrial fibrillation after transient ischaemic attack or minor stroke. Lancet 342:1255–1262 3. Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A et al (2009) Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med 361:1139–1151 4. Diener HC, Connolly SJ, Ezekowitz MD, Wallentin L, Reilly PA, Yang S et al (2010) Dabigatran compared with warfarin in patients with atrial fibrillation and previous transient ischaemic attack or stroke: a subgroup analysis of the RE-LY trial. Lancet Neurol 9:1157–1163 Abbreviations O O l OA: Oral anticoagulation; AF: atrial fibrillation; GQH: stroke registry of the Institute of quality assurance Hesse (Geschäftsstelle für Qualitätssicherung, GQH); TIA: transient ischaemic attacks; oAK: oral anticoagulants; mRS: modified Rankin scale. 12. Stolz E, Hamann GF, Kaps M, Misselwitz B (2011) Regional differences in acute stroke admission and thrombolysis rates in the German federal state of Hesse. Dtsch Arztebl Int 108:607–611 13. Jauss M, Schutz HJ, Tanislav C, Misselwitz B, Rosenow F (2010) Effect of daytime, weekday and year of admission on outcome in acute ischaemic stroke patients treated with thrombolytic therapy. Eur J Neurol 17:555–561 13. Jauss M, Schutz HJ, Tanislav C, Misselwitz B, Rosenow F (2010) Effect of daytime, weekday and year of admission on outcome in acute ischaemic stroke patients treated with thrombolytic therapy. Eur J Neurol 17:555–561 Discussion warfarin Warfarin vs. aspirin Apixaban vs. aspirin Dabigatran vs. warfarin Apixaban vs. warfarin Rivoroxaban vs. warfarin Tanislav et al. BMC Res Notes (2015) 8:262 Page 5 of 6 Page 5 of 6 corresponded with a higher proportion of male subjects (41.8–48.1%). Competing interests The authors declare that they have no competing interests. Author details 1 1 Department of Neurology, Justus Liebig University, Klinikstrasse 33, 35392 Giessen, Germany. 2 Dresden International University, Dresden, Germany. 3 AOK Division of the Federal State of Hesse, Eschborn, Frankfurt, Germany. 4 Geschäftsstelle Qualitätssicherung Hessen (GQH), Eschborn, Frankfurt, Germany. 1 Department of Neurology, Justus Liebig University, Klinikstrasse 33, 35392 Giessen, Germany. 2 Dresden International University, Dresden, Germany. 3 AOK Division of the Federal State of Hesse, Eschborn, Frankfurt, Germany. 4 Geschäftsstelle Qualitätssicherung Hessen (GQH), Eschborn, Frankfurt, Germany. Even though EAFT and SIFA included a small number of patients with a relatively moderate burden of vascular risk factors, we considered these two ground-breaking trials, because both specifically addressed stroke patients in secondary prevention [2, 9]. In these trials the signifi- cant advantage of oAK over a therapy with platelet inhib- itors was demonstrated. In contrast, the vast majority of AF trials provided evidence for the safety and efficacy of OA in the general population of both stroke and non- stroke patients. The percentage of stroke patients ranged from 13 to 25% in these trials. Despite methodological limitations, specifically the small number of patients, these subgroup analyses indicated the efficacy of OA in patients with AF after having a stroke [4, 5, 22]. Indisput- ably, the participation of stroke patients in AF trials is crucial because they face higher risks of both recurrent stroke and bleeding complications [18]. Because of this higher risk of a worse outcome, it is difficult to extrapo- late the benefit of OA in secondary prevention from pri- mary prevention studies. Trials designed to specifically investigate patients within secondary stroke prevention would be ideal. Study sponsorship There was no funding nor any funders involved in the study. There was no funding nor any funders involved in the study. Received: 9 January 2014 Accepted: 17 June 2015 Received: 9 January 2014 Accepted: 17 June 2015 Received: 9 January 2014 Accepted: 17 June 2015 Conclusions Morocutti C, Amabile G, Fattapposta F, Nicolosi A, Matteoli S, Trappolini M et al (1997) Indobufen versus warfarin in the secondary prevention of major vascular events in nonrheumatic atrial fibrillation. SIFA (Studio Italiano Fibrillazione Atriale) Investigators. Stroke 28:1015–1021 10. Patel MR, Mahaffey KW, Garg J, Pan G, Singer DE, Hacke W et al (2011) Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med 365:883–891 10. Patel MR, Mahaffey KW, Garg J, Pan G, Singer DE, Hacke W et al (2011) Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med 365:883–891 11. Foerch C, Misselwitz B, Sitzer M, Berger K, Steinmetz H, Neumann-Haefelin T (2005) Difference in recognition of right and left hemispheric stroke. Lancet 366:392–393f 12. Stolz E, Hamann GF, Kaps M, Misselwitz B (2011) Regional differences in acute stroke admission and thrombolysis rates in the German federal state of Hesse. Dtsch Arztebl Int 108:607–611 OA: Oral anticoagulation; AF: atrial fibrillation; GQH: stroke registry of the Institute of quality assurance Hesse (Geschäftsstelle für Qualitätssicherung, GQH); TIA: transient ischaemic attacks; oAK: oral anticoagulants; mRS: modified Rankin scale. 11. Foerch C, Misselwitz B, Sitzer M, Berger K, Steinmetz H, Neumann-Haefelin T (2005) Difference in recognition of right and left hemispheric stroke. Lancet 366:392–393 Disclosures Disclosures All authors report no disclosures related to the manuscript. Compliance with ethical guidelines Compliance with ethical guidelines Conclusions Despite clear evidence of the efficacy of OA, there is an age-dependent variable when considering stroke patients for this treatment. There is a considerable difference in age of patient populations in clinical trials when compared with clinical practice. Results have shown that in clinical trials younger patients (70–72 years) are preferable; how- ever, in clinical practice, patients selected for OA are older (75.1 years) than those in trials, but also younger than those who are selected for alternative therapy (79.8 years). With regard to the vascular risk factors of hypertension and diabetes mellitus, we found that the proportions in recent clinical trials are comparable to those in clinical practice. Both patients and clinicians would benefit from further investigations into secondary stroke prevention when selecting patients suitable for OA. Currently evi- dence is generally collected from studied in the primary stroke prevention with considerably younger patients. 5. Easton JD, Lopes RD, Bahit MC, Wojdyla DM, Granger CB, Wallentin L et al (2012) Apixaban compared with warfarin in patients with atrial fibrillation and previous stroke or transient ischaemic attack: a subgroup analysis of the ARISTOTLE trial. Lancet Neurol 11:503–511 6. Granger CB, Alexander JH, McMurray JJ, Lopes RD, Hylek EM, Hanna M et al (2011) Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med 365:981–992 7. Hankey GJ, Patel MR, Stevens SR, Becker RC, Breithardt G, Carolei A et al (2012) Rivaroxaban compared with warfarin in patients with atrial fibrillation and previous stroke or transient ischaemic attack: a subgroup analysis of ROCKET AF. Lancet Neurol 11:315–322 8. Mant J, Hobbs FD, Fletcher K, Roalfe A, Fitzmaurice D, Lip GY et al (2007) Warfarin versus aspirin for stroke prevention in an elderly community popu- lation with atrial fibrillation (the Birmingham Atrial Fibrillation Treatment of the Aged Study, BAFTA): a randomised controlled trial. Lancet 370:493–503 8. Mant J, Hobbs FD, Fletcher K, Roalfe A, Fitzmaurice D, Lip GY et al (2007) Warfarin versus aspirin for stroke prevention in an elderly community popu- lation with atrial fibrillation (the Birmingham Atrial Fibrillation Treatment of the Aged Study, BAFTA): a randomised controlled trial. Lancet 370:493–503 9. Morocutti C, Amabile G, Fattapposta F, Nicolosi A, Matteoli S, Trappolini M et al (1997) Indobufen versus warfarin in the secondary prevention of major vascular events in nonrheumatic atrial fibrillation. SIFA (Studio Italiano Fibrillazione Atriale) Investigators. Stroke 28:1015–1021 9. 13. Jauss M, Schutz HJ, Tanislav C, Misselwitz B, Rosenow F (2010) Effect of daytime, weekday and year of admission on outcome in acute ischaemic stroke patients treated with thrombolytic therapy. Eur J Neurol 17:555–561 MK and CT designed the study. CT and SM calculated the results. CT, SM, BM, SS, NS and MK were involved in data analysis and interpretation. They proved und contributed for important intellectual content. All authors read and approved the final manuscript. 14. Tanislav C, Milde S, Schwartzkopff S, Sieweke N, Kramer HH, Juene- mann M et al (2014) Secondary stroke prevention in atrial fibrillation: a challenge in the clinical practice. BMC Neurol 14:195. doi:10.1186/ s12883-014-0195-y.:95-0195 Authors’ contributions MK and CT designed the study. CT and SM calculated the results. CT, SM, BM, SS, NS and MK were involved in data analysis and interpretation. They proved und contributed for important intellectual content. All authors read and approved the final manuscript. 14. Tanislav C, Milde S, Schwartzkopff S, Sieweke N, Kramer HH, Juene- mann M et al (2014) Secondary stroke prevention in atrial fibrillation: a challenge in the clinical practice. BMC Neurol 14:195. doi:10.1186/ s12883-014-0195-y.:95-0195 14. Tanislav C, Milde S, Schwartzkopff S, Sieweke N, Kramer HH, Juene- mann M et al (2014) Secondary stroke prevention in atrial fibrillation: a challenge in the clinical practice. BMC Neurol 14:195. doi:10.1186/ s12883-014-0195-y.:95-0195 Tanislav et al. BMC Res Notes (2015) 8:262 Page 6 of 6 15. Schmidt R, Ropele S, Ferro J, Madureira S, Verdelho A, Petrovic K et al (2010) Diffusion-weighted imaging and cognition in the leukoariosis and disability in the elderly study. Stroke 41:e402–e408 22. Diener HC, Eikelboom J, Connolly SJ, Joyner CD, Hart RG, Lip GY et al (2012) Apixaban versus aspirin in patients with atrial fibrillation and previ- ous stroke or transient ischaemic attack: a predefined subgroup analysis from AVERROES, a randomised trial. Lancet Neurol 11:225–231 from AVERROES, a randomised trial. Lancet Neurol 11:225–231 16. Kalra L, Yu G, Perez I, Lakhani A, Donaldson N (2000) Prospective cohort study to determine if trial efficacy of anticoagulation for stroke prevention in atrial fibrillation translates into clinical effectiveness. BMJ 320:1236–1239 23. Palm F, Kleemann T, Dos SM, Urbanek C, Buggle F, Safer A et al (2012) Stroke due to atrial fibrillation in a population-based stroke registry (Lud- wigshafen Stroke Study) CHADS(2), CHA(2) DS(2)-VASc score, underuse of oral anticoagulation, and implications for preventive measures. Eur J Neurol 20:117–123 17. Darkow T, Vanderplas AM, Lew KH, Kim J, Hauch O (2005) Treatment pat- terns and real-world effectiveness of warfarin in nonvalvular atrial fibrilla- tion within a managed care system. Curr Med Res Opin 21:1583–1594 24. Giralt-Steinhauer E, Cuadrado-Godia E, Ois A, Jimenez-Conde J, Rodri- guez-Campello A, Soriano C et al (2013) Comparison between CHADS2 and CHA2 DS2-VASc score in a stroke cohort with atrial fibrillation. Eur J Neurol 20:623–628 18. Fang MC, Chang Y, Hylek EM, Rosand J, Greenberg SM, Go AS et al (2004) Advanced age, anticoagulation intensity, and risk for intracranial hemor- rhage among patients taking warfarin for atrial fibrillation. Ann Intern Med 141:745–752 25. Authors’ contributions Hylek EM, Evans-Molina C, Shea C, Henault LE, Regan S (2007) Major hemorrhage and tolerability of warfarin in the first year of therapy among elderly patients with atrial fibrillation. Circulation 115:2689–2696 19. Hylek EM, Evans-Molina C, Shea C, Henault LE, Regan S (2007) Major hemorrhage and tolerability of warfarin in the first year of therapy among elderly patients with atrial fibrillation. Circulation 115:2689–2696 20. Jeerakathil T, Wolf PA, Beiser A, Hald JK, Au R, Kase CS et al (2004) Cerebral microbleeds: prevalence and associations with cardiovascular risk factors in the Framingham Study. Stroke 35:1831–1835 21. Connolly SJ, Eikelboom J, Joyner C, Diener HC, Hart R, Golitsyn S et al (2011) Apixaban in patients with atrial fibrillation. N Engl J Med 364:806–817 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
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https://openalex.org/W2757339220	https://www.nature.com/articles/s41598-018-25556-9.pdf	English	null	Reprogramming, oscillations and transdifferentiation in epigenetic landscapes	Scientific reports	2,018	cc-by	11,748	Bivash Kaity1, Ratan Sarkar2, Buddhapriya Chakrabarti3 & Mithun K. Mitra 1 Waddington’s epigenetic landscape provides a phenomenological understanding of the cell differentiation pathways from the pluripotent to mature lineage-committed cell lines. In light of recent successes in the reverse programming process there has been significant interest in quantifying the underlying landscape picture through the mathematics of gene regulatory networks. We investigate the role of time delays arising from multi-step chemical reactions and epigenetic rearrangement on the cell differentiation landscape for a realistic two-gene regulatory network, consisting of self-promoting and mutually inhibiting genes. Our work provides the first theoretical basis of the transdifferentiation process in the presence of delays, where one differentiated cell type can transition to another directly without passing through the undifferentiated state. Additionally, the interplay of time-delayed feedback and a time dependent chemical drive leads to long-lived oscillatory states in appropriate parameter regimes. This work emphasizes the important role played by time-delayed feedback loops in gene regulatory circuits and provides a framework for the characterization of epigenetic landscapes. The rise of transcription factor mediated induced pluripotency1–6 has given rise to increased efforts in order to understand the epigenetic landscape that underpins the process of cellular differentiation. Yamanaka and co-authors have shown that four transcriptions factors (TF), Sox2, Oct4, Klf4 and c-Myc are enough to repro- gram fully differentiated cells to the pluripotent state, in mouse fibroblast cells1, as well as in human fibroblasts2. A different program, using a combination of the TFs Sox2, Oct4, NANOG, and LIN28 has also been shown to induce pluripotency in fully differentiated human cells3–5.hf p p y yf The process of cellular differentiation has traditionally been thought of in terms of Waddington’s epigenetic landscape7, using the metaphor of a ball rolling down a hill, with the final valley representing the differentiated states, the top of the hill representing the pluripotent state, and the valley chosen by a cell from among similar ones as cell fate decisions. The discovery of the reprogramming pathway has led to an interest in mathemati- cally modeling the epigenetic landscape though the underlying Gene Regulatory Networks (GRN). Initial studies using a toy single gene regulatory network studied the properties of cellular differentiation using a self-activating gene8–12. Later studies have expanded this work to model more realistic two gene networks, that are self-activating and mutually inhibiting11,13–17. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1IIT Bombay, Department of Physics, Mumbai, 400076, India. 2Indian Institute of Science, Centre for High Energy Physics, Bangalore, 560012, India. 3University of Sheffield, Department of Physics and Astronomy, Sheffield, S3 7RH, UK. Correspondence and requests for materials should be addressed to M.K.M. (email: mithun@phy.iitb.ac.in) Reprogramming, oscillations and transdifferentiation in epigenetic landscapes Received: 20 November 2017 Accepted: 19 March 2018 Published: xx xx xxxx Bivash Kaity1, Ratan Sarkar2, Buddhapriya Chakrabarti3 & Mithun K. Mitra 1 Bivash Kaity1, Ratan Sarkar2, Buddhapriya Chakrabarti3 & Mithun K. Mitra 1 These models have shown that the cell can follow different pathways during the differentiation and reverse programming pathways, and provided a more nuanced understanding of the epige- netic landscape11,13–16. An experimental attempt to elucidate the reverse programming pathway was made by Nagy and Nagy18, in which they attempted to clarify the pathway of a cell undergoing reverse differentiation through the use of a controlled time-dependent chemical drive. Somatic fibroblast cells derived from mouse iPSC have the four pluri- potency factors (Sox2, Oct4, Klf4 and c-Myc) incorporated in a latent state. These were activated through a single chemical factor, doxycycline, which was supplied for different durations. They observed that when the doxycy- cline input was provided for a time of less than ~7 days (dPNR), the cell stayed in the fully differentiated somatic state. On the other hand, on providing the doxycycline input for a period greater than ~14 days (dCPS), the cell made a transition to the induced pluripotent state. For a chemical drive between dPNR and dCPS, the system was neither in the initial state, nor did it reach the final pluripotent state, and was stuck in a new uncharacterized state, referred to as the “Area 51” state. This provided concrete evidence that the path followed by the cell on the reverse differentiation pathway is not necessarily the same as that during the forward differentiation process. 1IIT Bombay, Department of Physics, Mumbai, 400076, India. 2Indian Institute of Science, Centre for High Energy Physics, Bangalore, 560012, India. 3University of Sheffield, Department of Physics and Astronomy, Sheffield, S3 7RH, UK. Correspondence and requests for materials should be addressed to M.K.M. (email: mithun@phy.iitb.ac.in) Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 1 www.nature.com/scientificreports/ Figure 1. (a) A schematic of gene regulatory network. The two transcription factors X and Y are self-activating, and mutually inhibiting; (b) The bifurcation diagram of the non-delayed system of equations Eq. 1. The dashed green lines correspond to the two values of the feedback parameter a that were analysed in subsequent section. The other parameters were fixed at a0 = 0.5, b = 1.0, k = 1.0, n = 4, and S = 0.5. The velocity vector plots in the (x, y) plane for these two points are shown in (c) Regime B and (d) Regime A. Figure 1. (a) A schematic of gene regulatory network. Bivash Kaity1, Ratan Sarkar2, Buddhapriya Chakrabarti3 & Mithun K. Mitra 1 We demonstrated a proof-of-concept of this time-delayed feedback regulation in the context of a single-gene time evolution, where we showed that a competition between the delay timescale and the timescale of the chemical drive can set up long-lived oscillations in the concentration of the transcription factor and inter- preted this as a possible explanation of the cells caught in limbo during the differentiation process. p p p g gf p In the current work, we present an analysis of the interplay of time-delayed feedback and a time-dependent chemical drive on a more realistic two-gene regulatory network. The underlying motif of the GRN consists of two self-activating and mutually inhibiting transcription factors, which is an extremely common motif observed in biological GRNs11,13–17. Examples include the the differentiation pathway of the Common Myeloic Progenitor (CMP) cell which differentiates into the Myeloid and Erythroid cell fates depending on the over-expression of the TF PU.1 or GATA1 respectively. The myeloid and Erythroid cell lines differentiate further to produce the different cell fates in the myeloid lineage30–34. While a full analysis of the reverse differentiation pathway involves multiple genes mediated by multiple TFs, we restrict our analysis to this smaller subset of two-gene mediated regulatory networks. We show that starting from a differentiated state, it is possible to follow the reverse pathway to reach the undifferentiated state. Depending on the interplay between the chemical drive and the delayed feedback, we observe long-lived oscillatory states in this two-TF GRN, which might provide an explanation of the unchar- acterized states observed in reverse differentiation experiments18. We also show that this same framework can provide a theoretical basis for the phenomenon of transdifferentiation, where one differentiated cell can switch to a different differentiated cell fate without passing through the undifferentiated or pluripotent state. To the best of our knowledge, this is the first study that provides a theoretical underpinning to the transdifferentiation process, which is well known in experimental contexts35–42.iif In subsequent sections, we first define the differential equations that govern the time evolution of the TFs in the presence of a time-dependent chemical drive and time-delayed feedback. We then present results in different parameter regimes, which show oscillatory states and transdifferentiation events. Further, we characterize the phase space of the model in terms of the drive time and the delay timescale as well as characterize the oscillations observed in greater detail. Bivash Kaity1, Ratan Sarkar2, Buddhapriya Chakrabarti3 & Mithun K. Mitra 1 Finally, we discuss the relevance of the current model in the context of generating a more detailed picture of epigenetic landscapes. Bivash Kaity1, Ratan Sarkar2, Buddhapriya Chakrabarti3 & Mithun K. Mitra 1 The two transcription factors X and Y are self-activating, and mutually inhibiting; (b) The bifurcation diagram of the non-delayed system of equations Eq. 1. The dashed green lines correspond to the two values of the feedback parameter a that were analysed in subsequent section. The other parameters were fixed at a0 = 0.5, b = 1.0, k = 1.0, n = 4, and S = 0.5. The velocity vector plots in the (x, y) plane for these two points are shown in (c) Regime B and (d) Regime A. In a previous work12, we argued that modeling of the underlying gene regulatory network requires careful consideration of the time delays associated with the feedback regulation of the transcription factors. The reverse differentiation process can take place over a duration of weeks, and is accompanied by a host of changes in the epigenetic markers that characterize the state of the cell, such as changes in histone protein expression levels19,20, as well as changes in the chromatin compaction characteristics21–29. The timescales associated with these physi- cal changes would impact the feedback regulation, and could have a critical impact in assessing the differentia- tion pathways. We demonstrated a proof-of-concept of this time-delayed feedback regulation in the context of a single-gene time evolution, where we showed that a competition between the delay timescale and the timescale of the chemical drive can set up long-lived oscillations in the concentration of the transcription factor and inter- preted this as a possible explanation of the cells caught in limbo during the differentiation process. In a previous work12, we argued that modeling of the underlying gene regulatory network requires careful consideration of the time delays associated with the feedback regulation of the transcription factors. The reverse differentiation process can take place over a duration of weeks, and is accompanied by a host of changes in the epigenetic markers that characterize the state of the cell, such as changes in histone protein expression levels19,20, as well as changes in the chromatin compaction characteristics21–29. The timescales associated with these physi- cal changes would impact the feedback regulation, and could have a critical impact in assessing the differentia- tion pathways. Results The time evolution equations incorporating a simultaneous chemical drive and delayed feedback is given by, τ τ τ τ τ τ = Θ − + − + − + + − − = Θ − + − + − + + − − dx dt a d t a x t S x t b S S y t kx t dy dt a d t a y t S y t b S S x t ky t ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (2) n n n n n n n n n n n n 0 1 1 1 1 2 0 2 2 2 2 1 (2) the first term represents the time dependent drive, which is modeled by the Heaviside theta function, with the parameter d setting the duration for which the input is provided. Feedback processes mediated by the TF x are characterised by a delay timescale τ1, while those mediated by the TF y are characterised by a delay timescale τ2. The parameters a1/2 and b1/2 controls the strength of the positive and negative feedback for the two transcription factors. In order to reduce the dimensionality of the parameter space, we shall assume them to be the same throughout the remainder of this work, i.e. a1 = a2 = a and b1 = b2 = b. The decay constant is assumed to be the same for both TFs for similar reasons. A brief discussion on the implications of asymmetric parameters is pre- sented in subsequent sections.hf q The nonlinear delay differential equations were solved using MATLAB (Mathworks, Inc.) using the dde23 solver. The solutions of the equations yields the trajectories of the system in the (x, y) concentration space. The fixed points and the bifurcation diagram of the non-delayed model was obtained by plotting the nullclines of the system of equations and subsequently performing a linear stability analysis. The trajectories obtained were ana- lyzed in the long-time limit to classify the phase space of the system. y g y p p y In the absence of a time-dependent drive and delayed feedback (Eq. 1), the behaviour of the system can be understood in terms of a bifurcation analysis of the phase space. The bifurcation diagram for this system is shown in Fig. 1(b). Results The phase space analysis shows that this two gene network is a multi-stable system, with a region of bistability for < ∼ . a a 0 77 c , and a region of multistability for a > ac. For a < ac, the system has two stable steady states (solid blue lines), separated by a central unstable state (dashed red line). The phase space velocities in this region are shown in Fig. 1(c), which clearly shows the two stable attractors separated by the central unstable state. For a > ac, the system undergoes a pitchfork bifurcation where central unstable state become stable and two new unstable states appear symmetrically on either side of the central steady state. The phase space velocity for this region is shown in Fig. 1(d). The central steady state concentrations of the two TFs are equal i.e., x = y, which in our model corresponds to the undifferentiated (or progenitor) state. The two terminal attractors, one with x = u, y ≈ 0 and another with x ≈ 0, y = u, correspond to the two differentiated states. Thus, in each of the differentiated states, one TF completely dominates over the other (  x y or  x y), with the weaker TF being effectively silenced. These then correspond to two distinct valleys of the epigenetic landscape which represent the two differ- entiated states. A comprehensive phase space and bifurcation analysis for this non-delayed model has been stud- ied previously13,15.ff We study the reverse differentiation process, starting from one of the differentiated steady state, and follow the time evolution of the system for different values of the drive duration and delay time. Since the bifurcation diagram shows two distinct dynamical behaviour regimes, i.e., for a > ac (Regime A), and a < ac (Regime B), we study the system at a representative point in both these regimes. The parameters chosen for this paper are marked with a dotted line in the bifurcation diagram Fig. 1(b). Long lived oscillatory states. We first report results for the set of model parameters given by, drive ampli- tude a0 = 0.5, the positive and negative feedback amplitudes a = 1.0, and b = 1.0, decay constant k = 1.0, Hill expo- nent n = 4, and S = 0.5. We shall refer to this representative point in parameter space as Regime A, as illustrated in the bifurcation diagram Fig. Results Two-gene networks have been studied in the literature as a model system to investigate the properties of epige- netic landscapes. The most common motif in a two gene network is where the transcription factor corresponding to each gene up-regulates its own production and down-regulates the production of the transcription factor associated with the other gene (Fig. 1a). It is possible to write down differential equations governing the time evolutions of the concentrations of the two transcription factors, = + + + − = + + + − dx dt a x S x b S S y kx dy dt a y S y b S S x ky (1) n n n n n n n n n n n n 1 1 2 2 = + + + − = + + + − dx dt a x S x b S S y kx dy dt a y S y b S S x ky (1) n n n n n n n n n n n n 1 1 2 2 (1) Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 2 www.nature.com/scientificreports/ where x and y denote the concentrations of the two transcription factors11,13. The feedback terms are modeled as Hill functions11, with exponent n controlling the steepness of the switch curve, while the parameter S controls the concentration at which the half-maximal point is reached. The first term corresponds to a positive feedback mechanism, with each TF up-regulating its own production, while the second term represents the mutual neg- ative feedback between the two components. Finally there is a decay term with the strength of the degradation process controlled by the decay parameter k. This model has been studied extensively11,13,16 as a representation of the cell differentiation process and has yielded insights into the epigenetic landscape as a cell differentiates.f ff In order to model the reverse differentiation process, we incorporate a time dependent chemical drive (along the lines of our earlier work12) as in experiments18. The feedback regulation depend on the concentrations of the TFs at some previous times, to account for the finite timescales of chromosome reorganization and other epige- netic state markers. We model these finite time processes through the incorporation of a time-delayed feedback. The degradation process is still assumed to be dependent on the instantaneous concentration. Results (a) d = 10: System stays in initial undifferentiated state; (b) d = 75: Sustained oscillations with the system spending more time in the vicinity of one state than the other; (c) d = 250: Sustained oscillations with the system spending approximately equal times in the vicinity of the two states; (d) d = 510: Successful reprogramming to the undifferentiated state. etween the initial differentiated state and the central progenitor state (1, 1) for times which are much larger than he characteristic timescales in the system (d,τ). y On increasing the drive time even further, the system comes out of this long lived oscillatory state and transi- tions to central progenitor state beyond a certain critical d. This is shown in Fig. 2 for d = 510. The system shows small oscillations as it starts from the initial differentiated state, but it quickly settles into undifferentiated state. This would then correspond to a successful completion of the reverse differentiation process. Thus in regime A, when the input is provided for a small time, the system stays in the differentiated state, while if the input if provided for large enough times, it successfully transitions into the progenitor state. In between, for intermedi- ate drive times, the system shows long lived oscillatory states where it is neither in the differentiated nor in the undifferentiated state, but oscillates between the two. The results of our two-dimensional GRN with this interplay of drive and delayed feedback thus closely mirrors the experimental observations of Nagy and Nagy18, with the identification of the undetermined state in the experiments to the oscillatory state predicted by our analysis. Transdifferentiation. While the results described for Regime A are robust for a > ac (see bifurcation analysis Fig. 1(b) for details), the system shows a different class of behaviour for a < ac. As a representative point in this region of parameter space, we choose a0 = 1.0, a = 0.4, b = 1.0, k = 1.0, n = 5, and S = 0.5 and follow time trajec- tories of the two TFs starting from one of the differentiated states, for different drive and delay times. The drive strength a0 and the Hill coefficient n are changed from the parameter set of Regime A to illustrate the robustness of our results. We shall refer to this representative point in parameter space as Regime B as shown in Fig. 1(b). Results 1(b). The central steady state (undifferentiated state) for this choice of parameters occurs at (x, y) = (1, 1) while the two differentiated stable steady states are at (x, y) = {(2, ε) or (ε, 2)} with ε ≈ 0. The time evolution of the system is shown in Fig. 2 for different values of the drive time d for a given value of the delay time τ1 = τ2 = τ = 500 starting from one of the differentiated states. f Figure 2(a) shows the time evolution of the concentrations of the two TFs when the drive time d(=10) is much less than the delay time τ(=500). In this regime τ  d , the concentrations show some initial small fluctuations around the initial conditions, which quickly die out leaving the system in the same state as it initially started with. Thus for small drive times, the system remains in the differentiated state it started out in. As we increase the time for which the drive is provided, the initial small fluctuations grow, until beyond a certain critical value, we observe sustained fluctuations in the concentration of the transcription factors. This is shown for two drive times, d = 75 and d = 250 in Fig. 2(b,c). As can be seen from the figures, for these intermediate drive times, the system oscillates Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 3 www.nature.com/scientificreports/ Figure 2. Time series of the concentrations of the two transcription factors (x/y) in regime A for four different drive times for τ = τ1 = τ2 = 500. (a) d = 10: System stays in initial undifferentiated state; (b) d = 75: Sustained oscillations with the system spending more time in the vicinity of one state than the other; (c) d = 250: Sustained oscillations with the system spending approximately equal times in the vicinity of the two states; (d) d = 510: Successful reprogramming to the undifferentiated state. Figure 2. Time series of the concentrations of the two transcription factors (x/y) in regime A for four different drive times for τ = τ1 = τ2 = 500. Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 Results Time series of the concentrations of the two transcription factors (x/y) in regime B for four different drive times for τ = τ1 = τ2 = 20. (a) d = 20: System stays in initial undifferentiated state; (b) d = 51: Sustained oscillations in concentration of TFs. The main panel shows a single TF (x) while the inset shows a magnified view of both the TFs in a narrow window of time; (c) d = 83: Sustained oscillations in concentration of TFs. The main panel shows a single TF (y) while the inset shows a magnified view of both the TFs in a narrow window of time; (d) d = 92: Transdifferentiation to the other differentiated state. transitions to the other differentiated state (0, 1.4) without reaching the central progenitor state. This dynamics is reminiscent of the phenomenon of transdifferentiation, which has been observed experimentally where cells can directly transition from one differentiated state to another35–42.hf f The time evolution observed in regime B is qualitatively different from the behaviour obtained in regime A. In regime B, the system never reaches the central progenitor state for any choice of the drive and delay times. Similarly, the transdifferentiated state is never observed for regime A. Further the oscillatory state is distinct in the two cases, with oscillations between the progenitor and the differentiated state in regime A (half oscillations) and between the two differentiated states in regime B (full oscillations).i f g To the best of our knowledge this is the first theoretical model that helps elucidate how delayed feedback pro- esses can give rise to transdifferentiation in gene regulatory networks. Phase diagrams. The different dynamical behaviours in Regimes A and B, characterised by time evolution plots can conveniently be represented as phase diagrams in the drive vs. delay time (d vs. τ) plane as shown in Fig. 4. Both situations, where the delay timescale for the two TFs are same i.e. (τ1 = τ2 = τ) and different (τ1 ≠ τ2) are considered. The long time behaviour can be classified into four phases - (i) the system stays in the initial differ- entiated state (I); (ii) the system reaches an oscillatory state (IIA or IIB, depending on the nature of oscillations); (iii) the system undergoes reverse differentiation to reach the pluripotent state (III); and (iv) the system reaches a transdifferentiated state (IV). Results The (unstable) progenitor state for this choice of parameters corresponds to (x, y) = (0.6, 0.6) while the two differen- tiated states are at (x, y) = {(1.4, ε) or (ε, 1.4)} with ε ≈ 0.hf y The time evolution results in regime B is shown in Fig. 3 for different d for time delay given by τ1 = τ2 = τ = 20. When the drive time is small, d = 20, the system shows early time transient fluctuations, before relaxing back to the initial state. This is shown in Fig. 3(a), which is similar to the behaviour observed in regime A for small drive durations, where the system does not undergo reverse differentiation, but continues to remain in its initial differentiated state. f On increasing the drive time, the system shows sustained oscillations, as shown in Fig. 3(b,c) for d = 61 and d = 84 respectively. Crucially in this case, the oscillations are between the two differentiated states and not between the initial differentiated state and the progenitor state, as in regime A described above. The oscillations are again long-lived in comparison to the drive and delay time scales of the system. The insets in panels (b) and (c) of Fig. 3 show a magnified view of the oscillations for both the transcription factors.t ) g gi p Finally Fig. 3(d) shows the time evolution of the system for d = 82. In this case, after initial transient oscilla- ons, the system stabilizes to a “flipped” state i.e. starting from one of the differentiated states (1.4, 0), the system Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 4 www.nature.com/scientificreports/ Figure 3. Time series of the concentrations of the two transcription factors (x/y) in regime B for four different drive times for τ = τ1 = τ2 = 20. (a) d = 20: System stays in initial undifferentiated state; (b) d = 51: Sustained oscillations in concentration of TFs. The main panel shows a single TF (x) while the inset shows a magnified view of both the TFs in a narrow window of time; (c) d = 83: Sustained oscillations in concentration of TFs. The main panel shows a single TF (y) while the inset shows a magnified view of both the TFs in a narrow window of time; (d) d = 92: Transdifferentiation to the other differentiated state. Figure 3. Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 Results In this regime upon increasing the drive time d > dPNR, the system necessarily passes through a region where the oscillatory state is stable before it can make the transition to the central progenitor state beyond the dCPS drive time. The width of oscillatory region remains constant with increasing τ1 (unlike the Δτ = 0 and 25 cases) for small values of τ1 and widens for large values with dPNR and dCPS diverging away from each other.fh g PNR CPS g g y We now turn to the analysis of the phase behaviour in regime B, i.e. transdifferentiation regime. This is shown for τ = τ1 = τ2 in Fig. 4(d). For small values of the drive time, the system remains in the initial undifferentiated state (I). On increasing the drive time, for small values of the delay time τ (50), the final long-term behaviour of the system shows extreme sensitivity to parameter values. The inset of Fig. 4(d) shows a magnified view of the phase map for τ < 50 in the region 300 < d < 350. As can be seen from the inset, the final state shows signatures of chaotic behaviour, where the long time state is either the initial state (I - green dots) or oscillatory state (IIB - yel- low dots), or transdifferentiated state (III - red dots) for slight variations in drive time d. The long time behaviour of the system was obtained by time marching the set of delay differential equations (Eq. 2) for a time t much greater than the delay and drive timescales of the system ( τ =  t d 10 { , } 4 ). While it is possible that the final state obtained from our simulations is not the true steady state, biologically relevant timescales would amount to the states observed after times which are of comparable magnitude to the experimental timescales, such as the time d for which the chemical drive is provided to the system18. In these parameter regimes, as shown by the phase map, this deterministic system shows chaotic behavior. For larger delay times (  τ 50), the transdifferenti- ated state is no longer stable (in the range of drive times investigated) and the system transits from the initial state (I) to an oscillatory state (IIB) beyond a certain threshold drive time dcr. Results f Figure 4(a), shows the phase behaviour of the system in regime A for τ1 = τ2 = τ. In the region with small delay and drive times τ < τcr, and d < dcr, the system stays in the initial differentiated state (I). For small delay times τ < τcr, but beyond a critical drive time d > dcr, the system transitions directly from the differentiated state (I) to the central progenitor state (III), with the critical drive duration for the reverse differentiation process increasing linearly with the delay time i.e. (dcr ∝ τ). For τ > τcr this direct transition from the initial differentiated to the final undifferentiated state is lost. In this region, the system initially passes from the initial state to an oscillatory state (IIA) – as demonstrated in Fig. 2(b,c). In line with the experimental results18, we denote this minimal drive time beyond which the initial state is no longer the stable solution as dPNR (the Point-of-No-Return). By keeping τ fixed and increasing d, the oscillatory state is observed to be stable till a second critical value of the drive time dCPS (Commitment-to-Pluripotent-State). Beyond this value of drive time i.e. d > dCPS the undifferentiated state is stable. The threshold for the point-of-no-return dPNR decreases with increasing time delay, implying that the oscillatory state sets in for lower values of the drive time for higher τ values. On the other hand, the threshold for crossing to the pluripotent state increases linearly with delay times (dCPS ∝ τ), such that the crossover to the cen- tral state requires a chemical drive for longer durations as τ is increased. Thus with increasing delay timescales, the regime of stability for sustained oscillations continues to increase widening the gap between the point of no return and commitment to pluripotency. Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 5 www.nature.com/scientificreports/ Figure 4. Phase space in the drive time (d) vs. the delay time (τ1) plane for (a) Regime A: τ2 = τ1; (b) Regime A: τ2 = τ1 + 25; (c) Regime A: τ2 = τ1 + 50; (d) Regime B: τ2 = τ1. The inset shows a magnified view of a small region of this phase space to illustrate the chaotic behaviour in this regime; (e) Regime B: τ2 = τ1 + 25; (f) Regime B: τ2 = τ1 + 50. Results All other parameters for regime A and regime B are as defined in the text. Figure 4. Phase space in the drive time (d) vs. the delay time (τ1) plane for (a) Regime A: τ2 = τ1; (b) Regime A: τ2 = τ1 + 25; (c) Regime A: τ2 = τ1 + 50; (d) Regime B: τ2 = τ1. The inset shows a magnified view of a small region of this phase space to illustrate the chaotic behaviour in this regime; (e) Regime B: τ2 = τ1 + 25; (f) Regime B: τ2 = τ1 + 50. All other parameters for regime A and regime B are as defined in the text. In Fig. 4(b) we show the phase behaviour in regime A for the situation where the delay times are unequal i.e. τ1 ≠ τ2 with a constant difference between the two delay times, i.e. Δτ = τ2 − τ1 = 25. Similar to the situation when delay times are equal, the system directly transits from the initial state (I) to the final progenitor state (III) for small value of τ1 beyond a critical value of drive time dcr. However unlike equal delay time scenario, the critical drive time at which this transition takes place dcr remains almost constant as τ1 is increased. Beyond a threshold τcr, this direct transition gives way to a two-step transition, in which stable state is the initial state for d < dPNR, the oscillatory state for dPNR < d < dCPS, and finally the progenitor state for d > dCPS. This is similar to the equal delay time scenario described earlier. Further, the boundaries dPNR and dCPS behave as before, with dPNR decreasing with increasing τ1 and dCPS increasing linearly with τ1. The critical value of τ1 at which this two-step transition take place is lower in this Δτ = 25 case than in the previous Δτ = 0 one. Finally in Fig. 4(c) we show the phase behaviour for the case of Δτ = 50. Interestingly, in this case, there is no value of the time delay τ1 for which the system makes a direct transition from the differentiated (I) to the undifferentiated state (III). Results The oscillatory state reappears for  τ 30 1 , and as in the Δτ = 25 case, the region of stability of the oscillatory solution increases with increasing τ1.h g The above analysis of the phase behaviour of the model in regime A and B shows that the mutual interplay of drive and delay timescales can give rise to extremely rich landscapes, with non-trivial dependence of the final steady state on the time for which the input is provided, as well as the delay timescales associated with both the TFs. An interesting question to ask is how these exotic dynamical states are realizable in a biological context.hf The observation of a long-lived oscillatory state and a transdifferentiated state is extremely robust that occurs for a range of parameters characterizing the system. In order to show that the introduction of drive and delay generically leads to these novel state, we plot the phase diagram in the parameter space of the positive feed back strength (a = a1 = a2) and the negative feedback strength (b = b1 = b2). This is shown in Fig. 5(a). As can be seen from the figure, beyond a certain critical value of the positive feedback strength a, the system is always in a regime, where an external input provided for a sufficiently long time can drive the system to a reprogrammed state. For very small values of a and small values of b, the system has a single stable state and hence this monosta- ble region is not of interest in a biological context. The time trajectories of the concentrations of the transcription factor beyond the critical a value is qualitatively similar to those shown in Fig. 2. The parameters corresponding to Fig. 2 is shown as a red square in the a–b plane. Conversely, below this critical a, an external input provided for a sufficiently long time always drives the system to a transdifferentiated state. The time trajectories in this region are qualitatively similar to those shown in Fig. 3, which point is denoted by a blue circle in the a–b plane.hf q y g p y p The system of delay differential equations characterizing the gene regulatory network were analyzed in the regime where the positive and negative feedback strengths for the two transcription factors were completely sym- metrical. Results The phase boundary demarcating these two regions shows a non-monotonic behaviour (saw-tooth-like pattern), increasing linearly until τ ~ 340, beyond which it drops sharply before continuing to rise with a different slope. p p y gf p Interestingly, this apparent randomness is lost when the the two delay timescales are no longer equal, Δτ = τ1 − τ2 ≠ 0. Figure 4(e) shows the phase behaviour in Regime B for Δτ = 25. For small values of τ1 (<20), the system transitions from the initial state to the transdifferentiated state beyond a critical drive time dcr. For   τ 20 30 1 , there exists range of values of d which show sustained oscillations. Below this range, the system stays in the initial state, while for larger drive times, the transdifferentiated state is stable. For   τ 30 40 1 , there is no oscillatory state, and the system again transitions directly from the initial state to the transdifferentiated state. Above this delay timescale range τ1 > 40, the oscillatory state reappears in the phase map, and the region of Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 6 www.nature.com/scientificreports/ Figure 5. (a) Parameter phase plane in the positive feedback (a) vs negative feedback (b) plane for the case of symmetric parameters, a = a1 = a2 and b = b1 = b2. The parameter point shown as a red square corresponds to the parameter set for the trajectories shown in Fig. 2, while the point shown as a blue circle corresponds to the parameter set for the trajectories shown in Fig. 3. (b) Parameter phase plane in the a1 vs. a2 plane for the case of asymmetric parameters for b = 1. The insets in the reprogramming region of the phase plane show the time trajectories of the transcription factors for a1 = 1.0, a2 = 1.2, d = 150 for the reprogramming to the central attractor and a1 = 1.0, a2 = 1.2, d = 50 for the long-lived oscillatory state. The insets in the transdifferentiation region of the phase plane show the time trajectories for a1 = 0.6, a2 = 1.2, d = 500 starting from two different initial states, one with  x y, which shows transdifferentiation, and the other with  x y, which does not. All other parameters for both panels are the same as those in Fig. 1. Results All results are shown for Δτ = 25. Figure 5. (a) Parameter phase plane in the positive feedback (a) vs negative feedback (b) plane for the case of symmetric parameters, a = a1 = a2 and b = b1 = b2. The parameter point shown as a red square corresponds to the parameter set for the trajectories shown in Fig. 2, while the point shown as a blue circle corresponds to the parameter set for the trajectories shown in Fig. 3. (b) Parameter phase plane in the a1 vs. a2 plane for the case of asymmetric parameters for b = 1. The insets in the reprogramming region of the phase plane show the time trajectories of the transcription factors for a1 = 1.0, a2 = 1.2, d = 150 for the reprogramming to the central attractor and a1 = 1.0, a2 = 1.2, d = 50 for the long-lived oscillatory state. The insets in the transdifferentiation region of the phase plane show the time trajectories for a1 = 0.6, a2 = 1.2, d = 500 starting from two different initial states, one with  x y, which shows transdifferentiation, and the other with  x y, which does not. All other parameters for both panels are the same as those in Fig. 1. All results are shown for Δτ = 25. stability of the oscillatory state increases with increasing τ1. While the boundaries between the different phases are sharply defined for regime B in this Δτ ≠ 0 condition, the signature of the chaotic behaviour remains in the appearance and disappearance of the stable oscillatory solution for different drive times. pp pp yf Finally, Fig. 4(f) show the results for regime B with Δτ = 50. In this case, for low values of τ1 (18), the oscil- latory state is stable for a region of drive times. In an intermediate regime   τ 18 30 1 , there is no stable oscilla- tory state, and the system transitions from the initial state to the transdifferentiated state beyond a threshold drive time. Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 Results In all four cases, the trajectories are coloured by the time values, as shown in the corresponding colorbars. Figure 6. Trajectory plots in the x/y(t) vs. x/y(t + τ) plane for (a) Oscillatory state in regime A (state IIA). The oscillations are between the differentiated states and the central undifferentiated state, which are represented as half-cycles in this phase plane. (b) Reprogramming to the undifferentiated state in regime A (state III). Note the decaying transient oscillations in both x and y, which converge to the central steady state. (c) Oscillatory state in regime B (state IIB). The oscillations are between the two differentiated states in this regime, which is represented by single limit cycle in this phase plane. (d) Transdifferentiated state in regime B (state IV). Note the unstable oscillations around the central state preceding transdifferentiation. In all four cases, the trajectories are coloured by the time values, as shown in the corresponding colorbars. strengths, only one of the differentiated states can be transition to the other one, while the second differentiated state stays stable even for large values of the drive. This is shown in the insets in the transdifferentiated region of the parameter space for initial conditions corresponding to the two differentiated states. If the initial differenti- ated state has  y x, then the stronger feedback strength of the TF corresponding to y(t) can cause transdifferen- tiation to the other stable state in the system. On the other hand, if the initial differentiated state has  x y, then in this regime, it does not undergo any transdifferentiation. The situation is exactly reversed in the region of the parameter space corresponding to large values of a1 and small values of a2. Here the initial state with  x y undergoes transdifferentiation, while the other differentiated state does not. Characterizing the oscillations. We now turn to characterizing the long-lived oscillatory state observed in regimes A and B. In Fig. 6 we show the trajectory of the system in the oscillatory state plotted in the x(t) vs. x(t + τ) plane. While a stable steady state corresponds to the trajectories evolving to a single point in this plane, steady oscillations evolve to a limit cycle. Figure 6(a) shows the trajectory of the system in this plane in regime A for the concentrations of both the TFs x(t) and y(t). Results It is possible for the cell to drive the differentiation process deterministically to one of the differentiated cell fates through an asymmetric change in the feedback strengths. Various experiments have shown that stochas- ticity play a role in the cellular differentiation process, which argues that the central attractor corresponding to the undifferentiated state changes its stability through a symmetric variation of the control parameters13,43,44. Nevertheless, biochemical control of the feedback strength parameters leading to an asymmetric landscape is a distinct possibility. In order to show, that our predictions of an oscillatory or a transdifferentiated state are inde- pendent of whether the control parameters are symmetric or asymmetric, we plot the phase diagram of the sys- tem, in the a1–a2 plane, where the positive feedback strength of the two transcription factors are allowed to vary independently. This is shown in Fig. 5(b). When a1 and a2 are both sufficiently large, the system is in a repro- grammed state, for sufficiently large drive times. The insets in this reprogrammed region of the parameter space shows typical time trajectories for intermediate and large drive times, which leads to oscillations and reprogram- ming respectively. Note that the central undifferentiated state in this case is not symmetric with respect to the two transcription factors. The asymmetry in the feedback strengths leads to a central state with x ≠ y, However, repro- gramming to this central state proceeds as in the case of symmetric parameters. For small values of a1 and large values of a2, the system is in the transdifferentiated regime. However, due to the asymmetry in the feedback Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 7 www.nature.com/scientificreports/ Figure 6. Trajectory plots in the x/y(t) vs. x/y(t + τ) plane for (a) Oscillatory state in regime A (state IIA). The oscillations are between the differentiated states and the central undifferentiated state, which are represented as half-cycles in this phase plane. (b) Reprogramming to the undifferentiated state in regime A (state III). Note the decaying transient oscillations in both x and y, which converge to the central steady state. (c) Oscillatory state in regime B (state IIB). The oscillations are between the two differentiated states in this regime, which is represented by single limit cycle in this phase plane. (d) Transdifferentiated state in regime B (state IV). Note the unstable oscillations around the central state preceding transdifferentiation. Discussion k ll Our work illustrates the richness of epigenetic landscapes in the presence of external drive and delayed feedback. Using a generic model of two self-activating and mutually inhibiting genes, we show that the reprogramming to the common progenitor state requires a balance of the drive and delay timescales, as well as appropriate positive and negative feedback strengths. Apart from the reprogrammed state, the system can find itself in long-lived oscillatory states or in a transdifferentiated state in different parameter regimes. An analysis of the phase diagrams in the delay - drive time plane provides a comprehensive picture of the final states of the reverse differentiation process. Additionally, the phase diagrams also provide a signature of chaotic behaviour in appropriate regimes, which have been hypothesized to play an important role in the cell-fate determination process45. In the chaotic regime, the model predicts that slight changes in the drive or delay parameters can drive the system from one state to another. The chaotic regime can thus provide a pathway for the control of cell fate determination. The phenom- enon of transdifferentiation and oscillatory states is extremely robust and persists in a wide range of parameters as has been shown in Fig. 5. While the chaotic regions provide a regime where small changes in the drive and delay can affect the final state, there also exist regions where the final state is insensitive to wide variations of the drive and delay time.i To the best of our knowledge, this work provides one of the first theoretical basis for understanding the phenomenon of transdifferentiation in the presence of delayed feedback. Transdifferentiation was first shown experimentally in mouse fibroblasts in 1987, where the transcription factor MyoD, belonging to the Myogenic Regulatory Factors (MRF) family was shown to induce transdifferentiation of mouse embryonic fibroblasts to myoblasts35. The therapeutic potential of transdifferentiation has led to attempts to direct the process towards functional outcomes. The first proof of such a functional transdifferentiation was carried out in mouse liver cells which transitioned to pancreatic-beta-cell-like cells that helped control the effects of hyperglycemia46. Transdifferentiation has been shown to be a valid strategy to alter somatic cell fates in humans as well, which was first demonstrated in the transdifferentiation of a human liver cell to human beta cells through the effect of a single gene PDX-147. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Probability of the concentration of any one transcription factor for different values of the drive time for (a) regime A with τ = 500. The inset shows the probability near one differentiated state (x = 2) as a function of the ration of the drive to delay times d/τ; and (b) regime B with τ = 20. Figure 7. Probability of the concentration of any one transcription factor for different values of the drive time for (a) regime A with τ = 500. The inset shows the probability near one differentiated state (x = 2) as a function of the ration of the drive to delay times d/τ; and (b) regime B with τ = 20. to estimate whether there is a systemic behaviour across a range of delay timescales τ, we plot the probability of finding the TF1 at the vicinity of the undifferentiated state, x = 2, against the non-dimensionalised drive time d/τ for a range of τ values. Remarkably, this probability appears to have a simple scaling behaviour dictated by a single parameter d/τ, viz. p(x = 2) = 1 − (d/τ). While the oscillations in regime B can be characterized by a similar method as shown in Fig. 7(b), the chaotic nature of the oscillatory and transdifferentiated state implies that there is no simple scaling relation with the drive time in this case, unlike in regime A. to estimate whether there is a systemic behaviour across a range of delay timescales τ, we plot the probability of finding the TF1 at the vicinity of the undifferentiated state, x = 2, against the non-dimensionalised drive time d/τ for a range of τ values. Remarkably, this probability appears to have a simple scaling behaviour dictated by a single parameter d/τ, viz. p(x = 2) = 1 − (d/τ). While the oscillations in regime B can be characterized by a similar method as shown in Fig. 7(b), the chaotic nature of the oscillatory and transdifferentiated state implies that there is no simple scaling relation with the drive time in this case, unlike in regime A. Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 Results As can be seen clearly from the time evolution of the trajectories, both the concentrations evolve to a steady limit cycle corresponding to the oscillatory region (IIA). The oscillations for each TF are between the initial differentiated state and the central undifferentiated state, which is manifested as two distinct limit cycles for the two TFs in the upper right and lower left quadrant. The corresponding trajectories for regime B are shown in Fig. 6(c). In this case, the oscillations are between the two undifferentiated states (IIB), and hence the limit cycles for the two TFs completely overlap, unlike the limit cycle in regime A. In contrast, we show the evolution of the trajectories in this plane for the reprogramming in regime A (Fig. 6(b)) and transdifferentiation in regime B (Fig. 6(d)). For successful reprogramming, initial transient oscillations between the initial state and the central state decay to the central stable state for both the TFs. For transdifferentiation, the concentrations of the two TFs perform small oscillations around the undifferentiated state for a short period of time (blue limit cycle), beyond which the limit cycle becomes unstable and the TFs reach the other differentiated state. The oscillations enable the trajectories to mix, ensuring that the history of the initial state is lost, which triggers the switch to the trans- differentiated state. f While the oscillatory state is stable for dPNR < d < dCPS, the nature of oscillations changes as d is varied. A quantitative estimate of the difference in the oscillatory state can be obtained by comparing the residence time distributions of the two transcription factors for different drive and delay times. We plot the residence time dis- tribution as a function of the concentration for both regime A (Fig. 7(a)) and regime B (Fig. 7(b)). As can be seen clearly in Fig. 7(a), on increasing drive times, the peak in the probability distribution shifts from a single peak at x = 2 for low values of d to a bimodal distribution at higher drive times (d = 250), and back again to an unimodal distribution, but centered around the undifferentiated state at x = 1 at even higher drive times, d = 510. In order Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 8 Discussion k ll The implications of successful transdifferentiation in medical applications has led to a surge of experimental work using a combination of strategies including the forced expression of specific transcription factors and combination of defined factors with microRNAs or small molecules to achieve targeted functional transdifferentiation of one somatic cell type to another36–42,48–50. An understanding of the underlying dynamics that characterizes the transdifferentiation process has however been lacking, including the question of whether cells need to necessarily pass through partially reprogrammed states in order to convert to a different cell type.hf y p g p y p gf yp The mechanism proposed in this paper articulates one possible pathway for transdifferentiation, in the context of somatic cell types of similar lineages51. We show that it is possible to achieve transdifferentiation of one somatic cell type to another without passing through the undifferentiated state. Our analysis also provides an explanation for this process in terms of the underlying bifurcation diagram of the model - transdifferentiation arises when the feedback is tuned such that the central progenitor state is unstable. Interestingly, we observe that the transdiffer- entiation process is preceded by unstable oscillations, as has been seen in a different modeling approach52. A dif- ferent mechanism has been proposed where transdifferentiation proceeds through a distinct stable intermediate state or a series of unstable intermediate states53. The incorporation of delays, as in the current work, proposes a new pathway to transdifferentiation, where unstable oscillations around the central state serve as a precursor to the transdifferentiated state. It would be interesting to study the transdifferentiation process in the presence of delayed feedback, in a landscape with multiple lineage branching points in order to characterize the pathway in these more complex situations.h p The interplay of delayed feedback and chemical drive also leads to sustained oscillations in the levels of tran- cription factors. While we predicted the presence of sustained oscillations in the one gene network reported 9 www.nature.com/scientificreports/ previously12, this study conclusively highlights the importance of these oscillations for a more realistic two-gene network. Additionally, oscillations in the concentrations of transcription factors show different characteristics, (a) oscillations between the differentiated and undifferentiated state in one regime (IIA), and (b) oscillations between the two differentiated states in a different regime (IIB). The two oscillatory states may play different roles in the cellular context. Discussion k ll It has been shown experimentally that oscillations in the Notch effector gene Hes1 regulates the maintenance of neural progenitor cell types54,55. It has also been reported that Stella shows heteroge- neous expression levels and dynamic equilibrium in embryonic stem cells (ESC), which is responsible for the ESC existing in a metastable state where they can shift between Inner Cell Mass (ICM) and epiblast like phenotypes56. Oscillations have been reported to be a possible feature of epigenetic landscapes52,57. We show that a possible route to these oscillations is through the incorporation of delayed feedback, and this can potentially explain novel states observed during the reprogramming process18. The feedback loops involved in gene regulatory networks are typ- ically composed of multi-step reactions which introduce a characteristic delay time scale. Such delayed feedback loops have been shown to play an important role in both experimental and theoretical contexts58–61. While further experiments are needed to investigate whether the novel states seen in the doxycycline induced reprogramming process by Nagy and Nagy18 are indeed due to oscillations or some other mechanism, this work demonstrates the importance of oscillatory states in general when time delayed feedback circuits are incorporated in the modeling of gene regulatory networks.f g g y In this paper we consider a deterministic model of epigenetic landscapes, and consider the effects of time delays and chemical drive. In cellular contexts, it is also important to re-interpret our results in the presence of stochastic fluctuations, which can play a role in cell fate determination62–69. Noise induced transitions between different cell types can play an important role in gaining a comprehensive understanding of the differentiation process. The analysis of stochastic fluctuations in these highly nonlinear and non-autonomous delay differential equations present a significant challenge, and the results from an extensive analysis of the role of noise in these systems shall be presented in a forthcoming publication. y p g p The present work provides a framework for the analysis of reprogramming experiments and underscores the importance in accounting for delayed feedback in building a comprehensive picture of the epigenetic landscape. 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Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 10 www.nature.com/scientificreports/ Author Contributions M.K.M. and B.C. conceived the study, analysed data, and wrote the manuscript. B.K. performed simulations, analysed data, and wrote the manuscript. R.S. performed initial simulations. All authors reviewed the manuscript. Acknowledgementsi g M.K.M. acknowledges financial support from Ramanujan Fellowship, DST, INDIA (13DST052) and the IIT Bombay Seed Grant (14IRCCSG009). B.C. acknowledges the Biophysical Sciences Institute, and Institute of Advanced Studies, Durham University for financial support at the time the work was done. g M.K.M. acknowledges financial support from Ramanujan Fellowship, DST, INDIA (13DST052) and the IIT Bombay Seed Grant (14IRCCSG009). B.C. acknowledges the Biophysical Sciences Institute, and Institute of Advanced Studies, Durham University for financial support at the time the work was done. Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 11 www.nature.com/scientificreports/ Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPOrtS \| (2018) 8:7358 \| DOI:10.1038/s41598-018-25556-9 12
https://openalex.org/W2917499921	https://aura.abdn.ac.uk/bitstream/2164/12516/1/Kar2019_Article_AssociationOfSmokingAndCardiom.pdf	English	null	Association of smoking and cardiometabolic parameters with albuminuria in people with type 2 diabetes mellitus: a systematic review and meta-analysis	Acta diabetologica	2,019	cc-by	9,267	Acta Diabetologica (2019) 56:839–850 https://doi.org/10.1007/s00592-019-01293-x Acta Diabetologica (2019) 56:839–850 https://doi.org/10.1007/s00592-019-01293-x REVIEW ARTICLE Association of smoking and cardiometabolic parameters with albuminuria in people with type 2 diabetes mellitus: a systematic review and meta-analysis sh Kar1,2 · Clare Gillies1 · Mintu Nath1 · Kamlesh Khunti1 · Melanie J. Davies1 · Samuel Seidu1 Received: 23 November 2018 / Accepted: 23 January 2019 / Published online: 24 February 2019 © The Author(s) 2019 Abstract Aims Smoking is a strong risk factor for albuminuria in people with type 2 diabetes mellitus (T2DM). However, it is unclear whether this sequela of smoking is brought about by its action on cardiometabolic parameters or the relationship is independ- ent. The aim of this systematic review is to explore this relationship. whether this sequela of smoking is brought about by its action on cardiometabolic parameters or the relationship is independ ent. The aim of this systematic review is to explore this relationship. Methods Electronic databases on cross-sectional and prospective studies in Medline and Embase were searched from Janu- ary 1946 to May 2018. Adult smokers with T2DM were included, and other types of diabetes were excluded. Results A random effects meta-analysis of 20,056 participants from 13 studies found that the odds ratio (OR) of smokers developing albuminuria compared to non-smokers was 2.13 (95% CI 1.32, 3.45). Apart from smoking, the odds ratio of other risk factors associated with albuminuria were: age 1.24 (95% CI 0.84, 1.64), male sex 1.39 (95% CI 1.16, 1.67), duration of diabetes 1.78 (95% CI 1.32, 2.23), HbA1c 0.63 (95% CI 0.45, 0.81), SBP 6.03 (95% CI 4.10, 7.97), DBP 1.85 (95% CI 1.08, 2.62), total cholesterol 0.06 (95% CI − 0.05, 0.17) and HDL cholesterol − 0.01 (95% CI − 0.04, 0.02), triglyceride 0.22 (95% CI 0.12, 0.33) and BMI 0.40 (95% CI 0.00–0.80). When the smoking status was adjusted in a mixed effect meta-regression model, the duration of diabetes was the only statistically significant factor that influenced the prevalence of albuminuria. In smokers, each year’s increase in the duration of T2DM was associated with an increased risk of albuminuria of 0.19 units (95% CI 0.07, 0.31) on the log odds scale or increased the odds approximately by 23%, compared to non-smokers. Prediction from the meta-regression model also suggested that the odds ratios of albuminuria in smokers after a diabetes duration of 9 years and 16 years were 1.53 (95% CI 1.10, 2.13) and 5.94 (95% CI 2.53, 13.95), respectively. Conclusions Continuing to smoke and the duration of diabetes are two strong predictors of albuminuria in smokers with T2DM. Abstract Keywords Type 2 diabetes mellitus · Albuminuria · Smoking Keywords Type 2 diabetes mellitus · Albuminuria · Smoking Abbreviations T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus HbA1c Glycosylated haemoglobin HDL High-density lipoprotein LDL Low-density lipoprotein SBP Systolic blood pressure DBP Diastolic blood pressure Introduction Smokers with T2DM are disproportionately affected by pre- mature cardiovascular events. A recent systematic review of over 1 million people revealed that smokers with T2DM were approximately 50% more likely to die prematurely with cardiovascular events, compared to non-smokers [1]. However, the precise underlying cause for this heightened cardiovascular mortality remains unexplored. Smoking exacerbates insulin resistance, and adversely affects some Managed by Giuseppe Pugliese. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00592-019-01293-x) contains supplementary material, which is available to authorized users. * Debasish Kar d.kar@sheffield.ac.uk; d.kar@nhs.net Extended author information available on the last page of the article Abstract With a global surge in younger smokers developing T2DM, smoking cessation interventions at an early stage of disease trajectory should be promoted y p p Methods Electronic databases on cross-sectional and prospective studies in Medline and Embase were searched from Janu- ary 1946 to May 2018. Adult smokers with T2DM were included, and other types of diabetes were excluded.f Results A random effects meta-analysis of 20,056 participants from 13 studies found that the odds ratio (OR) of smokers developing albuminuria compared to non-smokers was 2.13 (95% CI 1.32, 3.45). Apart from smoking, the odds ratio of other risk factors associated with albuminuria were: age 1.24 (95% CI 0.84, 1.64), male sex 1.39 (95% CI 1.16, 1.67), duration of diabetes 1.78 (95% CI 1.32, 2.23), HbA1c 0.63 (95% CI 0.45, 0.81), SBP 6.03 (95% CI 4.10, 7.97), DBP 1.85 (95% CI 1.08, 2.62), total cholesterol 0.06 (95% CI − 0.05, 0.17) and HDL cholesterol − 0.01 (95% CI − 0.04, 0.02), triglyceride 0.22 (95% CI 0 12 0 33) and BMI 0 40 (95% CI 0 00 0 80) When the smoking stat s as adj sted in a mi ed effect meta regression ( , ), ( , ), ( , ), ( , 2.62), total cholesterol 0.06 (95% CI − 0.05, 0.17) and HDL cholesterol − 0.01 (95% CI − 0.04, 0.02), triglyceride 0.22 (95% CI 0.12, 0.33) and BMI 0.40 (95% CI 0.00–0.80). When the smoking status was adjusted in a mixed effect meta-regression model, the duration of diabetes was the only statistically significant factor that influenced the prevalence of albuminuria. In smokers, each year’s increase in the duration of T2DM was associated with an increased risk of albuminuria of 0.19 units (95% CI 0.07, 0.31) on the log odds scale or increased the odds approximately by 23%, compared to non-smokers. Prediction from the meta-regression model also suggested that the odds ratios of albuminuria in smokers after a diabetes duration of 9 years and 16 years were 1.53 (95% CI 1.10, 2.13) and 5.94 (95% CI 2.53, 13.95), respectively. Conclusions Continuing to smoke and the duration of diabetes are two strong predictors of albuminuria in smokers with T2DM. With a global surge in younger smokers developing T2DM, smoking cessation interventions at an early stage of disease trajectory should be promoted. Introduction Smokers with T2DM are disproportionately affected by pre- mature cardiovascular events. A recent systematic review of over 1 million people revealed that smokers with T2DM were approximately 50% more likely to die prematurely with cardiovascular events, compared to non-smokers [1]. However, the precise underlying cause for this heightened cardiovascular mortality remains unexplored. Smoking exacerbates insulin resistance, and adversely affects some However, the precise underlying cause for this heightened cardiovascular mortality remains unexplored. Smoking exacerbates insulin resistance, and adversely affects some (0121 840 Acta Diabetologica (2019) 56:839–850 cardiometabolic risk factors in T2DM including HbA1c, HDL cholesterol and arterial blood pressure [2]. Surpris- ingly, however, smoking cessation does not appear to con- fer any substantial cardiovascular risk reduction for up to 10 years in people with diabetes, compared to 3 years in people without [3]. Indeed, the World Health Organization (WHO) Multinational Study of Vascular Disease in Diabe- tes (MSVDD) demonstrated that the risk of cardiovascular mortality in people with diabetes remains up to 50% higher in recent quitters (1–9 years), compared to non-smokers [4]. This incongruous relationship between smoking cessation and mortality suggests that there may be some additional risk factor/s that contribute to a higher cardiovascular risk in recent quitters, which might not be reversed by short-term abstinence from smoking. “AND”. Additionally, we searched the references of the included studies to identify further suitable studies for inclusion. We followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocol (PRISMA- P) 2015 guidelines [10] (Fig. 1). We published the proto- col in the International Prospective Register for Systematic Reviews (PROSPERO) database (CRD 42018090637). The full search strategy is in Supplementary material 1. The inclusion criteria were studies reporting urinary albumin excretion (UAE) in adults (> 18 years) with T2DM. T2DM was defined as a condition affecting people’s blood sugar level which was then diagnosed by healthcare pro- fessionals and treated with diet, lifestyle interventions, oral medication or injectable therapy. People with type 1 diabetes mellitus (T1DM), steroid-induced diabetes, diabetes insipi- dus, and late auto-immune diabetes of adults (LADA) were excluded, but maturity-onset diabetes of the young (MODY) were included. Smokers were defined as self-reported ciga- rette smokers for at least a year after being diagnosed with T2DM. Materials and methods Data extraction was conducted using a predesigned data extraction template—study name, year of publication, coun- try of study, study design, number of participants, mean age, smoking status of the participants, and presence or absence of albuminuria (Table 1). Introduction For this study, albuminuria was defined as urinary albumin creatinine ratio (ACR) > 20 mg/gm or > 2.5 mg/ mmol in male or > 3.5 mg/mmol in female (KDIGO— Kidney Disease Improving Global Outcome guidelines http://kdigo.org) [11]. Total cholesterol and HDL were converted to mmol/l if they were reported in mg/dl (mg/ dl = ÷ 38.67 mmol/l), and TG (mg/dl = ÷ 88.57). HbA1c was expressed in both IFCC unit (mmol/mol) and DCCT unit (%). Blood pressure was expressed in mm of Hg and BMI was expressed as kg/m2. Albuminuria is an early indicator of both micro-, and macrovascular involvements in diabetes [5, 6] and the pro- gression of albuminuria is a reliable marker for the extent of vascular perturbation [7]. Aggressive management of tradi- tional risk factors such as glucose, blood pressure and lipid profile has not shown consistent benefit particularly when proteinuria is already established [8]. On the other hand, multifactorial interventions including smoking cessation at an early stage of disease trajectory have shown promising potential for the reversal of microalbuminuria and improved cardiovascular outcome [9]. However, conventional risk stratification score derived from the HbA1c, blood pressure and lipid profile may underestimate the influence of life style factors such as obesity and smoking on albuminuria dur- ing this crucial stage of disease trajectory. With a global surge of younger people developing metabolic syndrome and T2DM, it is pivotal to explore how best they can be protected from albuminuria which not only heralds incipient diabetic nephropathy but also poses a higher risk for premature car- diovascular complications. The aim of this systematic review and meta-analysis is to elucidate how smoking impacts upon the prevalence of albuminuria and how this relationship is influenced by cardiovascular risk factors such as age, male sex, duration of diabetes, HbA1c, blood pressure, lipid pro- file and body mass index (BMI). Studies in English language or translated into the Eng- lish language were accepted for inclusion. Observational prospective and cross-sectional studies were included for this review. Two investigators (DK and CLG) independently screened the articles using the inclusion and exclusion cri- teria. Any disagreement between the two investigators was resolved either by consensus or by consulting with a third investigator (SS). Included studies were selected by review- ing the titles and abstracts on electronic databases search. Additionally, hand searches were carried out from the refer- ences of the included studies. Search strategy and selection criteria For this systematic review and meta-analysis, we conducted a comprehensive search on Medline and Embase electronic databases from their inceptions to May 2018. The keywords used for the searches were: “type 2 diabetes”, “smoking”, “microalbuminuria” or “macroalbuminuria” or “albumi- nuria” or “proteinuria” in the title, abstract and keywords; the result was then combined using the Boolean operator Study-level data were also compiled for HbA1c, TC, HDL cholesterol, triglyceride, BMI, SBP and DBP. Continu- ous data were expressed as mean ± SD (standard deviation). For cross-sectional studies prevalence data, and for prospec- tive studies baseline data, were extracted. In prospective 1 3 841 Acta Diabetologica (2019) 56:839–850 1 3 dies, if albuminuria was absent at the baseline but was esent at follow-up, then the follow-up data were obtained. tracting data from all studies at just one time point, owed both cross-sectional studies and cohort studies to be combined in the meta-analyses. The study team us Newcastle–Ottawa Tool for the assessment of the qua observational studies to assess the quality of included s [12]. A random effect meta-analysis was conducted to Electronic database search Medline (n=1282) Embase (n=925) Title & abstract screening (n=2207) Screening Eligibility Identification Studies included (n=30) Duplicate removed (n=88) Excluded (n=2057) Does not meet the eligibility criteria Selected for full text review (n=150) Excluded (n=92) No T2DM No UAE data available Smoking status not mentioned Studies met eligibility criteria (n=58) Excluded (n=28) Not cross-sectional or prospective study (n=18) Studies not in English (n=2) Review articles (n=4) Conference abstract (n=4) Included 1 PRISMA flow chart Electronic database search Medline (n=1282) Embase (n=925) Excluded (n=2057) Does not meet the eligibility criteria Excluded (n=92) No T2DM No UAE data available Smoking status not mentioned Excluded (n=28) Not cross-sectional or prospective study (n=18) Studies not in English (n=2) Review articles (n=4) Conference abstract (n=4) Studies included (n=30) Fig. 1 PRISMA flow chart Fig. 1 PRISMA flow chart be combined in the meta-analyses. The study team used the Newcastle–Ottawa Tool for the assessment of the quality of observational studies to assess the quality of included studies [12]. A random effect meta-analysis was conducted to assess be combined in the meta-analyses. The study team used the Newcastle–Ottawa Tool for the assessment of the quality of observational studies to assess the quality of included studies [12]. Search strategy and selection criteria A random effect meta-analysis was conducted to assess studies, if albuminuria was absent at the baseline but was present at follow-up, then the follow-up data were obtained. Search strategy and selection criteria Extracting data from all studies at just one time point, allowed both cross-sectional studies and cohort studies to 1 3 1 3 Acta Diabetologica (2019) 56:839–850 842 1 3 Table 1 Characteristics of the included studies (baseline data for prospective studies unless stated otherwise)i Study included name/ID Study design Country Mean age (years) Sex (% male) Number of participants (n) Smoking status (n) Albuminuria (n) Mean dura- tion of DM Mean HbA1c Mean SBP S NS Q Yes No (years) mmol/mol(%) (mm of Hg) Chuahirunet al., 2003 [50]/08 Prospective USA 45 55 33 13 20 NS NS NS NS 92 (10.6) NS Chuahirunet al., 2004 [51]/09 Prospective USA 45 54 84 31 53 NS 46 38 5 95 (10.8) 115 Chuahirun et al., 2004 [60]/09 Prospective USA 49 50 157 69 88 NS 112 45 5 54 (7.06) 132 Ikeda et al., 1996 [52]/15 Cross-sectional Japan 62 100 142 81 40 21 58 84 NS 62 (7.8) 137 Tseng et al., 2010 [53]/28 Prospective Taiwan 58 55 519 199 320 NS 240 279 10 64 (8.0) 132 Voulgari et al., 2011 [54]/29 Prospective Greece 56 50 193 73 NS 120 193 NS NS 61 (7.75) 143 Phistkul et al., 2008 [55]/23 Prospective USA 47 52 91 39 52 NS 91 NS 4 59 (7.53) 145 Hsu et al., 2010 [56]/14 Prospective Taiwan 54 100 509 191 243 75 314 195 4 66 (8.2) 129 Baggio et al., 2002 [37]/02 Cross-sectional Italy 58 73 96 48 48 NS 96 NS 11 65 (8.1) NS Cederholm et al., 2005 [57]/06 Cross-sectional Sweden 67 59 31,037 4532 26,505 NS 4811 26,226 8 51 (6.85) 147 Savage et al., 1995 [58]/26 Cross-sectional USA 58 61 931 264 230 439 402 531 9 103 (11.6) NS Okhuma et al., 2016 [59]/21 Cross-sectional Japan 65 100 2770 760 559 1451 NS NS 19 57 (7.40) 130 Prashanth et al., 2010 [60]/25 Cross-sectional Oman NS 51 447 85 362 NS 163 284 10 70 (8.55) NS Corradi et al., 1993 [61]/10 Cross-sectional Italy NS 100 90 44 46 NS 46 44 NS 60 (7.65) 162 Anan et al., 2007 [62]/01 Cross-sectional Japan 45 18 55 20 35 NS NS NS 5 60 (7.65) 129 Yoem et al., 2016 [63]/31 Cross-sectional Korea 63 100 629 314 90 225 455 174 9 58 (7.44) 126 Forsblom et al., 1998 [64]/11 Prospectivea (follow-up data) Finland 58 61 108 36 54 NS 31 59 9 95 (10.8) 152 Tomlinson et al., 2006 [65]/27 Cross-sectional China 53 100 496 196 300 NS NS NS 3 63(7.94) 133 Kanauchi et al., 1998 [66]/16 Cross-sectional Japan 65 46 155 44 111 NS 78 77 13 56 (7.3) NS Gambaro et al., 2001 [67]/12 Prospective Italy 65 55 273 72 134 67 107 203 13 75 (9.0) NS West et al., 1980 [68]/30 Cross-sectional USA NS NS 973 323 421 229 416 557 7 NS 137 Klein et al., 1993 [69]/17 Cross-sectional USA NS NS 376 53 200 123 58 318 NS NS NS Bruno et al., 1996 [70]/04 Cross-sectional Italy 69 43 1521 NS NS NS 756 765 11 64 (8.05) NS Bruno et al., 2003 [71]/05 Prospective Italy 68 38 1103 149 708 222 426 677 10 65 (8.1) 154 Bentata et al., 2016 [72]/03 Prospective Morocco 65 NS 671 81 590 NS 520 151 8 68 (8.4) NS Gerstein et al., 2000 [43]/13 Cross-sectional Canada 65 63 3503 538 N/A 1777 1128 2375 11 58 (7.46) 142 Kohler et al., 2000 [73]/18 Cross-sectional USA 51 32 1044 NS NS NS 244 760 0.3 76 (9.1) NS Nilsson et al., 2004 [74]/20 Cross-sectional Sweden 65 54 40,648 4512 36,136 NS 5578 35,070 8 48 (6.55) 144 Parving et al., 2006 [76]/22 Cross-sectional Denmark 61 50 24,151 NS NS NS NS NS 8 58 (7.5) NS Pijls et al., 2001 [75]/24 Cross-sectional Netherlands 64 49 335 NS NS NS NS NS 6 NS 143 1 Acta Diabetologica (2019) 56:839–850 843 the odds of having albuminuria between smokers and non- smokers. Search strategy and selection criteria Further random effects meta-analyses models were fitted to compare participants with and without albuminuria for other risk factors (age, sex, duration of type 2 diabetes systolic and diastolic blood pressure, total cholesterol, HDL cholesterol, triglyceride, BMI and HbA1c), with categori- cal outcomes fitted as odds ratios and continuous variables as difference in mean values. To explore the relationship between smoking and albuminuria, meta-regression analy- ses were carried out. To investigate the influence of dura- tion of diabetes on the risk of albuminuria between smokers and non-smokers further, we used the mixed effect meta- regression model to predict the odds ratio and corresponding 95% confidence intervals of albuminuria, among smokers compared to non-smokers for the duration of type 2 diabetes ranging from 4 to 20 years. Results The plot demonstrates that the differences in outcomes between smokers and non-smokers were consistent for most studies suggesting that other factors were unlikely to contribute to the variation in the risk of albuminuria Table 2 Relationship of cardiometabolic risk factors and albuminuria before adjusting for smoking status Variables Mean difference 95% confidence interval p value Age 1.24 0.84–1.64 < 0.001 Male sex 1.39 1.16–1.67 0.003 SBP 6.03 4.10–7.97 < 0.001 DBP 1.85 1.08–2.62 < 0.001 HbA1c 0.63 0.45–0.81 < 0.001 Duration of dia- betes 1.78 1.32–2.23 < 0.001 Total cholesterol 0.06 − 0.05 to 0.17 0.31 HDL cholesterol − 0.01 − 0.04 to 0.02 0.47 Triglyceride 0.22 0.12–0.33 < 0.001 Body mass index 0.40 − 0.00 to 0.80 0.05 Table 2 Relationship of cardiometabolic risk factors and albuminuria before adjusting for smoking status Fig. 3 A radial plot of random effects meta-analysis showing the standardized differences in observed outcomes (zi) between smokers against their corresponding precision (xi). The plot demonstrates that the differences in outcomes between smokers and non-smokers were consistent for most studies suggesting that other factors were unlikely to contribute to the variation in the risk of albuminuria associated with albuminuria: each year increase in the duration of T2DM was associated with an increased log of odds of albuminuria on an average by 0.19 units (95% CI 0.07–0.31), or it increased the odds approximately by 21% (Fig. 4). After 9 years of diabetes, the odds of albuminuria in smokers was approximately 50% higher 1.53 (1.10–2.43) compared to non-smokers. The odds ratio rose further to almost three times at 12-year duration 2.74 (1.74–4.30) and almost six times after 16 years 5.94 (2.58–15.05). The predicted mean odds ratio of albuminuria among smokers compared to non-smokers conditional on a range of the dura- tion of diabetes are presented in Supplementary material 4. The visual exploration of the funnel plot showed slight asymmetry of the plot suggesting possible publication bias. However, outcomes from the Egger’s test showed the evi- dence was not statistically significant (p = 0.063) (Supple- mentary material 2). Results A total of 2207 studies were identified by electronic data- base searches. After removing the duplicates, 2119 articles were screened for eligibility; 150 of them were accepted for abstract review, and 58 of them were included for full- text review. Overall, 30 studies (20 cross-sectional and ten prospective observational) with a total of 113,140 people with T2DM were included. The mean age of the study participants was 58 years, and 51% of them were male. Amongst the study participants, 11% were smokers, 60% were non-smokers, and 4% were quitters. Smoking status was unavailable for 25% of the study participants. The prevalence of albuminuria in the included studies was 14%. The mean duration of T2DM was 8 years; the mean HbA1c was 63 mmol/mol (7.9%), and the mean SBP was 125 mmHg. The outcomes from the random effects meta- analysis of 13 studies on 4313 smokers and 15,743 non- smokers showed that the pooled odds ratio of albuminuria in smokers, compared to non-smokers was 2.13 (95% CI 1.32–3.45; p = 0.002; Fig. 2), indicating a statistically sig- nificant increased risk of albuminuria in smokers. The heterogeneity between studies was assessed using the I2 statistic, which represents the total proportion of study variation that is due to heterogeneity rather than sampling error/chance [13]. Publication bias among studies was assessed by visual inspection of the funnel plot and the Egg- er’s test. The type 1 error to determine the level of statistical significance was set at p = 0.05. All statistical analyses were carried out using the metafor package (version 2.0.0) in the R statistical software environment and Cochrane Collabora- tion Review Manager version 5. i Except for one study, the radial plot suggested that the outcomes for most of the studies were consistent, regard- ing the effects of smoking and the variation in the risk of albuminuria (Fig. 3). Fig. 2 Forest plot showing an odds ratio of albuminuria in smokers compared to non-smokers Fig. 2 Forest plot showing an odds ratio of albuminuria in smokers compared to non-smokers 3 Acta Diabetologica (2019) 56:839–850 844 Fig. 3 A radial plot of random effects meta-analysis showing the standardized differences in observed outcomes (zi) between smokers against their corresponding precision (xi). Results Further meta-analyses demonstrated that cardiometabolic factors associated with albuminuria (OR, 95% CI) were: age 1.24 (95% CI 0.84–1.64, p < 0.001); male sex 1.39 (95% CI 1.16–1.67; p = 0.003); SBP 6.03 (95% CI 4.10–7.97, p < 0.001); DBP 1.85 (95% CI 1.08–2.62, p < 0.001); dura- tion of T2DM 1.78 (95% CI 1.32–2.23, p < 0.001); BMI 0.40 (95% CI 0.00–0.80, p = 0.05); total cholesterol 0.06 (95% CI − 0.05 to 0.17; p = 0.31); HDL − 0.01 (95% CI − 0.04 to 0.02; p = 0.47); triglyceride 0.22 (95% CI 0.12–0.33; p < 0.001) and HbA1c 0.63 (95% CI 0.45–0.81; p < 0.001) (Table 2) (Supplementary material 3). 3 Discussion This systematic review summarises the relationship between smoking and albuminuria in people with T2DM, and whether this relationship is influenced by other confounding variables such as age, sex, the duration of T2DM, HbA1c, BMI, HDL and total cholesterol, systolic and diastolic blood pressure. The meta-analysis suggests that smoking is a strong predictor of albuminuria in people with T2DM. The meta-regression, on the other hand, concedes that apart from the duration of T2DM, none of the above confounding variables has any statistically significant influence on albu- minuria, when adjusted for smoking status. There is a linear relationship between smoking and the duration of T2DM with albuminuria. Smokers with T2DM have 21% increased annual risk of albuminuria, compared to non-smokers. Therefore, smoking cessation at an early stage of disease trajectory is likely to be one of the most effective interven- tion strategies to prevent the development of albuminuria in smokers with T2DM. Meta-regression analyses found most moderator variables were not associated with the study effect except the duration of diabetes showing a significant association (p = 0.001). We observed that the inclusion of duration of diabetes as a mod- erator variable reduced the residual heterogeneity although there was still evidence of residual heterogeneity (Q statis- tic = 10.09, p = 0.002); the estimate of residual heterogeneity (τ2) reduced from 0.69 (95% CI 0.38–3.84) based on the ran- dom effect meta-analysis model to 0.23 (95% CI 0.10–2.13) based on the mixed effect meta-regression model (Table 3) (Supplementary material 3). Therefore, the time to diabetes as a moderator variable accounted for almost 60% of the heterogeneity.i The statistically significant residual heterogeneity sug- gested that other moderators not investigated in this study might be important. Discussion The duration of T2DM was positively This is the first systematic review and meta-anal- ysis exploring the relationship between smoking and 1 3 3 Acta Diabetologica (2019) 56:839–850 845 Table 3 Relationship of cardiometabolic risk factors with albuminuria after adjusting for smoking status Statistically significant variable that influenced the relationship between smoking and albuminuria was the duration of T2DM (highlighted in bold font) Moderator variables Overall effect size (Z) Heterogeneity (τ2) p value Age 0.75 (− 0.084–0.18) 0.70 (0.33–6.44) 0.46 Male sex 0.27 (− 0.02–0.03) 0.79 (0.36–6.81) 0.78 HbA1c 1.43 (0.1–0.65) 0.76 (0.30–4.94) 0.15 HDL − 0.50 (− 47.78 to 28.83) 9.93 (1.66–100) 0.61 Total cholesterol 0.92 (− 1.36 to 3.75) 1.74 (0.56–15.78) 0.35 Triglyceride − 1.14 (− 0.51 to 0.14) 0.01 (0–1.28) 0.25 Duration of diabetes 3.18 (0.07–0.31) 0.23 (0.10–2.13) 0.001 SBP 1.09 (− 0.29 to 0.101) 1.26 (0.44–10.22) 0.27 DBP 0.26 (− 0.13 to 0.17) 2.05 (0.66–18.43) 0.79 BMI 2.48 (0.15–1.30) 0.74 (0.36–6.86) 0.93 Statistically significant variable that influenced the relationship between smoking and albuminuria was the duration of T2DM (highlighted in bold font) Fig. 4 Predicted odds ratio (OR) of albuminuria in smokers compared to non-smokers with duration of type 2 diabetes based on the outcome of the logistic mixed model. The solid line shows the predicted mean and dashed line shows the corresponding 95% confi- dence interval. The OR below the horizontal dotted line is not statistically significant (p > 0.05). The plot also shows the observed OR of individual studies (points) where the point sizes are proportional to the inverse of the corresponding standard errors hand, taking into consideration all the above confounding variables, concluded that the duration of diabetes is the most important predictor of albuminuria in smokers with T2DM. albuminuria, and how other cardiometabolic parameters influence this relationship. Although multiple studies have shown smoking augments the risk of albuminuria in peo- ple with type 1 diabetes [14, 15], its role in T2DM remains undetermined. T2DM, as opposed to T1DM, is one of the components of metabolic syndrome. In addition to hypergly- caemia, it is often accompanied by obesity, hypertension and dyslipidaemia [16]. All these risk factors are closely associ- ated with albuminuria [17], and therefore, the relationship between smoking and albuminuria is much more intricate in T2DM, compared to T1DM. Previous studies have shown that smokers have a higher urinary albumin excretion rate, which might have been independent of glycaemic effects [18]. Discussion Taking all these evi- dence into account, this systematic review emphasises that to effectively manage the prevalence and the progression of albuminuria in T2DM, the most effective strategy would be a multifactorial intervention where smoking cessation is one of the key components. choice of drugs seems to be a determinant factor of albu- minuria. Insulin sensitizers have shown better efficacy in halting the prevalence and progression of albuminuria com- pared to insulin and its secretagogues. In BARI-2D trial, the researchers have shown that insulin, and its secretagogues are more likely to cause an increased prevalence of albumi- nuria and coronary artery disease, compared to insulin-sen- sitizing drugs [22]. However, it will be interesting to know if this outcome is influenced by the choice of drugs or people who were on insulin had poorer glycaemic control. p g y Irrespective of hypertension, treatment with angiotensin- converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) has shown promising prospect of halting the prevalence and progression of albuminuria [23]. However, studies have shown that this reno-protective effect of ACEI and ARBs can be revoked in smokers [24], suggesting that renin–angiotensin axis blockade is less effective to prevent the progression of albuminuria in smokers. Raised triglyc- eride, and raised total and LDL cholesterol, with low HDL cholesterol are the hallmarks of dyslipidaemia in T2DM [25], but in smokers the predominant abnormality in lipid profile seems to be lower HDL cholesterol [26]. Smoking downregulates the hepatic and endothelial lipoprotein lipase activities [27] and tampers with the reverse cholesterol trans- port pathway [28]. As a consequence, they have lower HDL cholesterol compared to their non-smoker counterparts. Smoking cessation, on the other hand improves lipid profile particularly the HDL cholesterol, despite moderate weight gain [29, 30], which in turn halts the progression of albumi- nuria [31]. Conversely, isolated and piecemeal management of glucose, blood pressure and lipid profile did not show consistent efficacy to prevent the prevalence or progres- sion of albuminuria in smokers with diabetes [9, 32]. These observations are suggestive of an independent relationship between smoking and albuminuria mediated by a constella- tion of underlying pathophysiological processes. The findings of this systematic review have significant clinical implications. The World Health Organization (WHO) estimates that by 2030, a staggering number of 366 million people will suffer from T2DM worldwide. Discussion Meta-analyses in this systematic review concluded that there is a close association between smoking and albumi- nuria in people with T2DM. Meta-regression, on the other Early detection of albuminuria at the stage of microal- buminuria, and multifactorial intervention including smok- ing cessation, are advocated in all the guidelines across the globe, including the European Association of Study on Diabetes (EASD) and the American Diabetes Association (ADA) [19]. This recommendation is based on the obser- vation that once the daily urinary albumin excretion rate reaches the level of proteinuria (urinary albumin excre- tion > 300 mg/day), no interventions appears to be effective in reversing it [20, 21]. Addressing other anthropometric and metabolic risk factors including hip–waist ratio, BMI, HbA1c, blood pressure and lipid profiles remain at the centre of this intervention strategy. For glycaemic management, the 1 846 Acta Diabetologica (2019) 56:839–850 Although smokers have lower BMI than non-smokers, nonetheless they have more visceral adiposity and lower insulin sensitivity [41]. Smoking cessation, on the other hand, despite causing moderate weight gain, is associated with the reversal of visceral adiposity and an improve- ment in insulin sensitivity [30]. But this reversal takes longer in people with T2DM, compared to people without [3, 42]. Therefore, short-term abstinence may not yield any meaningful benefit in smokers with T2DM. The Heart Outcomes Prevention Evaluation (HOPE) study examined the factors that influence the prevalence and progression of albuminuria in people with and without diabetes. This study demonstrated that smoking, hypertension, older age, abdominal adiposity, vascular disease and left ventricular hypertrophy were significantly associated with albumi- nuria, in people with and without diabetes. However, in people with diabetes, the most significant determinants of albuminuria were the duration of diabetes, HbA1c and the use of insulin. People with diabetes were 1.16 times more likely to develop albuminuria after a diabetes dura- tion of 10.4 years (irrespective of their HbA1c), the risk of albuminuria increased by 8% for each 0.9% increase in the HbA1c, and the people with albuminuria were 1.3 times more likely to be on insulin compared to people who had normoalbuminuria. Sex, dyslipidaemia, creatinine, and BMI were not independently associated with albuminuria after adjustment of other factors [43]. 3 Discussion Amongst them, 60 million will be between 20 and 44 years, and 180 million will be between 45 and 64 years [44]. Young smokers with T2DM are at a higher risk of albuminuria as they will live longer with the condition. This study showed that the risk of albuminuria was simi- lar in smokers and non-smokers up to around 8.5 years of T2DM duration, and then the risk increased approxi- mately by 20% annually. Albuminuria marks the onset of microvascular complications which is often associated with retinopathy, neuropathy and macrovascular involve- ment [45]. Several studies have shown a rapid rise in the prevalence of albuminuria and cardiovascular complica- tions in younger patients with T2DM, compared to T1DM, despite having similar glycaemic control [46, 47]. Poor lifestyle choices including smoking have been attributed to this disparate response of glycaemic control in T2DM, as opposed to T1DM. Therefore, this study emphasises Several mechanisms have been proposed to explain the albuminuria in smokers with T2DM. They include increased blood pressure, altered intrarenal haemodynamics such as activation of the sympatho-adrenergic pathway, activation of the renin–angiotensin–aldosterone axis and the endothelin system [33–35]. In addition, smoking directly causes tubulo- interstitial disease [36] and causes neuro-endocrine disrup- tion, vascular endothelial damage and metabolic deregula- tions which adversely affect renal structure and function [18, 37, 38]. Therefore, addressing hyperglycaemia, hypertension and dyslipidaemia without smoking cessation may not halt the prevalence and progression of albuminuria in smokers with diabetes. Nicotine and other toxic metabolites in cigarettes appear to be handled differently in people with and without diabe- tes [39]. Nicotine infusion acutely increases insulin resist- ance in people with T2DM but not in people without [40]. 1 3 Acta Diabetologica (2019) 56:839–850 847 that smokers, particularly the younger smokers should be encouraged to quit soon after the diagnosis and persuaded to remain abstinent. Funding This research was partially funded by the East Midlands Collaboration for Leadership in Applied Health Research and Care (Project 17). One of the strengths of this study is that it included all the major studies available on the electronic databases from their inception and included 30 studies with 113,400 partici- pants. The quality of the papers was determined by the New- castle–Ottawa scale, which is a validated tool, and the review process followed PRISMA protocol [10], which is consid- ered to be the gold standard. Availability of data and material The corresponding author has all the data and materials. Availability of data and material The corresponding author has all the data and materials. Conclusion Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, 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. Albuminuria is one of the earliest biochemically measurable risk factors in T2DM, which heralds incipient micro- and macrovascular complications. It is a substantial milestone in the trajectory of disease progression and is independently associated with cardiovascular and all-cause mortality. This study reiterates that smoking is a strong predictor of albumi- nuria; the longer the duration of T2DM, the higher the risk. With a rapidly changing global prevalence of T2DM with an anticipated surge of younger people with T2DM [48], and an approximately 70% of them already having complica- tions [49], it is important to raise awareness about the effect of smoking and duration of T2DM on albuminuria, and its impact on cardiovascular mortality. Future research should be focused on elucidating the relationship between smok- ing cessation, and the progression of albuminuria in people with T2DM, particularly the length of abstinence required to reverse the risk of albuminuria. Albuminuria is one of the earliest biochemically measurable risk factors in T2DM, which heralds incipient micro- and macrovascular complications. It is a substantial milestone in the trajectory of disease progression and is independently associated with cardiovascular and all-cause mortality. This study reiterates that smoking is a strong predictor of albumi- nuria; the longer the duration of T2DM, the higher the risk. Discussion Publication bias was addressed by conducting the appropriate sensitivity test, which did not show any significant bias. On the other hand, the weaknesses of the study were that it was based mainly on cross-sectional, or the baseline data of prospective studies, and therefore, no temporal relationship between smoking and albuminu- ria can be confirmed. Second, most of the included stud- ies used self-reported smoking behaviour which might not be accurate. There was also considerable heterogeneity in the included studies, and therefore, the findings may not be generalisable. Although between-study heterogeneity was investigated, meta-regression models lacked statistical power to assess associations between the effect size and study-level covariates. References With a rapidly changing global prevalence of T2DM with an anticipated surge of younger people with T2DM [48], and an approximately 70% of them already having complica- tions [49], it is important to raise awareness about the effect of smoking and duration of T2DM on albuminuria, and its impact on cardiovascular mortality. Future research should be focused on elucidating the relationship between smok- ing cessation, and the progression of albuminuria in people with T2DM, particularly the length of abstinence required to reverse the risk of albuminuria. 1. Pan A, Wang Y, Talaei M, Hu FB (2015) Relation of smoking with total mortality and cardiovascular events among patients with diabetes mellitus: a meta-analysis and systematic review. Circula- tion 132(19):1795–1804 1. Pan A, Wang Y, Talaei M, Hu FB (2015) Relation of smoking with total mortality and cardiovascular events among patients with diabetes mellitus: a meta-analysis and systematic review. 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CLG and MN contributed to statistical analy- ses of the data. DK wrote up the manuscript, and all the co-authors contributed to it. KK and MJD were involved in overall supervision and worked in advisory roles in all aspects of the research. Ethics standard statement Not applicable. Consent for publication All the authors have approved the final manu- script and consented for publication. Compliance with ethical standards Conflict of interest DK, CLG and MN have no competing interests. MJD reports personal fees from Novo Nordisk, Sanofi-Aventis, Lilly, Merck Sharp & Dohme, Boehringer Ingelheim, AstraZeneca, Janssen, Servier, Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceu- ticals International Inc. She has also received grants from Novo Nor- disk, Sanofi-Aventis, Lilly, Boehringer Ingelheim, Janssen outside the submitted work. 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Affiliations Debasish Kar1,2 · Clare Gillies1 · Mintu Nath1 · Kamlesh Khunti1 · Melanie J. Davies1 · Samuel Seidu1 76. Parving HH, Lewis JB, Ravid M, Remuzzi G, Hunsicker LG, DEMAND investigators (2006) Prevalence and risk factors for microalbuminuria in a referred cohort of type II diabetic patients: a global perspective. Kidney Int 69(11):2057–2063 1 Diabetes Research Centre, Univerisity of Leicester, Leicester, UK Debasish Kar1,2 · Clare Gillies1 · Mintu Nath1 · Kamlesh Khunti1 · Melanie J. Davies1 · Samuel Seidu1 Clare Gillies clg13@leicester.ac.uk Mintu Nath mn193@leicester.ac.uk Kamlesh Khunti kk22@leicester.ac.uk Melanie J. Davies melanie.davies@uhl‑tr.nhs.uk Samuel Seidu sis11@leicester.ac.uk 1 Diabetes Research Centre, Univerisity of Leicester, Leicester, UK 2 Academic Unit of Diabetes and Endocrinology, University of Sheffield, Sheffield, UK Clare Gillies clg13@leicester.ac.uk Mintu Nath mn193@leicester.ac.uk Kamlesh Khunti kk22@leicester.ac.uk Melanie J. Davies melanie.davies@uhl‑tr.nhs.uk Samuel Seidu sis11@leicester.ac.uk 1 Diabetes Research Centre, Univerisity of Leicester, Leicester, UK 2 Academic Unit of Diabetes and Endocrinology, University of Sheffield, Sheffield, UK 1 3 3
https://openalex.org/W2106596862	https://europepmc.org/articles/pmc2777001?pdf=render	English	null	Genevestigator V3: A Reference Expression Database for the Meta-Analysis of Transcriptomes	Advances in bioinformatics	2,008	cc-by	3,738	Tomas Hruz,1 Oliver Laule,2 Gabor Szabo,2 Frans Wessendorp,2 Stefan Bleuler,3 Lukas Oertle,2 Peter Widmayer,1 Wilhelm Gruissem,2 and Philip Zimmermann2 1Institute of Theoretical Computer Science, ETH Zurich, 8092 Zurich, Switzerland 2Department of Biology, ETH Zurich, 8092 Zurich, Switzerland 3Computer Engineering and Networks Laboratory, ETH Zurich, 8092 Zurich, Switzerland Correspondence should be addressed to Philip Zimmermann, phz@ethz.ch Received 7 April 2008; Accepted 28 May 2008 Recommended by Yves Van de Peer Recommended by Yves Van de Peer The Web-based software tool Genevestigator provides powerful tools for biologists to explore gene expression across a wide variety of biological contexts. Its ﬁrst releases, however, were limited by the scaling ability of the system architecture, multiorganism data storage and analysis capability, and availability of computationally intensive analysis methods. Genevestigator V3 is a novel meta- analysis system resulting from new algorithmic and software development using a client/server architecture, large-scale manual curation and quality control of microarray data for several organisms, and curation of pathway data for mouse and Arabidopsis. In addition to improved querying features, Genevestigator V3 provides new tools to analyze the expression of genes in many diﬀerent contexts, to identify biomarker genes, to cluster genes into expression modules, and to model expression responses in the context of metabolic and regulatory networks. Being a reference expression database with user-friendly tools, Genevestigator V3 facilitates discovery research and hypothesis validation. Copyright © 2008 Tomas Hruz 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. Hindawi Publishing Corporation Advances in Bioinformatics Volume 2008, Article ID 420747, 5 pages doi:10.1155/2008/420747 Hindawi Publishing Corporation Advances in Bioinformatics Volume 2008, Article ID 420747, 5 pages doi:10.1155/2008/420747 Hindawi Publishing Corporation Advances in Bioinformatics Volume 2008, Article ID 420747, 5 pages doi:10.1155/2008/420747 Resource Review Genevestigator V3: A Reference Expression Database for the Meta-Analysis of Transcriptomes Tomas Hruz,1 Oliver Laule,2 Gabor Szabo,2 Frans Wessendorp,2 Stefan Bleuler,3 Lukas Oertle,2 Peter Widmayer,1 Wilhelm Gruissem,2 and Philip Zimmermann2 1Institute of Theoretical Computer Science, ETH Zurich, 8092 Zurich, Switzerland 2Department of Biology, ETH Zurich, 8092 Zurich, Switzerland 3Computer Engineering and Networks Laboratory, ETH Zurich, 8092 Zurich, Switzerland Tomas Hruz,1 Oliver Laule,2 Gabor Szabo,2 Frans Wessendorp,2 Stefan Bleuler,3 Lukas Oertle,2 Peter Widmayer,1 Wilhelm Gruissem,2 and Philip Zimmermann2 1Institute of Theoretical Computer Science, ETH Zurich, 8092 Zurich, Switzerland 2Department of Biology, ETH Zurich, 8092 Zurich, Switzerland 3Computer Engineering and Networks Laboratory, ETH Zurich, 8092 Zurich, Switzerland 2. Construction The large number of simultaneous users, the increasing load of server side data processing, and the need to implement advanced analysis tools required the choice of a technology that shares the load between server and client machines. In fact, some analyses such as the clustering of large data matrices require considerable CPU time, frequently overloading a single machine if multiple clustering jobs are sent to it simultaneously. At the same time, we looked for a technology that minimizes hardware and software adaptations on the user side. by Java 2 Swing technology and a custom communication protocol between the Java client and the server farm. The Genevestigator V3 client application is a signed applet loaded and running within the Internet browser. New versions of the client are distributed on the ﬂy from the server once they are available. A careful analysis of possible technologies revealed that a combination of Java and relational database (RDB) technolo- gies would represent the optimal combination to handle the complex problem of providing a large centralized database and complex, highly interactive analysis tools to users in the biology community. We therefore veriﬁed the penetration of Java (and its various versions) in the Genevestigator user base [7], to be sure that a suﬃciently powerful version of Java is installed on most user computers. The task of the application server is to cache and quickly provide the compressed data to the client computer, where they could be processed with visualization and clustering algorithms. The application server provides also the data- mining functionality with presampling algorithms to quickly retrieve the data according to biologically meaningful rank- ing functions. p Genevestigator V3 comprises four main components: (1) a database, (2) an application server, (3) a Java client application, and (4) a website for user support and man- agement. More speciﬁcally, Genevestigator V3 is a mul- titier Java/Java/Mysql system in which a thick Java client communicates with a farm of server machines running a Tomcat application server which, in turn, communicates with a distributed system of database instances to retrieve the queried data (see Figure 1). Except for a number of speciﬁc cases, most of the scientiﬁc computing is done on the client side of the application, which provides a highly interactive and integrated view of the analyzed data. 1. Background A load-balancing subsystem ensures that the clients can communicate with a scalable cluster of application servers. Moreover, the application servers can be transparently conﬁgured to work with a database cluster of an arbitrary size. therefore developed Genevestigator V3, which is based on a new framework of the original Genevestigator concept. In particular, we Browser Genevestigator application Plugins Tools Util Base applet Custom RPC protocol (http) Tomcat Cache services Cache Genevestigator services JDBC JDBC DB SQL server Browser Genevestigator application Plugins Tools Util Base applet Custom RPC protocol (http) Tomcat Cache services Cache Genevestigator services JDBC JDBC DB SQL server (i) curated and quality controlled more than 20,000 Aﬀymetrix expression microarrays from human, mouse, rat, Arabidopsis, and barley, (ii) manually curated pathway reaction networks for mouse and Arabidopsis (see Table 1), (ii) manually curated pathway reaction networks for mouse and Arabidopsis (see Table 1), (iii) developed an architecture to distribute the analysis task between the client and the server such that computationally intensive algorithms can run on the client computer, (iii) developed an architecture to distribute the analysis task between the client and the server such that computationally intensive algorithms can run on the client computer, (iv) designed a database model which provides high scalability and organism-independent modeling, (v) performed major improvements in existing analysis tools, (v) performed major improvements in existing analysis tools, Genevestigator services (vi) implemented a more eﬃcient and ﬂexible workﬂow including workspace storage and ﬁgure export, and (vi) implemented a more eﬃcient and ﬂexible workﬂow including workspace storage and ﬁgure export, and (vii) developed a set of new analysis tools for biomarker identiﬁcation, clustering, biclustering, and pathway analysis. Figure 1: System architecture of Genevestigator. The white spaces show the possible boundaries between computers. The application server modules are running in a Tomcat web container and communicate with the database through the JDBC protocol. A thick Java applet reads data from an application server and a cache subsystem through a custom RPC protocol tunneled over http/https. A load-balancing subsystem ensures that the clients can communicate with a scalable cluster of application servers. Moreover, the application servers can be transparently conﬁgured to work with a database cluster of an arbitrary size. 1. Background expression arrays in their biological context remains a major challenge for biologists. Additionally, the contextual information (sample metadata) of published experiments is not readily available in a systematic form, but frequently in plain text descriptions, published articles, or in author’s personal notes. Collecting and annotating sample metadata using accepted ontologies and combining them with the large amount of published expression data opens new perspectives in the analysis of transcriptomes across many cell types, tissues, organs, and conditions. Additionally, it allows to create large reference expression databases representing a virtual mirror of life’s biological processes. Systems biology explores interactions between components of a biological system. Frequently, the term systems biology is used in the context of mathematical modeling of cellular networks in unicellular organisms or in speciﬁc cell types of multicellular organisms. A less prevalent but fundamental approach is the modeling of networks at the organism level by including quantitative information about where, when, and why speciﬁc components are active. Dissecting the organism network (in contrast to the cellular network) of multicellular organisms helps to understand mechanisms that drive development, the functions of system components, and regulatory networks that trigger responses to environ- mental cues or to genetic perturbations. In [5, 6], we presented a novel approach to assemble public expression data for mouse and Arabidopsis into context-related proﬁles (metaproﬁles) and provided easy online access to this database. The earlier versions of Genevestigator, however, oﬀered only a limited number of analysis tools and the original architecture did not allow it to scale with the rapidly increasing number of users and gene expression information from multiple organisms. We To build such networks at multicellular scale, both expression data and contextual information have to be analysed simultaneously. Although gene expression data and statistical tools are now widely available [1–4], the simultaneous analysis of hundreds or thousands of gene Advances in Bioinformatics 2 Browser Genevestigator application Plugins Tools Util Base applet Custom RPC protocol (http) Tomcat Cache services Cache Genevestigator services JDBC JDBC DB SQL server Figure 1: System architecture of Genevestigator. The white spaces show the possible boundaries between computers. The application server modules are running in a Tomcat web container and communicate with the database through the JDBC protocol. A thick Java applet reads data from an application server and a cache subsystem through a custom RPC protocol tunneled over http/https. 3. Content and Tools (3) Clustering Analysis, and (4) Pathway Projector. Each toolset comprises several tools that focus on a speciﬁc type of analysis. Besides signiﬁcantly improving the Meta-Proﬁle analysis tools, the Biomarker Search, Clustering Analysis, and Pathway Projector toolsets largely comprise novel tools that were not available in previous Genevestigator releases. More details about these toolsets are provided on the Genevestiga- tor website (https://www.genevestigator.ethz.ch/). In brief; In order to build the Genevestigator reference expression database, we collected hundreds of Aﬀymetrix experiments from the public domain, controlled the quality of the data using several Bioconductor packages (see [6]), and man- ually annotated them according to systematic ontologies. As of March 2008, a compendium of more than 20,000 Aﬀymetrix arrays from human, mouse, rat, Arabidopsis, and barley has been made available for meta-analysis through Genevestigator. This gives users the opportunity to look at how genes are expressed in a very wide variety of contexts. For example, the human dataset currently com- prises expression monitoring across more than 330 diﬀerent conditions, including 89 chemicals, 64 diﬀerent diseases, 20 growth factors, 17 infection causes, and 36 diﬀerent tumor types (see Table 1 in Supplementary Material available online at doi:10.1155/2008/420747). An overview of the number of microarrays, pathways, and biological contexts for which expression data is available is shown in Table 1. In Genevestigator, the biological context is embedded into a time-space-response architecture and is given by the terms “Anatomy” (space), “Development” (time), and “Stimulus” or “Mutation” (external or internal perturbations). p g g The Meta-Proﬁle analysis toolset includes several tools that provide information about when, where, and in response to what genes of interest are expressed. Speciﬁcally, they visu- alize gene expression across experiments or biological con- texts such as anatomy, development, stimulus, and mutation. The Biomarker Search toolset allows users to easily identify genes that are speciﬁcally expressed in a set of biological states (anatomy or development) or speciﬁcally up- or downregulated in response to a set of perturbations (stimulus or mutation). An explanation on how to use these tools is provided on the website. The clustering tools in Genevestigator V3 group genes that show similarity in their summarized expression levels (i.e., in their metaproﬁles). These tools can also be used to cluster array-level proﬁles. Two types of methods are currently provided in the Clustering Analysis toolset: (1) hier- archical clustering, and (2) biclustering. 2. Construction This is enabled The database system behind the application server was carefully designed to accommodate multiorganism data and to provide the possibility to switch quickly between diﬀerent organisms. Another challenge was posed by the experiment classiﬁcation database subsystem, which stores the measurement data according to ontologies representing the sample experimental context, in particular the anatomy part, developmental stage, stimulus applied, and genetic background. In a further database subsystem, metabolic and regulatory pathways are mapped to multiple organisms and microarray data. 3 Advances in Bioinformatics Advances in Bioinformatics Table 1: Currently available data. Overview of the number of quality controlled and annotated arrays, partitioned into types of biological contexts for which expression data is available in the database (status: March 2008). For mouse and Arabidopsis, the number of curated pathway reactions is indicated. Table 1: Currently available data. Overview of the number of quality controlled and annotated arrays, partitioned into types of biological contexts for which expression data is available in the database (status: March 2008). For mouse and Arabidopsis, the number of curated pathway reactions is indicated Human Mouse Rat Arabidopsis Barley Size of arrays 10 k 20 k 47 k 12 k 40 k 8 k 31 k 22 k 22 k Number of Aﬀymetrix arrays 1109 3786 2782 3071 1967 2146 858 3110 706 Anatomy – total of nodes in anatomy tree 97 174 185 150 133 108 63 43 23 – central nervous system 33 46 51 39 16 29 20 – cell culture / primary cells 13 39 29 19 21 10 9 – other anatomy parts 51 89 104 92 96 69 34 Development – number of stages available 9 9 8 12 12 6 7 10 6 Stimulus (external perturbations) – total number of treatments 81 295 243 122 81 107 55 168 39 – chemical treatments 13 97 32 43 34 45 25 40 3 – diseases, biotic stresses 23 61 34 12 12 4 0 18 24 – other external perturbations∗ 45 137 175 67 35 58 30 110 12 Mutation (internal perturbations) – number of mutant models or cell lines 1 4 25 117 100 5 2 167 17 Reaction networks – Number of reaction nodes — — — 945 — — — 1199 — ∗Includes: surgery, stress, hormone, growth factors and cytokines, exercise, caloric restriction, fasting, peptides, several tumor types, light irradiation, and many more external perturbations. 3. Content and Tools Both methods have been shown to reveal biologically signiﬁcant associations between biological components or gene networks [8, 9]. An overview of the Genevestigator V3 data- and work- ﬂow is shown in Figure 2. Thanks to its novel software architecture and the complete redesign of workﬂows using usability criteria, Genevestigator V3 allows to work easily with several organisms and multiple lists of genes at the same time. Four toolsets are available to analyze these tran- scriptomes: (1) Meta-Proﬁle analysis, (2) Biomarker Search, The Pathway Projector toolset allows the visualization of expression data on a pathway or network level. Several conceptual features distinguish it from most currently avail- able pathway analysis tools. First, all pathways were veriﬁed 4 Advances in Bioinformatics Biological experiment Anatomy Development Stimulus Mutation Microarray data Sample annotation Public repositories Curation & quality control Genevestigator database Application server Client Java application Analysis output (a) 47 1752 900 2605 3458 4311 5164 6017 6870 7723 8576 98108_at 95927_f_at Open access/registration User manual Community pages Share with community Results New hypothesis Validation Export publish-ready ﬁgures Publish (b) Figure 2: Data ﬂow and analysis cycle. Schematic overview of Genevestigator V3. (a) Data ﬂow through the Genevestigator system. Microarray experiments are performed by the research community and either stored in public repositories or sent directly to Genevestigator. Data and annotations are curated and quality controlled by Genevestigator curators and stored in the Genevestigator system (see Figure 1). (b) Expression analysis cycle. The user starts at the Genevestigator website, from where the client application is launched. Four toolsets allow diﬀerent types of analysis. Results obtained are used either to conﬁrm an existing hypothesis or to create new ones that can be validated in the laboratory. Figures from Genevestigator can be exported in a variety of formats. The website provides user support and a community exchange platform. Biological experiment Anatomy Development Stimulus Mutation Microarray data Sample annotation Public repositories Curation & quality control Genevestigator database Application server Client Java application Analysis output (a) Microarray data Sample annotation Share with community Publish (b) (a) Figure 2: Data ﬂow and analysis cycle. Schematic overview of Genevestigator V3. (a) Data ﬂow through the Genevestigator system. Microarray experiments are performed by the research community and either stored in public repositories or sent directly to Genevestigator. Data and annotations are curated and quality controlled by Genevestigator curators and stored in the Genevestigator system (see Figure 1). (b) Expression analysis cycle. 3. Content and Tools The user starts at the Genevestigator website, from where the client application is launched. Four toolsets allow diﬀerent types of analysis. Results obtained are used either to conﬁrm an existing hypothesis or to create new ones that can be validated in the laboratory. Figures from Genevestigator can be exported in a variety of formats. The website provides user support and a community exchange platform. manually from the literature and modeled into a single reaction network, in which each reaction is represented only once, even if it is shared between several pathways. The user can ﬁnd locations within the network using classically deﬁned pathway terms. Nevertheless, network analysis focuses on identifying expression modules within the global reaction network, irrespective of classical pathway boundaries. For example, the user can start with a single reaction or pathway and create subnetworks by extending them with neighbor reactions or pathways according to their expression. A second distinguishing feature is the projection of expression metaproﬁle data onto these networks. The user can create virtually any type of comparison selected from the database and project it onto the network, for instance, compare one state of development with another, a tissue against another tissue, or a treatment against its control set. can be further analyzed by switching to the Meta-Proﬁle or Clustering analysis toolsets. Another major improvement is that all organisms are integrated into the same user client interface, allowing the user to study genes from several organisms in parallel and in the same session. Besides these analysis tools, several novel usability features have been added to the client, such as saving workspaces, exporting ﬁgures in Web or print resolution, saving pathway views, or exporting lists of genes. Users can submit their workspaces or pathway views to be placed on the community page of the Website. This allows other users to view the corresponding results without having to upload arrays or genes again, or to redesign network structures. Additionally, a role-based access control system allows conﬁdential data to be visualized in the context of all other experiments in the database, without being seen by nonauthorized users. Upon request, conﬁdential data from laboratories are quality-controlled and entered into the database by Genevestigator curators to ensure comparability and systematic annotation. Acknowledgments Matthias Hirsch-Hoﬀmann managed server infrastructure. The project was supported by funding from ETH Zurich. 5. Conclusion In summary, Genevestigator V3 provides a large and continuously growing reference database of systematically annotated and high-quality microarray data from several organisms. Powerful but user-friendly tools for the meta- analysis of transcriptomes allow to visualize gene expression through a large library of biological contexts, facilitating the modeling of gene regulatory networks and the iden- tiﬁcation of speciﬁc markers. The tool is accessible at http://www.genevestigator.ethz.ch. 4. Utility and Discussion In contrast to the previous versions, Genevestigator V3 integrates all the diﬀerent analysis tools. For example, marker genes identiﬁed using the Biomarker Search tool It is generally accepted that the integration of multiple data types will be essential to model biological systems. Transcriptomics data from single experiments provide an 5 Advances in Bioinformatics informative but selective view of biological processes. There is an increasing evidence, however, that mining microarray data simultaneously from many diﬀerent experiments and organisms make this type of data highly informative and valuable for functional genomics and systems biology appli- cations. The Genevestigator V3 discovery tool and reference database therefore covers a broad range of academic and commercial research interests. Until now, most researchers have used Genevestigator in molecular biology research for discovery or experimental validation. More recent tools in Genevestigator V3 now allow researchers to develop applications in plant and animal genomics, toxicogenomics, biomarker discovery, crop biotechnology, disease and clinical research, as well as pathway prioritization. Its multiorganism capabilities allow researchers to perform cross-species studies to conﬁrm orthologs and facilitate gene function discovery. [6] O. Laule, M. Hirsch-Hoﬀmann, T. Hruz, W. Gruissem, and P. Zimmermann, “Web-based analysis of the mouse transcrip- tome using Genevestigator,” BMC Bioinformatics, vol. 7, article 311, 2006. [7] T. Hruz, W. Gruissem, M. Hirsch-Hoﬀmann, and P. Zim- mermann, “Reducing Java Internet project risks: a case study of public measurement of client component functionality in the user community,” in Proceedings of the 4th International Conference on Principles and Practices of Programming in Java (PPPJ ’06), pp. 199–202, Mannheim, Germany, August- September 2006. [8] M. B. Eisen, P. T. Spellman, P. O. Brown, and D. Botstein, “Clus- ter analysis and display of genome-wide expression patterns,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 25, pp. 14863–14868, 1998. [9] A. Preli´c, S. Bleuler, P. Zimmermann, et al., “A systematic comparison and evaluation of biclustering methods for gene expression data,” Bioinformatics, vol. 22, no. 9, pp. 1122–1129, 2006. Genevestigator tools are made available in three forms: OPEN-ACCESS, CLASSIC, and ADVANCED. For academic users, the ﬁrst two are freely available, while access to ADVANCED is made available for a moderate subscription fee (in the range of reagent kits that are frequently used in the lab) to support our costs of curation and development. The tools and features available for each form are described on the Genevestigator website. References [1] A. Brazma, H. Parkinson, U. Sarkans, et al., “ArrayExpress— a public repository for microarray gene expression data at the EBI,” Nucleic Acids Research, vol. 31, no. 1, pp. 68–71, 2003. [2] T. Barrett, T. O. Suzek, D. B. Troup, et al., “NCBI GEO: mining millions of expression proﬁles—database and tools,” Nucleic Acids Research, vol. 33, database issue, pp. D562–D566, 2005. [3] N. E. Olson, “The microarray data analysis process: from raw data to biological signiﬁcance,” NeuroRx, vol. 3, no. 3, pp. 373– 383, 2006. [4] R. C. Gentleman, V. J. Carey, D. M. Bates, et al., “Bioconductor: open software development for computational biology and bioinformatics,” Genome Biology, vol. 5, no. 10, p. R80, 2004. [5] P. Zimmermann, M. Hirsch-Hoﬀmann, L. Hennig, and W. Gruissem, “GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox,” Plant Physiology, vol. 136, no. 1, pp. 2621–2632, 2004.
https://openalex.org/W2132227650	https://www.scielo.br/j/ibju/a/7NTgbrWdwtN9LCztVpRJ9jy/?lang=en&format=pdf	English	null	Quantification of tumor extension in prostate biopsies: importance in the identification of confined tumors	International Braz J Urol	2,003	cc-by	2,952	TUMOR QUANTIFICATION IN PROSTATE BIOPSIES Clinical Urology International Braz J Urol Official Journal of the Brazilian Society of Urology Vol. 29 (6): 497-501, November - December, 2003 Vol. 29 (6): 497-501, November - December, 2003 QUANTIFICATION OF TUMOR EXTENSION IN PROSTATE BIOPSIES – IMPORTANCE IN THE IDENTIFICATION OF CONFINED TUMORS KÁTIA R.M. LEITE, MIGUEL SROUGI, RUY G. BEVILACQUA, MARCOS DALL’OGLIO, CÁSSIO ANDREONI, JOSÉ R. KAUFMANN, LUCIANO NESRALLAH, ADRIANO NESRALLAH, LUIZ H. CAMARA-LOPES Laboratory of Surgical and Molecular Pathology, Syrian Lebanese Hospital, Sao Paulo, Discipline of Urology, Paulista School of Medicine, Federal University of Sao Paulo (UNIFESP), and Department of Surgery, University of Sao Paulo School of Medicine (USP), SP, Brazil ABSTRACT Objective: To assess the importance of quantifying the adenocarcinoma in prostate biopsies when determining the tumor’s final stage in patients who undergo radical prostatectomy. To identify the best methodology for obtaining such data. Patients and Methods: Prostate biopsies from 132 patients were examined, with determina- tion of Gleason histological grade and tumor volume in number of involved fragments, tumor extent of the fragment mostly affected by the tumor and the total percentage of tumor in the specimen. Theses parameters were statistically correlated with the neoplasia’s final stage following the evalua- tion of radical prostatectomy specimens. Results: An average of 12 and a median of 14 biopsy fragments were evaluated per patient. In the univariate analysis the Gleason histological grade, the largest tumor extent in one fragment and the total percentage of tumor in the specimen were correlated with tumor stage of the surgical speci- men. In the multivariate analysis, the Gleason histological grade and the total percentage of tumor were strongly correlated with the neoplasia’s final stage. The risk of the tumor not being confined was 3 for Gleason 7 tumors and 10.6 for Gleason 8 tumors or above. In cases where the tumor involved more than 60% of the specimen, the risk of non-confined disease was 4.4 times. Among 19 patients with unfavorable histological parameters, Gleason > 7 and extension greater than 60% the tumor final stage was pT3 in 95%. Conclusion: When associated to the Gleason histological grade, tumor quantification in pros- tate biopsies is an important factor for determining organ-confined disease, and among the methods, total percentage of tumor is the most informative one. Such data should be included in the pathologi- cal report and must be incorporated in future nomograms. Key words: prostate neoplasms; biopsy, needle; pathology; quantitative evaluation; neoplasms staging Int Braz J Urol. 2003; 29: 497-501 Key words: prostate neoplasms; biopsy, needle; pathology; quantitative evaluation; neoplasms stagin Int Braz J Urol. 2003; 29: 497-501 TUMOR QUANTIFICATION IN PROSTATE BIOPSIES sequential transversal sections being performed ev- ery 3 mm, designed from the proximal region towards the distal one. Between 10 and 12 sections from each lobe were included for histological study. The lymph nodes from the fat related to the resection of the iliac chain were dissected and sections representative of each nodular structure were included for study. The specimens of radical prostatectomy underwent the usual processing with inclusion in paraffin. Sections of 4 to 6 µm were stained by hematoxylin-eosin, and analyzed by one only pathologist as well (KRML). cal grade. Currently the literature has discussed the importance of quantifying the tumor in the prostate biopsy (2-4). Such assessment can be made in sev- eral ways, such as the measurement of the neoplasia in millimeters, the analysis of percentage of tumor in every fragment, the percentage of the most involved fragment and the number of fragments that are infil- trated by the neoplasia. The objective of this study is to assess the importance of quantifying the carcinoma in prostate biopsies, when determining the tumor final stage. The Gleason histological grade was used for evaluating the histological differentiation. The assess- ment of tumor extent was performed with the aid of the grid card, as described by Humphrey & Vollmer (6). The invasion of adipose tissue and the peripros- tatic neurovascular plexus was considered as involve- ment of extra-prostatic tissue and, therefore, non or- gan-confined disease. The staging system was TNM 2002 (7). PATIENTS AND METHODS The study comprises the retrospective analy- sis of prostate biopsies from 132 patients with mean age of 63 years, who underwent radical prostatectomy between January 1999 and March 2001. The mean number of analyzed fragments was 12, the median 14, ranging from 5 to 14. The mean and the median values for fragment length was 13 mm, ranging from 7 to 18 mm. Biopsies were fixed in formalin 10%, embedded in paraffin, stained by hematoxylin-eosin and analyzed by one only pathologist (KRML). The Gleason histological grade was used for evaluating the histological differentiation and for statistical analysis, and was divided in 3 groups, ≤ 6, 7, and > 7. For quantifying the tumor, the following were ana- lyzed: 1) Relationship between the number of posi- tive fragments over the total of biopsied fragments, 2) Percentage of a fragment that is more involved by the neoplasia, 3) Total percentage of tumor in the frag- ments, that is, the arithmetic mean between the per- centages of each isolated fragment. Non-parametric analyses (Mann-Whitney) were performed for assessing the significance between the biopsy variables and the neoplasia’s final stage. The qui-square test was used for evaluating the Gleason score and the status of the surgical speci- men. The multivariate logistic regression determined the relative risk of non organ-confined disease for the multiple variables. The tests were performed in the software SPSS version 11 (SPSS Inc. Chicago, IL). INTRODUCTION tial radiotherapy being indicated for low grade, small and absolutely confined tumors. With that purpose, the pre-operative nomogram of Partin et al. (1) is fre- quently used by surgeons, oncologists, and radiothera- pists. In the equation, one must consider the serum level of PSA, clinical staging and Gleason histologi- The selection of the best treatment for the prostate cancer patient depends basically on the sta- tus of the primary tumor. Curative therapies are indi- cated exclusively for confined tumors, with intersti- 497 TUMOR QUANTIFICATION IN PROSTATE BIOPSIES RESULTS The number of biopsied fragments ranged from 5 to 14, with mean of 12 and median of 14. The num- ber of fragments that were positive for tumor ranged from 1 to 11 with mean and median values of 3. The Gleason histological grade ranged from 5 to 9, with the mean of 6.7 and median of 6. The relation between the number of positive fragments and the total biopsied ranged from 7 to 100, with a mean of 29 and median of 25. While analyzing the extent of the fragment that was most involved by the tumor, the numbers ranged from 5% to 100%, with the mean of 57% and median of 60%. The total percentage of tumor in multiple frag- ments of biopsy ranged from 0.4% to 100%, with a mean of 35% and median of 25% (Table-1). The specimens of radical prostatectomy were fixed in buffered formalin 10% for a period of 4 to 16 h. Each gland was submitted to histological study in accordance to the previously described recommen- dations (5). After weighting and measuring the gland, thin transversal sections were performed in the surgi- cal margins related to the bladder neck and the pros- tate apex. The seminal vesicles were sectioned in the base and longitudinal sections were submitted to his- tological examination. The entire gland was included for study after having their margins painted with In- dia ink. The right and left lobes were separated, with 498 TUMOR QUANTIFICATION IN PROSTATE BIOPSIES Table 1 – Pre-operative clinical and pathological charac- teristics of the 132 patients under study. Patients (N) Mean Age ± SD Gleason Score (%) 5 –6 7 8 – 9 Mean ± SD Median % Positive Fragments Mean ± SD Median Total % of Tumor Mean ± SD Median % of Tumor in 1 Fragment Mean ± SD Median 132 63 + 8.4 67 (50.8) 26 (19.7) 39 (29.5) 6.7 + 1.2 6 29 + 19.4 25 35 + 29.2 25 57 + 28.8 60 Table 1 – Pre-operative clinical and pathological charac- teristics of the 132 patients under study. Results from the multivariate analysis showed that there was statistical significance only for total percentage of tumor and Gleason histological grade concerning the tumor’s final stage. The risk of non- confined disease was 3 times higher for Gleason 7 tumors and 10.6 times for adenocarcinomas with Gleason > 7 (p < 0.0001). RESULTS The risk of involvement of extra-prostatic tissue was 4.4 times higher for those tumors that occupied more than 60% of the specimen (p = 0.002). Nineteen cases were considered unfavorable, since they presented Gleason > 7 and total percent- age above 60%. Ninety five percent of these tumors were classified as pT3. DISCUSSION Our results show the power of tumor quanti- fication for determining the final stage of prostate adenocarcinomas. The current nomograms of Partin et al. (1) and the recently validated nomogram of Graefen et al. (8) include in the equation one single biopsy information (Gleason histological grade), with- out considering the tumor volume. The first studies concerning the quantifica- tion of prostate adenocarcinoma demonstrated the value of the number of fragments that were involved by tumor for identifying non-confined tumors. Ac- cording to those, the probability of extra-prostatic tumor extension varies from 7 to 38% when a single biopsy fragment is involved by tumor, and if this num- The univariate analysis demonstrated a sta- tistically significant difference for confined and non- confined tumors concerning larger tumor extension in one single biopsy fragment, total percentage of tumor and Gleason histological grade. Results are shown in Table-2. Table 2 – Univariate relation between pre-operative factors and final pathological stage . Median and range. Gleason Score % Positive Fragments Total % of Tumor % of Tumor in 1 Fragment pT2 6 (5 - 9) 21 (7 - 75) 20 (0.4 - 90) 40 (5 - 100) pT3 8 (5 - 9) 29 (7 - 100) 40 (1.1 - 100) 80 (10 - 100) p Value < 0.0001 0.058 < 0.0001 0.001 The qui-square test was used to calculate the significance of the Gleason score. For the other variables, the Mann-Whitney non- parametric test was used. ariate relation between pre-operative factors and final pathological stage . Median and range. The qui-square test was used to calculate the significance of the Gleason score. For the other variables, the Mann-Whitney non- parametric test was used. * The qui-square test was used to calculate the significance of the Gleason score. For the other variables, the Mann-Whitney non- parametric test was used. 499 TUMOR QUANTIFICATION IN PROSTATE BIOPSIES ber is 4 or above the percentage of non-confined tu- mor ranges from 47 to 100% (9-11). Currently, the sextant biopsies with representation of only 6 fragments is considered insufficient for diag- nosing prostate tumors (16), and are being replaced by wider representations of the gland, thus our data are important for directing new analyses. Rubin et al. had already demonstrated the relation between different methods for quantifying the prostate carcinoma in biopsies and adverse patho- logical aspects of the surgical specimen (12). DISCUSSION In univariate analysis, they showed that the probability of a tumor being no longer confined was 77% for tumors that involve more than 80% of a single bi- opsy fragment. Subsequently, Gao et al. confirmed the importance of such determination for low risk patients. While studying 62 patients, they showed that 38% of the tumors were no longer confined when there was an involvement of 25% or more in the extent of a single biopsy fragment (13). In our casuistry, the higher percentage of tumor in one single biopsy frag- ment was significant for determining the final stage only in univariate analysis. The median of the percent- age of tumor in one fragment was 40% in confined tumors and 80% in non-confined tumors (p = 0.001). In addition to tumor quantification, we con- firmed the importance of the Gleason histological grade for identifying the final status of the tumor. We demonstrated a risk of non-confined disease that is 3 times higher for Gleason 7 tumors and 10.6 times for tumors with Gleason grade equal or higher than 8. An interesting event was the identification of a group that we called unfavorable, whose histologi- cal grade was higher than 7 and total percentage of tumor was higher than 60%. Nineteen patients had tumors with such characteristics, and 95% of them were classified as pT3 after radical prostatectomy. We concluded that tumor quantification in prostate biopsy is important for identifying non-con- fined tumors, and among the studied parameters, the total percentage of tumor was the most informative one, along with the Gleason histological grade. We suggest that this data is incorporated to the patho- logical report and that it is considered in the design of new nomograms. In multivariate analysis, we demonstrated the value of total percentage of tumor in biopsies, together with Gleason histological grade for predicting of the tumor’s final status. The median of the total percent- age of tumor in biopsies was 20% for confined tu- mors and 40% for non-confined tumors (p < 0.0001). More interestingly, the logistic regression analysis demonstrated a risk 4.4 times higher of non organ- confined disease for tumors involving 60% of biop- sies or more. Freedland et al. DISCUSSION (14,15), were able to stratify patients with intermediate risk (Gleason 7 and/ or PSA of 10 to 20 ng/ml) and high risk (Gleason higher than 7 and/or PSA above 20 ng/ml) in sub- groups when they considered tumor extent in the bi- opsies. For patients with intermediate risk, the indexes of biochemical recurrence following radical prostate- ctomy were significantly higher in patients with in- volvement of more than 20% of the biopsy specimen. For high-risk patients, those with involvement of more than 55% of the specimen had higher indexes of bio- chemical recurrence following surgery. Correspondence address: Dr. Katia Ramos Moreira Leite Rua Dona Adma Jafet, 91 São Paulo, SP, 01308-050, Brazil Fax: + 55 11 3231-2249 E-mail: katiaramos@uol.com.br TUMOR QUANTIFICATION IN PROSTATE BIOPSIES TUMOR QUANTIFICATION IN PROSTATE BIOPSIES of tumor stage and volume at radical prostatectomy. J Urol. 2000; 163: 174-8. 5. Bostwick DG, Foster CS: Examination if radical pros- tatectomy specimens: Therapeutic and prognostic sig- nificance. In: Foster W, Bostwick D (eds.), Pathol- ogy of Prostate, Series Major Problems in Pathol- ogy. Philadelphia, WB Saunders Co. 1998; vol. 34, p. 172. 12. Rubin MA, Bassily N, Sanda M, Montie J, Strawderman MS, Wojno K: Relationship and signifi- cance of greatest percentage of tumor and perineural invasion on needle biopsy in prostatic adenocarcinoma. Am J Surg Pathol. 2000; 24: 183-9. 6. Humphrey PA, Vollmer RT: Intraglandular tumor ex- tent and prognosis in prostatic carcinoma: aplication of a grid method to prostatectomy specimens. Hum Pathol. 1990; 21: 799-804. 13. Gao X, Mohideen N, Flanigan RC, Waters B, Mojcik EM, Leman CR: The extent of biopsy involvement as an independent predictor of extraprostatic extension and surgical margin status in low risk prostate cancer: implications for treatment selection. J Urol. 2000; 164: 1982-6. 7. AJCC CANCER Staging Manual, Chicago, Lippincott Raven. 2002, 6th. ed. 8. Graefen M, Karakiewicz PI, Cagiannos I, Quinn DI, Henchall SM, Grygiel JJ, et al.: International valida- tion of a preoperative nomogram for prostate cancer recurrence after radical prostatectomy. J Clin Oncol. 2002; 20: 3206-12. 14. Freedland SJ, Csathy GS, Dorey F, Aronson WJ: Clini- cal utility of percent prostate needle biopsy tissue with cancer cutpoints to risk stratify patients before radical prostatectomy. Urology. 2002; 60: 84-8. 9. Peller PA, Young DC, Marmaduke DP, Marsh WL, Badalament RA: Sextant prostate biopsies: a histo- pathologic correlation with radical prostatectomy specimens. Cancer. 1995; 75: 530-8. 15. Freedland SJ, Aronson EJ, Csathy GS, Kane CJ, Amling CL, Presti JC, et al.: Comparison of percent- age of total prostate needle biopsy tissue with cancer to percentage of cores with cancer for predicting PSA recurrence after radical prostatectomy: results from the search database. Urology. 2003; 61: 742-7. 10. Wills ML, Sauvageot J, Partin AW, Gurganus R, Epstein JI: Ability of sextant biopsies to predict radi- cal prostatectomy stage. Urology. 1998; 51: 759-64. 16. Chab TY, Chan DY, Stutzman KLRE, Epstein JI: Does increases needle biopsy sampling of prostate detect a higher number of potentially insignificant tumors? J Urol. 2001; 166: 2181-4. 11. REFERENCES 1. Partin A, Kattan MW, Subong EN, Walsh PC, Wojno KJ, Oesterling JE, et al.: Combination of prostate spe- cific antigen, clinical stage, and Gleason score to pre- dict pathological stage of localized prostate cancer: a multi-institutional update. JAMA. 1997; 277: 1445- 51. 2. Graefen M, Haese S, Pichlmeier U, Hammerer PG, Noldus J, Butz K, et al.: A validated strategy for side specific prediction of organ confined prostate cancer: A tool to select for nerve sparing radical prostatec- tomy. J Urol. 2001; 165: 857-63. 3. Epstein JI, Potter SR: The pathological interpretation and significance of prostate needle biopsy findings: Implications and current controversies. J Urol. 2001; 166: 402-10. Our data confirm those from the literature with an important differential that is the number of analyzed fragments. The mean and median of biop- sies analyzed per patient were respectively 12 and 14, twice as those analyzed by Freeland et al. (14). 4. Epstein JI, Yang XJ: Reporting Cancer. Influence on prognosis and treatment. In: Epstein J.I. (ed.), Pros- tate Biopsy Interpretation. Philadelphia, Lippincott Williams & Wilkins. 2002; pp. 142-53. 500 Received: August 15, 2003 Accepted after revision: November 3, 2003 TUMOR QUANTIFICATION IN PROSTATE BIOPSIES Sebo TJ, Bock BJ, Cheville JC, Lohse C, Wollan P, Zincke H: The percent of cores positive for cancer in prostate needle biopsy specimens is strongly preditive Received: August 15, 2003 Accepted after revision: November 3, 2003 501 501
https://openalex.org/W4394890322	https://www.frontiersin.org/articles/10.3389/fmed.2024.1384026/pdf?isPublishedV2=False	English	null	The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries	Frontiers in medicine	2,024	cc-by	7,083	TYPE Original Research PUBLISHED 17 April 2024 DOI 10.3389/fmed.2024.1384026 TYPE Original Research PUBLISHED 17 April 2024 DOI 10.3389/fmed.2024.1384026 The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries OPEN ACCESS EDITED BY Janet Sultana, Mater Dei Hospital, Malta REVIEWED BY Julie Monk, Monash University, Australia Frits Lekkerkerker, Consultant, Amsterdam, Netherlands CORRESPONDENCE Annalisa Landi al@benzifoundation.org RECEIVED 08 February 2024 ACCEPTED 27 March 2024 PUBLISHED 17 April 2024 CITATION Landi A, Mimouni Y, Giannuzzi V, Schaefer F, Altavilla A, Gibson S and Julkowska D (2024) The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries. Front. Med. 11:1384026. doi: 10.3389/fmed.2024.1384026 OPEN ACCESS EDITED BY Janet Sultana, Mater Dei Hospital, Malta REVIEWED BY Julie Monk, Monash University, Australia Frits Lekkerkerker, Consultant, Amsterdam, Netherlands CORRESPONDENCE Annalisa Landi al@benzifoundation.org EDITED BY Janet Sultana, Mater Dei Hospital, Malta REVIEWED BY Julie Monk, Monash University, Australia Frits Lekkerkerker, Consultant, Amsterdam, Netherlands CORRESPONDENCE Annalisa Landi al@benzifoundation.org RECEIVED 08 February 2024 ACCEPTED 27 March 2024 PUBLISHED 17 April 2024 CITATION Landi A, Mimouni Y, Giannuzzi V, Schaefer F, Altavilla A, Gibson S and Julkowska D (2024) The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries. Front. Med. 11:1384026. doi: 10.3389/fmed.2024.1384026 Annalisa Landi1, Yanis Mimouni2, Viviana Giannuzzi1, Franz Schaefer3, Annagrazia Altavilla4,5, Spencer Gibson6 and Daria Julkowska2 on behalf of EJP RD Annalisa Landi1*, Yanis Mimouni2, Viviana Giannuzzi1, Franz Schaefer3, Annagrazia Altavilla4,5, Spencer Gibson6 and Daria Julkowska2 on behalf of EJP RD Landi A, Mimouni Y, Giannuzzi V, Schaefer F, Altavilla A, Gibson S and Julkowska D (2024) The creation of an adaptable informed consent form for research purposes to overcome national and institutional bottlenecks in ethics review: experience from rare disease registries. Front. Med. 11:1384026. d i 10 3389/f d 2024 1384026 1Fondazione per la Ricerca Farmacologica Gianni Benzi Onlus, Bari, Italy, 2European Joint Programme on Rare Diseases Coordination, INSERM, Paris, France, 3Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine, University of Heidelberg, Heidelberg, Germany, 4Teddy European Network of Excellence for Paediatric Research, Pavia, Italy, 5Espace Ethique PACA-Corse Assistance Publique - Hôpitaux de Marseille (AP-HM), Hôpital de la Timone, Marseille, France, 6Department of Genetics and Genome Biology, University of Leicester, Leicester, United Kingdom Introduction of harmonization in ethical evaluations across the European Union (EU) can lead to inconsistent ethical reviews for the same study at diﬀerent research sites (13–15). The new European Clinical Trials Regulation introduces a streamlined process, allowing for a single study submission and review by one designated Ethics Committee per country (16). Nevertheless, this change, aimed at simplifying the review process, unfortunately, does not extend to observational studies. In recent years, there is growing awareness and consensus in the Rare Diseases (RD) community about the need for secondary use of healthcare data for research purposes (1, 2). Systematic data collection, access and sharing practices are needed to foster RD research and many initiatives are ongoing to address these topics (3, 4). Furthermore, as anticipated above, the advent of the GDPR (7) also has not solved this fragmentation since it is implemented at the national and local Ethics Committee levels with a certain degree of variance in interpretation. Thus, additional legislative eﬀorts are required to guarantee comparable ethical standards among sites (17). For all these reasons, non-interventional multicenter research projects face challenges to comply with the applicable requirements and to obtain approval from all competent Ethics Committees involved. Nevertheless, the use of health data in the RD ﬁeld may be aﬀected by the low prevalence of the diseases and the data collection heterogeneity (5). As health data reuse is being scaled up, there is an urgent need to reconcile the beneﬁts of data access and sharing with privacy rights and constraints, as well as with ethical and regulatory requirements (6). The European General Data Protection Regulation (GDPR), Regulation (EU) 2016/679 (7), enables a new legal framework for data protection in Europe and provides diﬀerent legal bases for processing personal data. Nevertheless, some issues might be triggered especially within the “research exception” option (8). In the RD ﬁeld, the European Reference Networks (ERNs) have struggled in setting an Informed Consent Form (ICF) for registries that is acceptable to Ethics Committees across the EU. A generic ICF originally developed for the Clinical Patient Management System (18), an online platform for transnational clinical consultations among ERN Members which contained a section on data handling in registries, was not accepted by all Ethics Committees to which it was submitted. Introduction Moreover, age-appropriate information for pediatric participants and/or assent forms were not developed by most ERNs, since the assent is not a legally mandated requirement but requested only by individual Ethics Committees according to local provisions. In the framework of the European Joint Programme on Rare Diseases (EJP RD), an Informed Consent Facilitation Group was established to support the ERNs addressing these challenges. The Group was composed of members of the EJP RD Advisory Regulatory and Ethics Board, the independent Ethics Advisor team, the EJP RD Coordination team, clinical experts from the ERNs, and experts in pediatrics. All the authors of this manuscript are part of the Informed Consent Facilitation Group. In RD research, also when involving children, it is crucial to ensure clear and explicit consent for data processing and its subsequent reuse (9). This necessitates, in the case of minors, not only obtaining the authorization of their parents/legal representatives but also seeking the assent of the minors themselves tailored to the unique needs of minors (10). This dual requirement of consent and assent aligns with various international laws and guidelines that emphasize the involvement of children in health- related decision-making. Harmonized tools for the standardization of practices need to be further developed to help in the assessment procedures at national level when processing special categories of data (6). While the use of personal data including health data is regulated around the world by data protection laws allowing citizens to control the use of their personal data, the large diversity of regulations and healthcare landscapes across and even within countries results in challenges for researchers in processing and sharing data in collaborative research activities (11). The group examined the current practices and regulations surrounding informed consent, incorporating insights from the feedback provided by various Ethics Committees, with the aim to develop an adaptable ICF for research purposes to be used for ERN registries. Key ethics and data protection challenges encountered during ethics review processes were identiﬁed during the review. To address these challenges, a harmonized framework for informed consent was developed. This framework is uniquely designed to be adaptable, allowing for necessary modiﬁcations to ﬁt national and local requirements. In the ﬁeld of RD research, multinational collaboration is often essential due to the limited prevalence of these conditions, which makes them a formidable challenge for any single country or region. COPYRIGHT © 2024 Landi, Mimouni, Giannuzzi, Schaefer, Altavilla, Gibson and Julkowska. 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. Background: The lack of harmonization of evaluation criteria by Ethics Committees in the European Union (EU) has led to inconsistent ethics reviews received by research sites participating in multicenter non-interventional studies. The European General Data Protection Regulation (GDPR) appears to be implemented at national level with a substantial degree of variance in interpretation. The European Reference Networks (ERNs) were struggling in setting an Informed Consent Form (ICF) for registries, allowing reuse of data for research purposes. The aim of this work is to develop an adaptable ICF for research purposes to be used in ERN registries. Methods: To work on this challenge, a team was established within the European Joint Programme on Rare Diseases (EJP RD) to develop a patients’ registry ICF template allowing easy adaptation to ERNs, country, and site-level speciﬁcities. ERN and patients’ representatives validated the choice of developing a GDPR- compliant template for research purposes. The feedback received from 34 Ethics Committees on the Clinical Patient Management System ICF, including the submission of patients’ data to the ERN registries and the EU consent regulatory framework were analyzed along with existing ontologies for data access and reuse. An adaptable ICF was developed following iterative cycles of consultation and review by clinicians, research experts, ethics and regulatory advisors, and patients’ representatives. The development of pediatric material for minor participants was also undertaken. Results and Conclusion: Research oriented ICF templates for adults and for parents/legal representatives of patients were released in 26 national languages. 01 Frontiers in Medicine Frontiers in Medicine frontiersin.org Landi et al. 10.3389/fmed.2024.1384026 10.3389/fmed.2024.1384026 This adaptable ICF aims to foster, according to patients’ preferences, the reuse of registries data for research purposes in compliance with the applicable laws and standards. Pediatric material is being ﬁnalized to collect minors’ assent. ICF machine-readability is also progressing to enhance data discovery and facilitate its access and reuse conditions. informed consent, rare diseases, research, ethics review, registries Frontiers in Medicine frontiersin.org Analysis of the Clinical Patient Management System ICF submitted to the Ethics Committees Separate material for pediatric participants (i.e., assent forms and informative material for minors) was developed only by few ERN registries. Only one out of 38 Ethics Committees reviewing the general ICF requested age-appropriate material for minors. Despite this, the Informed Consent Facilitation Group recognized the importance of developing pediatric material for minor participants in the ERN registries and started with the development of material for adolescents. This development process was guided by a participatory methodology, adhering to data protection norms and children’s rights, and was inﬂuenced by the principles outlined in the draft guide by the Council of Europe (25). The initial feedback received following the submission of the Clinical Patient Management System ICF to 38 concerned Ethics Committees is shown in Figure 1. To note, for many countries more sites were involved, and so more than one Ethics Committee was concerned in the revision process. The Group performed an in-depth reading and analysis of the initial ICF developed for the Clinical Patient Management System to process patients’ registry data for research purposes, the opinion letters that the European Rare Kidney Disease Reference Network members received from 34 out of the 38 Ethics Committees to which the ICF was submitted, the amended ICFs that followed the implementation of Ethics Committees’ comments as well as the other registry ICFs developed by individual ERNs. It was highlighted that within the same country diﬀerent feedback was found for the same ICF. Moreover, the EU rules (including GDPR), guidelines and standards applicable to the consent were detected and consulted (7, 10, 16, 19–24). Starting from the adaptable ICF for adults, along with existing materials from ERNs, including assent forms and informational content for minors, an assent form template speciﬁcally for adolescents aged 12–17 was created during three Pediatric Expert Patients Training Courses, the ﬁrst of which consisted of ﬁve sessions, organized under the EJP RD. This approach ensured that the information provided is concise, transparent, and understandable, presented in a format accessible to adolescents, and utilizes clear, straightforward language. Finally, an analysis of the compliance of the ICF with the GDPR was performed to identify the information to be provided to data subjects, as listed in Article 13. During the training courses, attended by approximately 26 pediatric patients, participants completed a questionnaire to assess their understanding of the objectives of patients’ registries, the relevance of children’s rights in data protection, and the GDPR rules. Analysis of the Clinical Patient Management System ICF submitted to the Ethics Committees They were given an overview of children’s rights and data protection rules. Emphasis was placed on the concept of the “right to an open future.” This principle posits that children should be shielded from making certain irreversible decisions, ensuring that their future options remain open until they reach adulthood and can make informed choices. This concept is especially pertinent in the context of research involving personal health data in rare diseases, where the implications of data use can signiﬁcantly impact a child’s future. Materials and methods Materials and methods Development of the adaptable ICF template A meeting with ERN representatives was scheduled on 15 December 2020 to discuss two alternative approaches: (1) to develop a new version of the ICF focused on fully GDPR compliant reuse of ERN registry clinical data for research purposes and allowing adaptations at diﬀerent levels, or (2) to create a dual ICF covering both the primary and secondary use of the ERN registry clinical data. Based on the consultation, the ﬁrst approach was agreed. Participants were given ICF templates designed for adults and instructed to use a color-coding system for their feedback: terms diﬃcult to understand were to be marked in red; clear information deemed relevant for giving consent or assent was to be highlighted in green; and parts deemed understandable, irrelevant, better placed elsewhere, or that needed rephrasing, were to be highlighted in pink. Additionally, participants were encouraged to provide open-ended feedback and to suggest improvements, e.g., information that could be better explained through visual aids. This interactive approach aimed to reﬁne the consent forms to be as clear and relevant as possible. A ﬁrst draft of the adaptable ICF template was developed starting from the previous version of the Clinical Patient Management System ICF. Then, the draft went through several rounds of revisions and adaptations by various experts. Iterative cycles of consultation and review by ERN clinicians and researchers, ethics and regulatory advisors, and patients’ representatives, including Young Persons Advisory Groups (YPAGs) were undertaken. Around 34 experts provided their contribution during the cycles of consultation. The comments were addressed by the Group and the updated ICF versions were consolidated until their ﬁnalization. Figure 2 shows the diﬀerent steps and the timeline for the development of the adult version of the ICF template. Based on the course outcomes, a draft version of the assent form for adolescents was created and shared with the participants for review as well as a glossary. Concurrently, similar revision processes were undertaken with some existing YPAGs to further validate the assent form. Finally, the creation of informative material and assent form for children was also deemed necessary and planned as a next step. The template aimed to give subjects the chance to consent to the participation in the registry while providing them additional choices related to the reuse of their data. Both EJP RD and external experts revised the ICF before ﬁnalization and translation from English to 26 other European languages. Introduction Such research, distributed across various laboratories and clinics worldwide (12), faces the complexity of diverse ethical standards and procedures. This diversity arises also because each Ethics Committee may operate under its own set of rules and require diﬀerent documents and contents for approval. This lack 02 frontiersin.org Landi et al. 10.3389/fmed.2024.1384026 Frontiers in Medicine Development of the adaptable ICF template Figure 3 shows the timeline for the development of the pediatric material. 03 frontiersin.org Landi et al. 10.3389/fmed.2024.1384026 FIGURE 1 Initial feedback distribution by type from ethics committees across countries. FIGURE 2 Timeline for the development of the adult version of the ICF template. Scouting of existing ontologies of aligning the terms and codes from these ontologies and matrix FIGURE 1 FIGURE 1 Initial feedback distribution by type from ethics committees across countries. FIGURE 2 Timeline for the development of the adult version of the ICF template. FIGURE 2 Timeline for the development of the adult version of the ICF template. Timeline for the development of the adult version of the ICF template. Frontiers in Medicine Development of the adaptable ICF template Thirty-four out of the initial 38 Ethics Committees, from 12 countries, provided a wide range of responses at the time of the analysis following the submission of the Clinical Patient Management System ICF for approving the reuse of patients’ registry data for research purposes and after the preliminary feedback shown in Figure 1: 10 stated that ethics approval was not required for this type of activity, 17 accepted the Clinical Patient Management System ICF after formal ethical review without any changes, 6 accepted an amended version of the ICF following the inclusion of additional information that made it fully compliant with the GDPR, and 1 requested a full application procedure according to the clinical trials requirements, including the provision of informative materials and assent form for minors with compulsory review by patients’ organizations. The documents produced for the latter Ethics Committee were also used by two other sites in the same country (Netherlands). Feedback from three other Ethics Committees was still pending at the time of analysis while one site rejected its participation in the registry. The adaptable ICF template was designed speciﬁcally for research purposes. It addresses the collection, access, storage, and potential future (re)use of data within the ERN registries, but it does not cover the primary use of health data in the Clinical Patient Management System. The structure of the template includes a primary consent section for data inclusion in the ERN registries for speciﬁc research objectives. In addition to this primary consent, participants can select ﬁve optional consent choices according to their preferences: use of data to support commercial projects; transfer of data to non-EU countries; linking of data to existing databases/registries; possibility for the patient to be recontacted for any research project or clinical study; will to be informed about any incidental ﬁnding. These optional consent choices were chosen by the Group and agreed with the experts involved during the consultation phases considering the main reasons for which participants might refuse to participate in the registry (e.g., in case of data transfer outside the EU countries) and choices that might be not applicable for all ERNs (e.g., incidental ﬁndings only for ERNs handling genetic data). Results The missing information identiﬁed during the analysis and possible improvements of the existing form were discussed with the Informed Consent Facilitation Group members, with EJP RD data experts and ERN representatives. Scouting of existing ontologies of aligning the terms and codes from these ontologies and matrix with corresponding terms and phrases in the ICF. The outcomes of this initial investigation were then shared with ontology experts and Information Technology developers from the EJP RD, followed by proposing ways to incorporate these alignments into data models and tools, to enable a machine-readable ICF. As an additional test of functionality of the proposed alignment, we established various “consent proﬁles,” based on patients’ preferences expressed in the consent form and attempted to represent them in a machine-readable format. The terms of the Data Use Ontology (26), the Informed Consent Ontology (27), and the Automatable Discovery and Access Matrix (28) were assessed to see if they could be used to adequately encode the consent and data access and reuse conditions deﬁned in the adaptable ICF. Such an encoding would greatly assist with the digitalization of a participants consent in a potentially interoperable format (i.e., that can be used by various Information Technology systems). The process consisted 04 frontiersin.org Landi et al. 10.3389/fmed.2024.1384026 FIGURE 3 Timeline for the development of the pediatric material. Timeline for the development of the pediatric material. Timeline for the development of the pediatric material. Frontiers in Medicine Scouting of existing ontologies The review of informed consent and data utilization frameworks examined ontologies and engaged in experts’ consultation resulting in the identiﬁcation of 67 diﬀerent codes. These codes corresponded to speciﬁc terms or expressions found within the ICF: 44 of these codes belonged to the Informed Consent Ontology, 22 to the Data Use Ontology and one code pertained to the Disease Ontology, speciﬁcally referring to the disease under study. The identiﬁed codes from the Data Use Ontology and the Informed Consent Ontology were grouped into several thematic categories. The ﬁrst category, the Data Use and Sharing Permissions, encompassed terms related to the permissions for using and sharing data. These terms deﬁne speciﬁc conditions under which data may be accessed and used, particularly in research contexts. The terms of the second category, Consent and Legal Compliance, relate to the informed consent processes, legal obligations, and ethical compliance in data collection and use. The third Patient Engagement and Communication category includes terms emphasizing the importance of patient engagement, understanding the consent forms, and facilitating the eﬀective communication regarding data use. Finally, the terms of the Data Management and Security category deal with data management practices and security measures to protect data integrity and conﬁdentiality. Each category represents a diﬀerent aspect of data handling in medical research, encompassing ethical considerations, legal compliance, patient interaction, and data protection. The ﬁnal versions of the adaptable ICF templates were released, following diﬀerent rounds of consultations, with translations into 26 European national languages and made publicly available on the EJP RD website (29). Development of the adaptable ICF template Most modiﬁcations requested by the Ethics Committees were related to compliance with GDPR and in particular: information relating to the duration of the data storage; the (geographical) location of the registry; the data subjects’ rights; the name and contact details of the data controller and the Data Protection Oﬃcer (DPO); and the reference to the supervisory authority to exercise data subjects’ rights. Importantly, these optional consents are not a prerequisite for participation in the ERN registries: they are designed to provide participants with a greater control over their data and to express their preferences. 05 frontiersin.org Landi et al. Landi et al. 10.3389/fmed.2024.1384026 Two versions of the ICF were developed. One for adult patients, the other for parents or legally designated representatives of minors or incapacitated individuals. consultation and conclusions were further discussed with pediatric participants. In the third training workshop, a child friendly glossary was developed. At the time of this manuscript submission, the assent form template for adolescents is in the process of ﬁnal consultation before the release of its ﬁnal version and the preparation of informative material and assent form for children considered as further step. To enhance the ﬂexibility and applicability of the ICF across diﬀerent contexts, the template was designed with modiﬁable sections. These adaptable areas were clearly marked to facilitate easy customization. This allows to tailor the ICF to the speciﬁc requirements of individual ERNs or sites, e.g., to describe the disease(s), indicate the type of collected data and the possible link with other registries or databases, provide information on where and for how much time the data is stored, how the research results exploiting the registry will be communicated, and the contact details of the reference person or entity to be contacted for inquiries, including information on the data controller and the DPO. The adaptable areas allow also to comply with any relevant national and local laws and with site speciﬁcities (e.g., information on insurance). This approach ensures that the ICF can be eﬀectively used in diverse settings while adhering to varying legal and regulatory standards. Development of the pediatric material for minors The ﬁrst pediatric workshop revealed a general lack of awareness of children and adolescents about the objectives, contents, procedures and uses of patient registries. Most of them also declared not to be aware of children’s rights and data protection principles. Those who declared to be aware of these issues were trained within YPAGs, patients or consultative groups. Children participants provided suggestions to enrich the developed assent form for adolescents by including (1) a lay glossary explaining some concepts (e.g., data controller, DPO, data protection authority, and commercial use), (2) child-friendly interactive elements and diagrams, and (3) ﬂowcharts or images explaining the ﬂow of personal data in the registry. Moreover, they recommended to post on the registry website more comprehensive child-friendly information about the adopted safeguards and data protection policies, and to post lay summaries with results of the studies carried out with the data of pediatric patients participating in the registries. They also underlined that children and adolescents must be protected against being engaged in certain irreversible choices. In this perspective, the question of the commercial use of data was raised and it was underlined the need to require an opinion from an Ethics Committee on a case-by-case basis, considering the best interests of the child. The undertaking of this ICF mapping exercise contributed to the reﬁnement of the Data Use Ontology through the elimination of subclasses that caused ambiguity for the users and the addition of a new optional consent category in the ICF (30). This enhancement enabled the inclusion of provisions for the reuse of data in commercial projects (e.g., industry sponsored drug development trials) and the disclosure of incidental ﬁnding. Ultimately, this analysis facilitated the creation of 32 unique “consent proﬁles” using the DUC proﬁle creator developed by EJP RD (31) that exploits the Common Conditions of Use Elements (32). This reﬂects the incorporation of ﬁve optional consent options within the ICF templates, thereby augmenting the granularity and ﬂexibility of consent documentation and data reuse in scientiﬁc research settings. The need to clearly diﬀerentiate research conducted with commercial sponsorship, as outlined in the adaptable ICF, from academic studies was highlighted from experts. In particular, it was discussed that no ﬁnancial beneﬁt is foreseen for data subjects and data could become property of the concerned company that could also be used for further commercial purposes and for patents. Frontiers in Medicine Discussion The adaptable ICF template released in the framework of EJP RD aims to foster the reuse of registry data for research purposes in compliance with the applicable relevant laws and standards as well as patient preferences. Within the second EJP RD pediatric training workshop, the assent form developed for adolescents was submitted to further 06 frontiersin.org Landi et al. Landi et al. 10.3389/fmed.2024.1384026 The relevance of this work consists, on one hand, in giving subjects the chance to consent to the main purposes of the registry while providing them with the choice about other data processing activities (e.g., the transfer of data to non-EU countries, the reuse of data for research projects with commercial sponsors or the possibility to be re-contacted to participate in other research projects). It aims to reinforce the concept of “granularity,” as stated in the GDPR and to create personalized “consent proﬁles” based on patients’ preferences on their data use. On the other hand, the concept of avoiding a “one-size-ﬁts-all” approach for the ICF was addressed. The focus was on developing an adaptable ICF that could conform to diverse national and local regulations and standards, allowing for straightforward text modiﬁcations. This concept was further expanded to the assent template, generated based on the recommendations from the young patient. The involvement of children in developing pediatric assent and their feedback highlighted the importance of the participatory methodology to take decisions and implement practices adequate to children needs, expectancies and rights. A greater involvement of children in developing speciﬁc child friendly tools in rare diseases should be promoted. along with patients’ representatives. One crucial ﬁnding from our work emphasizes the need for the technical development of these registries to incorporate data usage conditions within a tailored data governance framework, since the design phase. along with patients’ representatives. One crucial ﬁnding from our work emphasizes the need for the technical development of these registries to incorporate data usage conditions within a tailored data governance framework, since the design phase. Ensuring that RD registries meet high-quality standards in technical infrastructure, ethics, and data protection is imperative. To address potential risks, speciﬁc safeguard measures, such as conducting a Data Protection Impact Assessment under Article 35 of GDPR, should be implemented. This is particularly vital when dealing with vulnerable individuals, like children. Conclusion In conclusion, this work represents a signiﬁcant milestone that serves as a model in various research activities dependent on consent. It is highly valuable for the RD community and holds potential for extension and application to other disease communities. It might also be used to complement the information on informed consent included in the European Medicines Agency Guideline on registry-based studies (40). Additionally, the ICF developed here is currently being tested by other registries and research projects. Its successful implementation is expected to enable the secondary use of healthcare data in various other research endeavors, including initiatives like the European Rare Diseases Research Alliance (41); thereby broadening the scope and impact of this work. Another eﬀort is currently making the adaptable ICF machine- readable, leveraging on the ontology mapping and the created consent proﬁles. This work exploits the EJP RD created Data Use Condition tool (31) using the Common Conditions of Use Elements, that were partly derived from the development of this ICF, and on the use of the Open Digital Rights Language ontology (35) for semantic implementation. Enabling machine- readability of access and reuse conditions also considers ERN registry data access policy, data sharing agreement and data transfer agreement, jointly developed by EJP RD and the European Rare Disease Research Coordination and Support Action consortium (36). We aim through these approaches to enhance the ERN registries data discovery and display the access and reuse conditions when querying for RD information, and to facilitate the data submissions and access requests for researchers and data access committees following the successful implementation of the Data Use Oversight System (37) and the BBMRI Negotiator (38). We are also considering to eventually enable patients to use the ERN registries’ websites for exercising their rights. This includes developing age-appropriate information and tools, dynamically modifying consent, accessing their data, and having the ability to update their data. This goal is currently still a topic of discussion and has not been ﬁnalized. Data availability statement The original contributions presented in this study are included in this article/supplementary material. Further inquiries on the original data can be directed to the corresponding author. Discussion An unresolved question relates to the validity of informed consent regarding data reuse, especially in the context of the European Commission’s proposal for a European Health Data Space (39). This complexity is further compounded when considering the integration of ERN registries into this framework, because it raises intricate legal considerations surrounding data processing. The outcomes of this work not only aim to promote harmonized practices and facilitate the secondary use of health data in general, but also provide adaptable templates. Furthermore, these outcomes can assist the Registry Data Access Committees by oﬀering a more transparent view of patients’ preferences regarding the use of their data. It has been demonstrated that RD patients and parents are keen to make their samples and data available to researchers if this is done with respect and reciprocity (2, 11, 33, 34). We strongly believe that our tool could increase their willingness to share data and foster their active participation in the RD registries. Moreover, pediatric material has been developed to collect minors’ assent to be informed and agree on the use of their data. Considering their apparent lack of awareness of registries as research tools, more eﬀorts and education campaigns will be needed to inform pediatric patients about the value of RD registries and about their rights in the context of data use and protection. Frontiers in Medicine References Orphanet J Rare Dis. (2017) 12:64. doi: 10.1186/s13023-017-0617-1 16. European Parliament and Council of the European Union. REGULATION (EU) No 536/2014 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 April 2014 on clinical trials on medicinal products for human use, and repealing directive 2001/20/EC. Brussels: European Parliament and Council of the European Union (2014). 6. Landi A, Thompson M, Giannuzzi V, Bonifazi F, Labastida I, da Silva Santos L, et al. The “A” of fair – as open as possible, as closed as necessary. Data Intell. (2020) 2:47–55. doi: 10.1162/dint_a_00027 17. Giannuzzi V, Felisi M, Bonifazi D, Devlieger H, Papanikolaou G, Ragab L, et al. 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Acknowledgments We would like to express our sincere appreciation to the European Joint Programme on Rare Diseases (EJP RD) Advisory Regulatory and Ethics Board, independent Ethics Advisor team, Coordination team, ICF Facilitation Group, pediatric experts, Pillar 2 Information Technology developers, and ontology experts. We are also thankful to the European All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conﬂict of interest The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. This initiative has received funding from the European Union’s Horizon 2020 Research and Innovation program under grant agreement no. 825575. 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. Author contributions AL: Conceptualization, Formal analysis, Methodology, Writing – original draft, Writing – review & editing. YM: Conceptualization, Formal analysis, Methodology, Writing – review & editing. VG: Methodology, Writing – original draft, Writing – review & editing. FS: Conceptualization, Methodology, Supervision, Writing – review & editing. AA: Methodology, Writing – review & editing. SG: Formal analysis, Methodology, Our research highlights the importance of assembling a multidisciplinary team with diverse and complementary expertise when establishing and managing patient registries, i.e., experts in data management, regulatory compliance, ethics, and legal matters, 07 frontiersin.org Landi et al. 10.3389/fmed.2024.1384026 Writing – review & editing. DJ: Conceptualization, Methodology, Writing – review & editing. Writing – review & editing. DJ: Conceptualization, Methodology, Writing – review & editing. Reference Networks (ERNs) representatives, adult patients’ representatives, Young Persons Advisory Groups, and European Rare Disease Research Coordination and Support Action consortium Legal Working Group. Writing – review & editing. Frontiers in Medicine References Ethical considerations for clinical trials on medicinal products conducted with minors recommendations of the expert group on clinical trials for the implementation of regulation (EU) No 536/2014 on clinical trials on medicinal products for human use revision 1. Brussels: European Commission (2017). 21. i-Consent Consortium. Guidelines for tailoring the informed consent process in clinical studies. Valencia: FISABIO (2021). 21. i-Consent Consortium. Guidelines for tailoring the informed consent process in clinical studies. Valencia: FISABIO (2021). 22. 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https://openalex.org/W4220660375	https://jhumanitarianaction.springeropen.com/track/pdf/10.1186/s41018-022-00117-y	English	null	The National Health Cluster in Yemen: assessing the coordination of health response during humanitarian crises	Journal of international humanitarian action	2,022	cc-by	10,900	Backgroundh The Republic of Yemen was established on 22 May 1990 following the merger between the Yemen Arab Republic (North Yemen) and the People’s Democratic Republic of Yemen (South Yemen) (UNDP 2013; ACAPS 2016). The Houthi rebels seized the capital Sana’a in late 2014 and fi Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract Yemen has been facing political, economic and social challenges since 1990. The fragility of Yemen’s situation has led to a widespread conflict in 2015, resulting in the world’s largest humanitarian crisis. Amid the humanitarian catastro‑ phe and the collapsing health system, a platform for coordinating humanitarian health response, called the National Health Cluster, has expanded its operations across the country. The study aims to evaluate the performance of the National Health Cluster in Yemen between 2015 and 2019. A qualitative research design was employed, and ten semi-structured interviews with key Health Cluster stakeholders were conducted. The study applied the Active Learn‑ ing Network for Accountability and Performance in Humanitarian Action (ALNAP) guide to evaluating humanitarian action using the Development Assistance Committee (DAC) criteria. Six evaluation criteria were selected: relevance, effectiveness, efficiency, effects, connectedness and participation. Inputs from interviews were manually transcribed and then analysed using NVivo 12 software. The study results indicate that the Health Cluster in Yemen has contrib‑ uted to saving lives and strengthening the local health capacities in diseases surveillance. In addition, its positive effect was evident in improving the humanitarian health response coordination. Nevertheless, engaging health stakeholders, especially national organisations, was suboptimal. Exit strategies were lacking, while services to address mental health, non-communicable diseases, senior citizens and people with dis‑ abilities were not prioritised in the Health Cluster strategic plans and partners’ response. To ameliorate Health Clus‑ ter performance, revising its objectives and establishing a cluster-specific rapid response funding mechanism are pivotal. Furthermore, preparing the national health system for recovery and actively engaging all stakeholders in the Health Cluster’ response and strategic decisions would maximise its positive impact on Yemen’s health system and population. Keywords: Health Cluster, Yemen, Coordination, Conflict, Humanitarian health response, Evaluation, Health systems then marched towards the south to apprehend Presi- dent Hadi, who fled to the south and declared Aden the interim capital of Yemen in March 2015. The inter- nal conflict took an international perspective when the Yemeni president escaped Aden to Saudi Arabia and requested military assistance from neighbouring coun- tries and the United Nations (UN) Security Council to restore the legitimacy of his government. The 6-year con- flict has deteriorated Yemen’s overall health and develop- ment outlook (Action Against Hunger 2019; Moyer et al. Journal of International Humanitarian Action Journal of International Humanitarian Action Journal of International Humanitarian Action Al‑Awlaqi et al. Journal of International Humanitarian Action https://doi.org/10.1186/s41018-022-00117-y Al‑Awlaqi et al. Journal of International Humanitarian Action https://doi.org/10.1186/s41018-022-00117-y (2022) 7:9 Open Access The National Health Cluster in Yemen: assessing the coordination of health response during humanitarian crises Sameh Al‑Awlaqi1,2* , Fekri Dureab3,4 and Marzena Tambor1,5 *Correspondence: sameh.alawlaqi@gmail.com 2 School of Health and Related Research, The University of Sheffield, Sheffield, UK Full list of author information is available at the end of the article The Cluster Approachh 2019), turning the country into the largest humanitarian crisis in the world (OCHA 2019b). The concept of the Cluster Approach was first introduced in 2005 by the Emergency Relief Coordinator (ERC) of United Nations (UN) Office for the Coordination of Humanitarian Affairs (OCHA), Jan Egeland, to address the gaps in coordination and effectiveness in humani- tarian response (Stoddard et al. 2007). The Cluster Approach was one of the pillars of the so-called humani- tarian reform agenda, which came into effect following the inadequate and poorly coordinated humanitarian response to the crisis in Darfur and the Indian Ocean earthquake (OCHA 2007; International Council of Vol- untary Agencies (ICVA) 2013). There are eleven clusters specialised in different humanitarian disciplines, i.e. pre- venting emergencies and mitigating their risks, prepared- ness and response to disasters and working in recovery and reconstruction phases (Inter Agency Standing Com- mittee (IASC) 2015). The clusters’ titles and their lead agencies are listed in Table 1. Fragmentation of the health system has emerged as a natural consequence of the protracted crisis since 2015. Two completely independent ministries of health mani- fested this fragmentation: one in the northern territories controlled by the de facto authorities, i.e. the Houthis’ government, and another in the southern part of the country run by the legitimate, internationally recognised government (Michael 2019; Pulitzer Center 2019).h The war had a calamitous impact on the health sys- tem, with more than half of the health facilities either not operational or destructed (WHO 2017b; Yemen Health Cluster 2017). The Ministry of Public Health and Population (MoPHP) has failed to disburse health work- ers’ salaries for several years (MSF 2017; Yemen Health Cluster 2017; Al-Awlaqi 2019). Epidemics such as chol- era (declared the largest in the twentieth century) and diphtheria, as well as deteriorating levels of malnutrition and, most recently, COVID-19, burdened the collapsing health system (Dureab et al. 2020). Health Clusters exist in twenty-nine countries and ter- ritories with emergencies or protracted humanitarian cri- ses and two regional coordination mechanisms in Syria and the Pacific (Global Health Cluster 2021). Its mandate is to provide a coordinated response and avoid gaps and duplication of humanitarian response during emergen- cies (WHO 2015). Before the escalation of the conflict in 2015, mil- lions of Yemenis suffered from adverse social and eco- nomic situations; many were living below the poverty line, i.e. Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 2 of 14 Table 1 Humanitarian clusters and their lead agencies, adapted from the IASC Cluster Coordination reference module (Inter Agency Standing Committee (IASC) 2015) No. Title of Cluster Cluster Lead Agency 1 Emergency telecommunication World Food Programme (WFP) 2 Education United Nations Children’s Fund (UNICEF) and Save the Children 3 Early recovery United Nations Development Programme (UNDP) 4 Camp coordination and camp management International Organization for Migration (IOM)/United Nations High Commis‑ sioner for Refugees (UNHCR) 5 Water, sanitation, and hygiene UNICEF 6 Shelter International Federation of Red Cross and Red Crescent Societies (IFRC)/(UNHCR) 7 Protection UNHCR 8 Nutrition UNICEF 9 Logistics WFP 10 Health World Health Organization (WHO) 11 Food security WFP and Food and Agriculture Organization (FAO) The Cluster Approachh on less than $1.25 per day for basic needs such as food, shelter, or clothing (World Bank 2015; Euro- pean Union 2016). As of 2021, around two-thirds of Yemenis desperately need humanitarian aid (OCHA 2021b). Furthermore, the latest Global Hunger Index Report 2020 states that Yemen is one of eight countries with an “alarming level of hunger”, including various forms of life-threatening malnutrition among children (Grebmer et al. 2020). Methods and materialsh were heavily supported by the UN agencies, interna- tional non-governmental organisations (INGOs), and national non-governmental organisations (NNGOs) (OCHA 2019b). These organisations coordinate their humanitarian health response via the Health Cluster, led by WHO, the Cluster Lead Agency (CLA) (Yemen Health Cluster 2018). were heavily supported by the UN agencies, interna- tional non-governmental organisations (INGOs), and national non-governmental organisations (NNGOs) (OCHA 2019b). These organisations coordinate their humanitarian health response via the Health Cluster, led by WHO, the Cluster Lead Agency (CLA) (Yemen Health Cluster 2018). This research aims to evaluate the national Health Clus- ter performance during the conflict period (2015–2019) in Yemen by employing a qualitative research design. We conducted in-depth interviews with key informants who represented major health stakeholders in Yemen. To guide the interview and data analysis, we developed an analytical framework based on the Active Learning Net- work for Accountability and Performance in Humanitar- ian Action (ALNAP) guide on evaluating humanitarian action. ALNAP guide applies Organisation for Economic Co-operation and Development (OECD)’ Development Assistance Committee (DAC) criteria to fit the context of complex humanitarian emergencies. This evaluation guide combines best practices and provides a helpful instrument for evaluating humanitarian interventions for individual evaluators and organisations alike (ODI 2006). The Cluster Approach was first introduced in Yemen in 2009 following the resurgence of hostilities in the northern part of the country (Sa’adah wars), with the Nutrition Cluster being the first cluster to be activated (OCHA 2019a). In March 2010, the arrangement for establishing the Health Cluster was formalised and then activated in 2011 (OCHA 2010; Global Health Cluster 2018) Since the beginning of the recent hostilities in 2015, the Health Cluster expanded its operations in what is known as “subnational hubs” or “subclusters” in the following areas: Sana’a, Aden, Ibb, Hodeida and Sa’dah (until early 2019). These hubs cover the twenty-two governorates of Yemen (OCHA 2021a). To ensure that we cover the main methodological con- siderations in this research, we applied the Consolidated Criteria for Reporting Qualitative Research (COREQ)’ checklist, which lists the most commonly used steps in qualitative research design (Tong et al. 2007a, b). The checklist aligns with the Enhancing the Quality and Transparency of Health Research (EQUATOR)’ recom- mendations (EQUATOR Network 2015). The Health Cluster works with the MoPHP to deliver coordinated health response to the affected popula- tions throughout the country. The Health Cluster in Yemen In response to the failure of the national system to meet the increasing population health needs amid the escalation of conflict, many of the health systems’ functions in both sides of Yemen (north and south) Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Al‑Awlaqi et al. Journal of International Humanitarian Action Page 3 of 14 Analytical framework and data collection instrumentffi We used six criteria (relevance, effectiveness, efficiency, effects, connectedness and participation). We excluded other criteria for the reasons detailed in Table 2.h 2) To strengthen preparedness, surveillance and response to communicable diseases outbreaks and epidemics, including immunisation for vaccine-pre- ventable diseases The evaluation framework and criteria applied in this study were previously used in various humanitarian set- tings over the last two decades (ODI 2006). Specifically, they were applied in to evaluate the Cluster Approach at the global and national levels (Steets et al. 2010). 3) To deliver a principled and coordinated health response and promote an integrated approach with other sectors for a comprehensive humanitarian response with a focus on the most vulnerable districts We used the analytical framework to prepare an inter- view guide for semi-structured interviews. The interview topic guide was divided into six themes corresponding to the six evaluation criteria; each criterion has specific, mostly open-ended, questions. 4) To improve access to reproductive, maternal, new- born, child and adolescent health services for vulner- able populations, including IDPs and the poorest and deprived segments in the society We developed the topic guide in English and piloted it before commencing actual interviews. A few revisions were added, mainly on the relevance (i.e. the cluster objectives were explicitly mentioned to give more clarity to the informants) and on the efficiency criteria, i.e. ques- tions have been revised to reflect funding. When conducting this study in 2019, two prominent evaluations of the Cluster Approach at the global level were available (Stoddard et al. 2007; Steets et al. 2010). More recently, another two studies have attempted to determine the ideal level of coordination within the Cluster Approach in general and the role of the Health Cluster in the cholera response in Yemen, respectively (Clarke and Campbell 2018; Hopkins 2019). However, none of these studies evaluated the overall Health Clus- ter performance during the conflict period in Yemen. Methods and materialsh It has the following objectives (Yemen Health Cluster 2018): 1) To increase access of vulnerable populations, including internally displaced persons (IDPs), to MHSP covering life-saving and basic quality health services, through support to health system and community resilience Study population and sample selectionh The population of this study covered various Health Cluster stakeholders, including the Ministry of Health, UN agencies, INGOs, NNGOs, and international health experts who work or have previously worked in Yemen Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 4 of 14 Table 2 Description of ALNAP-DAC evaluation criteria used to design the study’s analytical framework (adapted from ALNAP guide 2006) Criteria Definition and comments Relevance It evaluates the synergies between the intervention, national priorities and local needs. It can be used in all evaluation types Effectiveness It indicates to what extent an intervention was successful in bringing about the intended results. It also implies timeli‑ ness and can be used in sectoral or organisational evaluations Efficiency It measures all resources (monetary and nonmonetary) required to achieve the desired output and compare alterna‑ tives to achieve the same output. Thus, it can be used in all evaluation types with adequate financial data Effects “Effects” is not a standard criterion, but it was intended to replace “impact”. “Impact” needs a comprehensive assess‑ ment that involves the affected population, which cannot be covered in this study. Alternatively, the study looked at various “effects” on the national health system, humanitarian response coordination, Cluster partners and a vulnerable population Connectedness It ensures that short-term interventions are linked with long-term plans and can be applied in evaluations that focus on coordination between organisations and stakeholders. It is adapted from the concept of “sustainability” Cross-cutting theme Participation Participation means that primary stakeholders (Cluster partners) must be fully engaged in all phases of developing and implementing health interventions Among the eight cross-cutting issues, participation was relevant to the case of Yemen as it reflects how active was the Cluster in engaging partners. In addition, it provides a glimpse of the coordination and synergies between the Health Cluster and its members and highlights the power dynamics within the Cluster Excluded ALNAP-DAC criteria Coverage Coverage was not selected because it needs identification of the people who benefited from humanitarian action and why they have benefited or excluded from that action (ODI 2006), which is beyond the scope of this study. However, as coverage has an indirect link with “effectiveness”, some aspects of the coverage, i.e. identifying vulnerable groups within the Health Cluster partners’ response, has been included in the study Coherence Coherence focuses on incorporating a humanitarian dimension into global policies. Study population and sample selectionh Measuring the coherence of the Health Cluster as a coordination mechanism against international policies is too broad and does not fit the purpose of this study Impact “Impact” was replaced by “effects”. Assessing impact needs a specific impact evaluation approach (Steets et al. 2009, 2010). It also requires collecting feedback from the affected population (Hallam 1998), which is beyond the research‑ er’s capacity. Previous evaluation reports of the Cluster Approach employed “effects” as a criterion instead of “impact” for comparable reasons (Steets et al. 2009) It evaluates the synergies between the intervention, national priorities and local needs. It can be used in all evaluation types It indicates to what extent an intervention was successful in bringing about the intended results. It also implies timeli‑ ness and can be used in sectoral or organisational evaluations It measures all resources (monetary and nonmonetary) required to achieve the desired output and compare alterna‑ tives to achieve the same output. Thus, it can be used in all evaluation types with adequate financial data “Effects” is not a standard criterion, but it was intended to replace “impact”. “Impact” needs a comprehensive assess‑ ment that involves the affected population, which cannot be covered in this study. Alternatively, the study looked at various “effects” on the national health system, humanitarian response coordination, Cluster partners and a vulnerable population It ensures that short-term interventions are linked with long-term plans and can be applied in evaluations that focus on coordination between organisations and stakeholders. It is adapted from the concept of “sustainability” Participation means that primary stakeholders (Cluster partners) must be fully engaged in all phases of developing and implementing health interventionsl Among the eight cross-cutting issues, participation was relevant to the case of Yemen as it reflects how active was the Cluster in engaging partners. In addition, it provides a glimpse of the coordination and synergies between the Health Cluster and its members and highlights the power dynamics within the Cluster Coverage was not selected because it needs identification of the people who benefited from humanitarian action and why they have benefited or excluded from that action (ODI 2006), which is beyond the scope of this study. However, as coverage has an indirect link with “effectiveness”, some aspects of the coverage, i.e. identifying vulnerable groups within the Health Cluster partners’ response, has been included in the study Coherence focuses on incorporating a humanitarian dimension into global policies. Study population and sample selectionh Measuring the coherence of the Health Cluster as a coordination mechanism against international policies is too broad and does not fit the purpose of this study “Impact” was replaced by “effects”. Assessing impact needs a specific impact evaluation approach (Steets et al. 2009, 2010). It also requires collecting feedback from the affected population (Hallam 1998), which is beyond the research‑ er’s capacity. Previous evaluation reports of the Cluster Approach employed “effects” as a criterion instead of “impact” for comparable reasons (Steets et al. 2009) Table 3 Characteristics of the recruited participants Organisation Gender Health Cluster (former coordinator) Male NNGO Male NNGO Female Nutrition Cluster Male Health Cluster Male WHO Male OCHA Male INGO Female INGO Male International health consultant (former coordinator in Yemen) Male Table 3 Characteristics of the recruited participants between 2015 and 2019. These different stakeholder groups were selected to ensure a broad and diversified perspective for the study (Hallam 1998). We followed a non-probability, purposive sampling, namely expert or stakeholder sampling, to identify key health stakeholders in Yemen. The selection of key informants was based on researchers’ previous national and international expertise in the Cluster Approach and humanitarian coordination. In evaluations and evidence- informed policy research, it is recommended to employ stakeholders sampling (Palys 2008).ii Sixteen key informants fit the definition of the study population (Cluster coordinators, MoPHP officials, rep- resentatives of national and international NNGOs and international humanitarian experts). Out of the tar- geted sixteen informants, ten participated in the study (Table 3). Among those who did not take part, four did not respond to invitation e-mails. Effectiveness Eff Effectiveness answers the question of whether the Health Cluster activities achieved their desired pur- pose at the right time. When questions about effec- tiveness were asked, four sub-themes emerged, i.e. the overall effectiveness, the timely response, the Min- istry of Health fragmentation and the multisectoral programming.hf Data collection The principal investigator conducted the interviews between March and April 2019. Eight interviews with key informants residing in Yemen were conducted online. Two participants were interviewed face to face. Each interview lasted around 45 to 60 min. The Despite the probability of introducing bias by the selected sampling technique, we attempted to include a diverse sample as possible among key health stakeholders to obtain a holistic picture about the Health Cluster in Yemen. Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Al‑Awlaqi et al. Journal of International Humanitarian Actio Page 5 of 14 interviews were audiotaped alongside manual notetak- ing. Audiotaped data were transcribed by manual typ- ing into word documents (verbatim). to the national health priorities and the needs of the affected population. These objectives, nevertheless, did not translate into timely response due to weak decision- making power within the Health Cluster itself, especially at the level of its subnational hubs. Eight interviews were conducted in the English lan- guage and two in the Arabic language, in line with the preferences of some participants. The latter was trans- lated into English by the principal investigator before the analysis. Another informant, an international health expert, indicated that these objectives were relevant yet limited in scope to life-saving interventions. “ …But I think these (the Cluster objectives) are more or less remaining in the field of life-saving, […] the needs are much more, much bigger than actually what all these actors together can deliver.” (Inform- ant #7) “ …But I think these (the Cluster objectives) are more or less remaining in the field of life-saving, […] the needs are much more, much bigger than actually what all these actors together can deliver.” (Inform- ant #7) Data analysis Th i i The principal investigator uploaded all transcripts into NVivo software version 12 for Macintosh. At the beginning of the analysis, six themes (called nodes or codes on NVivo), which corresponded to the six eval- uation criteria (relevance, effectiveness, efficiency, effects, connectedness and sustainability), were manu- ally created under “codes>nodes” tab within NVivo software by the researcher. The informants’ opinions about the objective of deliv- ering a principled and coordinated health response and promoting an integrated approach were mixed. Inform- ants from INGOs thought that there were bottlenecks in delivering a principled health response due to the politi- cal influence of the national authorities on the Health Cluster decision-making process, i.e. national authori- ties or parties to the conflict grant access to field areas only if the Health Cluster and its partners abided by the preconditions of these authorities. These precondi- tions, on some occasions, contradicted the humanitarian principles: An additional two themes (areas for improvement and challenges) were added to the list of themes that were not on the main list of themes as they were found relevant during the analysis. The study findings were analysed and fact-checked by the following: 1. Reflexivity: the author’s own experience in Yemen as a public health expert — and a previous cluster co- coordinator — was taken into consideration. “I think the Health Cluster is struggling […] in a politically polarised environment such as Yemen set- ting. (It was) so difficult to work in a fully principled way because sometimes a lot of humanitarian prin- cipals were compromised by the Health Cluster and partners in return to access”. (Informant#9) 2. The results were compared against the available body of literature (mostly grey literature) on coordination mechanisms in humanitarian settings.h 3. The anonymised results were checked by public health specialists from Yemen and other countries with previous experiences in humanitarian health response in Yemen. Ethical considerationsh The study obtained ethical approval from the School of Health and Related Research (ScHAAR) Ethics Com- mittee before interviews. After receiving a detailed par- ticipant information sheet, participants provided their written consent to participate in this study and to use their anonymised responses in future publications. The opinions of key informants on the overall effec- tiveness of the Health Cluster performance during the last 3 years were dissimilar. Cluster coordinators and respondents from INGOs, NNGOs and UN organisa- tions believed that the performance was unsatisfactory and needed further improvement (first group). The sec- ond group, nonetheless, represented by other UN agen- cies and international experts, witnessed a compelling performance. Efficiency (funding) According to ALNAP definition, efficiency assesses how project inputs that have monetary value were converted into results, taking into consideration whether the results were maximised for given inputs. It may entail com- parison with an alternative to assess the most efficient approach to implementing an intervention (ODI 2006).i “They (decision makers) were not present at Aden hub; they were in Sana’a. Remote management was difficult, and the response was difficult, slow or not up to the need”. (Information #10) “They (decision makers) were not present at Aden hub; they were in Sana’a. Remote management was difficult, and the response was difficult, slow or not up to the need”. (Information #10) Among those who witnessed satisfactory performance, prevention of deaths, proper coordination among the health stakeholders and clusters and provision of sub- sidised or free health services in the Health Cluster partners’ response were the areas the Health Cluster per- formance was evaluated as effective in addressing them: Due to the lack of adequate financial data, respondents could not comment on the specific budgets allocated for the Health Cluster nor were able to identify a more efficient alternative. Therefore, answers to this theme were focused on “funding” and whether it was sufficient, rather than “efficiency” in its economic interpretation. It also addresses the role of the Health Cluster in govern- ing the health sector’ funds via the Yemen Humanitarian Response Plan (YHRP). “I can say it with a very good feeling that we pre- vented massive deaths […] hundreds of thousands of deaths could happen if you have no health system and the country is actually in humanitarian crisis; this did not happen”. (Informant #7) Most informants indicated that there were no direct funds allocated for the Health Cluster to provide health response: “I think the Health Cluster has done a good job […] we developed minimum quality standards matching the minimum service package adopted by MoPHP. Those minimum quality standards also go and in line with the SPHERE standards”. (Informant#5) “There were no direct funds to Health Cluster, there were funds that went to the main stakeholders such as UNICEF, WHO or UNFPA, but the Cluster has no direct funds”. (Informant #1) Some informants from NNGO and UN organisa- tions also indicated that the Health Cluster coordination budget was not matching the needs of the population. Relevance Relevance is defined as compliance of the Health Cluster objectives with the national health priorities and the local health needs of the population (ODI 2006). Moreover, among those who believed that the per- formance was not satisfactory, many informants have Many key informants agreed that the Health Clus- ter’ objectives in Yemen were, to certain extent, relevant Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 6 of 14 authorities. There are many layers; too many coordi- nation and difficulties the health partners were fac- ing”. (Informant#5) authorities. There are many layers; too many coordi- nation and difficulties the health partners were fac- ing”. (Informant#5) identified specific gaps in the Health Cluster work. They criticised the interference of the Cluster Lead Agency (CLA) in the Health Cluster work and the unsatisfactory level of Health Cluster’ transparency in sharing its work plan and budget. Furthermore, the Health Cluster’ over- ambitious targets and the political influence of national authorities on the Health Cluster’ work were other sig- nificant drawbacks that negatively impacted the effective- ness of the Health Cluster. “Two Yemeni governments, South and North, two governments are controlling different geographi- cal areas and each claim they are controlling the whole of Yemen, which weakened the decision- making among partners when they tried to abide by policies of one or another entity, which affected the response”. (Informant #1) “I felt that sometimes the coordinators […] were already taken by parties to the conflict […] they were not representing the voices of all partners”. (Inform- ant#9) Effectsf Effects, derived from the term impact, consider all nega- tive and positive encounters resultant from the Health Cluster performance and activities during the appraised period. Responses from key informants regarding con- nectedness were diverse. These responses can be cat- egorised into three categories: (a) no exit strategy incorporated, (b) exit strategies were included to some extent with limitations and (c) a paradoxical effect on connectedness. Most key informants agreed that there were positive effects on the health system because the Health Cluster partners’ response allowed the health system to provide Minimum Health Service Package (MHSP), strength- ened the local capacity and advocated for adequate funding: Regarding the first category, a few informants from NNGOs and Health Cluster indicated that there were no exit strategies in the Health Cluster partners’ pro- jects, and that during emergencies — which is the case in Yemen — the transition to development is not a priority “I think without the Health Cluster, the situation in Yemen would have been much worse for the national health system”. (Informant #7) “If you compare between HeRAMS22016 and 2018, I can say there was a good improvement in the health system inputs […]. That is how the Health Clus- ter helped in maintaining the functionality of the national health system”. (Informant #5) “When we plan for humanitarian response, we do not think about sustainability because it is not a recovery intervention, it is just to provide first-line interventions to cover the acute needs, so talking about sustainability is difficult and not realistic”. (Informant#2) On the other hand, one of the adverse effects of Health Cluster on the health system, as emphasised by some key informants who represented interna- tional experts and NNGOs, was that it might have contributed to the shortage of senior MoPHP staff by recruiting them to work for the Cluster or various UN agencies. Others argued that the huge incentives will have a detrimental effect on health staff availability in the future: In the second category, i.e. exit strategies were included with limitations and challenges, the inform- ants (INGOs, international experts and the Clus- ters) mentioned examples from the Health Cluster partners’ experiences to support this statement. For instance, the ongoing capacity building activities of MoPHP staff at the facility and community levels dur- ing the conflict: “We are also responsible for the tragedy of the national health system, why? resource, the functionality of the health facilities, and so on”. (Informant #5) resource, the functionality of the health facilities, and so on”. (Informant #5) Efficiency (funding) Other informants among the INGOs confirmed that the Health Cluster could not mobilise resources on some occasions, especially in areas with cholera resurgence: Many informants commonly stated that the Health Cluster response was not timely. They attributed that to the division within the Ministry of Health and the lengthy bureaucratic procedures of the new and exist- ing political parties to get health projects approved. This division resulted in two different procedures correspond- ing the two sides of the country. These procedures have to be followed and coordinated at two parallel levels by the Health Cluster’ partners, which was very challenging: “The fund for the Health Cluster is still not enough, because it is not proportional to the existing gap or the programme”. (Informant #8) “Now we have a national authority for managing or coordination of the humanitarian assistance they call it NAMCHA1[…], I am talking about de facto “Now we have a national authority for managing or coordination of the humanitarian assistance they call it NAMCHA1[…], I am talking about de facto “After we agree with the Ministry of Health of both sides and with the partners […] (and) have a clear framework and plan, indicators, and activities, then we have to cost these activities. Based on what we call it severity matrix […], (It has) health sys- tem indicators, the availability of service, human 1 NAMCHA: National Authority for the Management and Coordination of Humanitarian Affairs Page 7 of 14 Page 7 of 14 Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Al‑Awlaqi et al. Journal of International Humanitarian Action Connectedness Connectedness addresses the inclusion of exit strategies in the Health Cluster partners’ response during the last 3 years in Yemen. Exit strategies are plans to sustain the health intervention once the donor funding is over and the overall health situation is improved, i.e. as a result of peace and stability. not going to take the Health Cluster interventions to sustainable solutions”. (Informant# 4) the active participation of national NGOs in the Health Cluster: “...I found the national NGOs’ role was not strong or not effective in the Cluster like (compared to) inter- national NGOs, maybe because of the low capacities […], the lack of confidence within their capacities or they thought their role was not important, as most of the discussions became dominant by international NGOs”. (Informant #2) Participation During interviews, the key informants were asked about their views regarding motivation and the level of engagement of health stakeholders in the Health Cluster activities such as meetings, joint response plans and pri- oritisation of affected areas. All informants agreed that there were four main reasons (Fig. 1) which motivated the partners to be active Health Cluster members, namely staying well- informed with the field situation, getting visible in national and international humanitarian platforms, and, most importantly, having priority access to humanitarian aid funds. f Many informants, except those who represented UN organisations and the Health Cluster, confirmed that the level of participation and representation of health stake- holders in the Health Cluster was very minimal, espe- cially the national NGOs and local health authorities. Informants emphasised that these NGOs were not given an equal chance to participate in the decision-making process of the Health Cluster. “I used to attend civil society organisations’ meet- ings, and I was surprised that many organisa- tions did not even know about the Cluster sys- tem”. (Informant #3) “I used to attend civil society organisations’ meet- ings, and I was surprised that many organisa- tions did not even know about the Cluster sys- tem”. (Informant #3) Effectsf […] ministries are increasingly getting weaker by the time, and we are contributing to this weakness. Most of our staff come from the Ministry, so we are actually depleting the Ministry of Health from its staff”. (Informant #7) “Whoever stays in the country, the human resource, they are now receiving incentives […] training, […] there is a capacity building. These things can be sustained even if the Health Cluster partners with- draw”. (Informant #5) The third category of informants from INGO, NNGOs, and international experts indicated that the Health Clus- ter partners’ response had, nonetheless, a paradoxical effect which adversely affected the connectedness aspect. Operating mobile health clinics outside health facilities for a longer time and paying high salaries or incentives to NNGOs and MoPHP staff were among the examples mentioned: “I think this intervention (humanitarian agen- cies paying monetary incentives for health workers) was a bit risky, because if the health worker used to receive high incentives and those humanitarian partners stopped their incentives after the war (when the funds ceased), I am afraid that health service provision in these health facilities would collapse […] even if the government pays their salaries regu- larly”. (Informant#2) “The government, yes, they were in favour of using mobile clinics in many locations for whatever rea- son, but no, […], this [ mobile clinics’ approach] is 2 HeRAMS: Health Resources Availability Monitoring System Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Al‑Awlaqi et al. Journal of International Humanitarian Action Page 8 of 14 Fig. 1 The main motivations of health stakeholders to actively participate in the Health Cluster meetings in Yemen Fig. 1 The main motivations of health stakeholders to actively participate in the Health Cluster meetings in Yemen Challengesh This theme emerged during the interviews. Two main categories of challenges were discussed during the inter- views; these were the following: Furthermore, some informants from national NGOs believed in a vicious cycle of underrepresentation, underfunding and poor capacity. These issues hindered A- Governance and capacity: fragmentation within MoPHP and the weak capacity of its staff, especially Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 9 of 14 (2022) 7:9 in management and planning capacities, were among the main challenges indicated by many respondents. Furthermore, the strong centralisation of the Health Cluster was a challenge, too, as it prevented timely decision-making within its peripheral subnational hubs. Finally, the limited financial resources of the Health Cluster hindered deploying a swift response to those who need it: areas can be organised under four headings: (1) strength- ening leadership, decentralisation, and effectiveness, (2) improving participation and inter-Cluster collaboration, (3) transition to development and lastly, (4) cross-cutting recommendations. 1. Leadership: First, more delegation and decision- making power should be given to the subnational hubs, including financial independence. Second, to deliver a principled response abided by humanitar- ian principles, the Health Cluster should maintain its full autonomy and impartiality without interfer- ence from CLA or local authorities. Third, strength- ening advocacy for unrestricted access to affected areas is paramount. 1. Leadership: First, more delegation and decision- making power should be given to the subnational hubs, including financial independence. Second, to deliver a principled response abided by humanitar- ian principles, the Health Cluster should maintain its full autonomy and impartiality without interfer- ence from CLA or local authorities. Third, strength- ening advocacy for unrestricted access to affected areas is paramount. “The problem is the centralisation. Which means that there are no decisionmakers at the sub-cluster level. The decisions come from the Cluster itself in Sana’a”. (Informant #10) Moreover, informants, mainly INGOs and NNGOs, had to undergo parallel coordination and authorisation pro- cedures at the two sides of the country, which was a time- consuming process that hindered their timely response. For instance, the newly emerged coordination structure within the de facto territories (NAMCHA) has delayed health pro- jects’ implementation because of the very lengthy bureau- cratic procedures it applies to get projects approved. Some informants stated that it is also crucial to clarify the role of the Health Cluster to the local authorities and stakeholders. Challengesh The informants expressed their need to have a strong, neutral and impartial cluster coordinator who can act with power and integrity, despite the enormous political pres- sure, to represent the best interests of the Health Cluster partners among the national authorities and international actors. “So, if you have a project for one year, can you imag- ine losing four months in coordination? Then […] when you go to the governorate you have to repeat the same process with NAMCHA branch, with the national security, with the governor office, with the governorate health office, with the (district) health office and so on. I think this is one of the most impor- tant things that we need to sort out to help the Health Cluster performing well”. (Informant #5) “We want strong (Health Cluster) leadership; strong leadership means we need fighters in this position (Cluster Coordinator) to fight on behalf of the group (Health Cluster partners)”. (Informant #9) 2. Participation: Participation and inter-Cluster col- laboration can be improved, according to inform- ants from the Health Cluster and INGOs, by strengthening inter-Cluster coordination mecha- nisms, especially with the Nutrition Cluster. Inform- ants, mainly INGOs, emphasised the importance of representing NGOs in the Health Cluster technical working groups and the Strategic Advisory Group (SAG). Moreover, in the opinion of some partici- pants, the Health Cluster should encourage and motivate the MoPHP to play a more active role in the Health Cluster plans and response. 2. Participation: Participation and inter-Cluster col- laboration can be improved, according to inform- ants from the Health Cluster and INGOs, by strengthening inter-Cluster coordination mecha- nisms, especially with the Nutrition Cluster. Inform- ants, mainly INGOs, emphasised the importance of representing NGOs in the Health Cluster technical working groups and the Strategic Advisory Group (SAG). Moreover, in the opinion of some partici- pants, the Health Cluster should encourage and motivate the MoPHP to play a more active role in the Health Cluster plans and response. B- Access and coverage of health services: The informants from INGOs, NNGOs and Health Cluster expressed that they had difficulty in accessing many deprived areas for various reasons, e.g. security issues, checkpoints and local authorities, which was one of their biggest challenges as it hindered the implementa- tion of health response.f Some informants mentioned the unaffordability of health service to many vulnerable populations, because of user fees and cost of treatment, as another tough challenge that hindered vulnerable people from receiving the treatment they need: 3. Transition to development: which is required to sustain the current health services and prepare the health system for the next phase, i.e. health system recovery. Participants, mainly from Health Cluster and international experts, urged that it is inevita- ble to invest in the public health sector and national NGOs to build their capacities and to improve the cost-effectiveness of health interventions, especially in areas with no current conflict. Some informants stated that there is a need to find an approach to 3. Transition to development: which is required to sustain the current health services and prepare the health system for the next phase, i.e. health system recovery. Participants, mainly from Health Cluster and international experts, urged that it is inevita- ble to invest in the public health sector and national NGOs to build their capacities and to improve the cost-effectiveness of health interventions, especially in areas with no current conflict. Some informants stated that there is a need to find an approach to “We note that in the North and the South- the de facto power [ in the North] and the legitimate gov- ernment in the South-, are putting conditions on the NGOs to access populations”. (Informant #9) NGOs to access populations”. (Informant #9) Discussion h d Third, based on the results of this study, it can be con- cluded that the Health Cluster performance would have benefited from robust decentralised structures at the sub- national hubs (Hopkins 2019). According to the inform- ants, although these subnational hubs were established during the conflict in 2017 (Yemen Health Cluster 2017), they lack decision-making power. Moreover, the Health Cluster did not sufficiently consider the North-South divide, i.e. two ministries of health, which might have necessitated a different approach. For example, establish- ing two national clusters with full autonomy ensures har- monised, equitable and timely health responses to those who need it in both areas. This study provides insights into the Health Clus- ter performance in Yemen between 2015 and 2019, intending to use these reflections to improve the coordination of the humanitarian health response in Yemen. Previous studies attempted to assess the ideal level of coordination within the clusters in general (Clarke and Campbell 2015) and the role of the Health Clus- ter, among other coordination platforms, in the cholera response in Yemen (Hopkins 2019). However, there were no specific evaluation studies on Yemen’s Health Cluster performance during the conflict period. Therefore, this study fills a unique niche within the literature concern- ing health coordination in fragile and conflict-affected settings with its primary focus on Yemen. In their study on the Cluster Approach, Clarke and Campbell, which included Yemen among seven other countries (Clarke and Campbell 2018), found that the participation of NNGOs in the Cluster was extremely weak, which repre- sented one of the main drawbacks of the Cluster system in these countries. Fourth, the Health Cluster capacity to deploy field assistance during an outbreak or an acute health emer- gency was severely restricted by the limited financial resources. Many key informants indicated that it is vital that the Health Cluster has its budget, independent of donors’ proposal windows, to respond to emergencies at their early stages. Then, additional projects with larger budgets can back the initial response once the donors are on board and all bureaucratic procedures to deploy more significant funds are finalised. The results of this study indicate that the Health Cluster in Yemen was (and still is) a vital coordina- tion mechanism for the humanitarian health response during the appraised period (2015–2019), effective in achieving most of its objectives and reaching many people in need. Areas for improvement According to the key informants, specific areas within the Health Cluster performance can be improved. These Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 10 of 14 retain senior and qualified ministerial staff in their positions within MoPHP. development” perspective, as many informants in the study indicated. Second, the results of this study show that the Health Cluster was partially effective in achieving its objectives and its intended purpose during the assessed period. 4. Cross-cutting recommendations: One inform- ant supported mainstreaming gender equality in the Health Cluster by recommending a female staff for the position of Cluster Coordinator. This inform- ant stated that considering gender issues within the Health Cluster management will ensure that mater- nal mortality, gender-based violence (GBV) and local cultural sensitivities that affect health are taken care of effectively. 4. Cross-cutting recommendations: One inform- ant supported mainstreaming gender equality in the Health Cluster by recommending a female staff for the position of Cluster Coordinator. This inform- ant stated that considering gender issues within the Health Cluster management will ensure that mater- nal mortality, gender-based violence (GBV) and local cultural sensitivities that affect health are taken care of effectively. The views were diverse; those who run and participate in the Health Cluster had a more critical view, perhaps because of their extensive involvement in the Health Cluster at the micro level. While those from UN organ- isations, in their views, may have reflected on the over- all achievement of the Health Cluster from the macro level, i.e. life-saving interventions, the number of people reached and other overall targets. f “We should consider that the Cluster Coordinator (is a female), at least. We should have more females because a big part of life-saving human- itarian interventions has to do with women”. (Inform- ant #7) Even though the Health Cluster’s interventions suc- cessfully prevented potential deaths and treated morbidi- ties, timely health response was a challenge due to many factors. The ongoing conflict, limited financial resources allocated for the Health Cluster and Yemen’s North- South divide, to name a few. Recommendations for policy and researchh The study recommends several actions to be taken from policy and research perspectives. It is crucial to under- stand that the implementation of these recommendations could be challenged by the political and security situation and by the global partners’ conflicting agenda for Yemen. However, these recommendations can act as an umbrella to streamline efforts to improve the Health Cluster per- formance in Yemen. Furthermore, the Health Cluster’s effects on the humanitarian coordination were described as posi- tive and considered by many informants. However, the humanitarian organisations, including the UN agen- cies and the Health Cluster itself, may have contributed to the shortage of competent senior staff at the ministe- rial level by recruiting them directly to work for the UN organisations. Moreover, some informants added that UN recruitment offers a more competitive remuneration package compared to the public sector. It is pivotal to refine the Health Cluster objectives and further align them with the national priorities and popu- lation emergent needs from a policy perspective. The key recommendations are establishing a rapid funding mech- anism managed by the Health Cluster, strengthening the local capacities and supporting the health system in tran- sition to recovery and development. The Health Cluster provided a platform for effective health coordination, attempted to build partners’ techni- cal capacities and prioritised them for funding opportu- nities. Nevertheless, power dynamics within the Health Cluster played a negative role on the national partners as NNGOs felt neglected and not involved in the strategic decision-making process compared to their international counterparts. Concerning the recommendations for research, the Health Cluster will benefit from further studies which involve measuring the effectiveness and cost-effective- ness of the health sector interventions coordinated by the Health Cluster. Impact evaluation would provide accurate figures on the medium-to-long-term impact of the Health Cluster, especially on the vulnerable com- munities and the national health system. Bringing les- sons learnt and success stories from other countries with similar contexts, especially those related to health system recovery and humanitarian-development nexus, might improve the Health Cluster strategic directions and align its objectives to population needs. The recommendations for policy and research are summarised in Table 4. Active engagement of national NGOs feeds into the broader strategy of the Global Health Cluster, which stresses the importance of adopting a “localisation” approach to strengthening the national capacities, i.e. Recommendations for policy and researchh investing in national organisations to expand and sustain health services in the long-term (WHO 2017a). However, there were limitations in addressing gender within the Health Cluster, with a persistent trend of limited involve- ment of females at the senior leadership, i.e. Cluster Coordinator’ position in the capital was occupied exclu- sively by males. Finally, the results of this study indicate that the focus of the Health Cluster on life-saving interventions and not prioritising plans for exit strategies or health sys- tem recovery might be an area that needs improvement. Establishing a more vital link between the current health interventions and the longer-term plans for health sys- tem recovery and transition to development, the so- called humanitarian-development nexus, is crucial and should be prioritised (Qirbi and Ismail 2017), especially in stable areas where there is no actual conflict, i.e. south- ern and eastern governorates. This approach, of ensuring connectedness, is in line with the Global Health Cluster, WHO and IASC recommendations (WHO 2016, 2017a; IASC 2019). This fits within the New Way of Working, a partnership approach specifically designed to strengthen the humanitarian-development-peace nexus by building Discussion h d ii Moreover, the study showed that the Health Cluster performance between 2015 and 2019 had many positive and negative effects at various levels of the national yet divided health system, humanitarian response coordina- tion, health stakeholders and the population in need. The Health Cluster partners’ response provided tremendous support to the health system and ensured the continu- ity of life-saving health services (Yemen Health Cluster 2017). In addition, financial incentives for health work- ers, which were included in many of the partners’ health First, the Health Cluster objectives were partially rel- evant to the context of Yemen between 2015 and 2019 and, to some extent, were in line with the national health priorities and the urgent health needs of the population. Nevertheless, these objectives were limited to life-saving interventions, and they lacked an explicit “transition to Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7 Page 11 of 14 collaborative partnerships among UN organisations, civil society organisations and governments to address the root cause of conflict and fragility and their conse- quences on the communities (OCHA 2017). programmes, were a significant step toward maintaining a minimum level of health system functionality. The role of the Health Cluster in supporting health management information system, i.e. strengthening the early warning system, has improved the surveillance capacity within the MoPHP. The same finding was con- firmed in another study about MoPHP preparedness to outbreaks in Yemen (Dureab and Jahn 2019). Limitations of the study • The Health Cluster subnational hubs were not ade- quately represented in this study. Key informants came from the leading national hubs in Aden and Sana’a. There was difficulty in accessing subnational coordinators due to communication issues. • The final sample size (10 interviews out of 16) might be small to draw representative conclusions. Nevertheless, poor phone and Internet connectiv- ity, unstable security status and North-South divide (and the sensitivity around it) contributed to not reaching sixteen participants. Moreover, making “generalisation” or “statistical inference” was not the primary aim of this study, nor making objectiv- ity judgments about the sample. Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 12 of 14 Al‑Awlaqi et al. Journal of International Humanitarian Action Table 4 Recommendations for policy and research Study outcome Recommendations Organisation Policy Research Relevance: The Health Cluster objectives, although they were relevant (to some extent) to the context in Yemen, they had short-term and emergency perspective. Some vulnerable groups and illnesses need specific activities to address them. - Cluster objectives can undergo a review process by all Cluster members to review their relevance and update them according to the gaps and needs. - Include specific objectives to emphasise health system strengthening and recovery outcomes. - Inclusion of objectives (or sub-objectives) to support people with special needs and mental health interventions. - Further research on the best approach to iden‑ tify relevant objectives and to measure their effectiveness and efficiency is needed. Health Cluster, WHO as a CLA Effectiveness: The Health Cluster was effective in delivering its activities according to objectives, but not on a timely basis. Neutrality and independence of the Health Cluster were not protected on some occasions. - There should be a mechanism to provide a rapid response within the authority and budget of the Health Cluster to provide a swift response to emergencies. - Equal representation and delegation of decision-making power to leaders of subna‑ tional hubs. - Strengthening the Health Cluster leadership and financial independence. - Clarifying the role of the Health Cluster in all operational areas. - Cost-effectiveness analysis of the health sector response coordinated by the Health Cluster would provide evidence to invest in the most effective interventions. Health Cluster, WHO as CLA, OCHA, IASC Efficiency (funding): The Cluster funds were enough to cover its basic coordination tasks, but not to respond to emerging outbreaks or displacement needs. Limitations of the study - The Health Cluster would benefit from having an independent financial mechanism to rapidly respond to emergencies at early stages. Health Cluster, WHO, OCHA, donors Effects: The Health Cluster gave suboptimal attention to MoPHP senior staff’ capacity, persons with disabilities, senior people, mental health, and chronic disease. - Finding an approach to “second” MoPHP senior staff while maintaining their salaries and benefits. - Establishing and expanding activities to tackle chronic diseases, geriatric, and mental health and to prioritise enough assistance to people with disabilities. - Impact evaluation research, which could include the perspective of the affected popula‑ tion within its design. IASC, Health Cluster, WHO, UNICEF and OCHA MoPHP Connectedness: - The inclusion of exit strategies to sustain health services after the end of donors’ support was not included in the Health Cluster’ partners responses. - The Health Cluster should encourage donors to include specific sections on exit strategies in health project proposals. - The Health Cluster shall work with MoPHP and all health stakeholders to draft and implement a health system recovery plan. - Research to evaluate projects and to docu‑ ment successful stories (good practices) in sustaining the health services after concluding aid funds. Health Cluster, SAG, UNFPA, OCHA, donors Participation: - Participation and equitable representation of all partners in the Health Cluster’ decision- making process was unsatisfactory. - The composition of SAG should be reviewed to ensure fair representation of all partners. - The process of decision-making within the Cluster should be clarified, standardised, agreed upon, and circulated to all partners upon their final collective consensus. - Regular partner surveys, with action points to follow the outcomes. - Research on the role of observers (e.g. MSF) and the private sector in the Health Cluster. Health Cluster, WHO, OCHA, MoPHP Al‑Awlaqi et al. Journal of International Humanitarian Action (2022) 7:9 Page 13 of 14 Page 13 of 14 • The researcher could not organise the interviews with the current MoPHP officials. However, two key informants were former senior MoPHP staff at the ministerial level. Cluster in Yemen. Overall, this study stresses the impor- tance of effective health coordination during emergen- cies to save lives and strengthen local health systems. The response coordinated by the Health Cluster was adequate, with room for improvement. Conclusions In this qualitative study, we attempted to retrospectively evaluate the Health Cluster performance in Yemen, cov- ering 2015–2019. The study fills an important gap in the scholarly, peer-reviewed work related to the Health References ACAPS (2016) Country profile of Yemen. Available at: https://www.acaps.org/ country/yemen/country-profile. Accessed 24 Nov 2018 Limitations of the study Despite the ongo- ing conflict, constrained access and division within the government, the Health Cluster’s response has succeeded to reach many populations in need and was in line, to a considerable extent, with the essential, life-saving health priorities. Moreover, despite the tremendous constraints faced, the Health Cluster has effectively coordinated a joint response with other clusters (Nutrition, WASH and FSL). For example, the cholera outbreak response and the integrated famine risk reduction response. Finally, the Health Cluster has worked closely with both govern- ments to maintain the minimum health service package. However, the package was not comprehensive enough to fully include mental health services or respond to specific population groups’ needs, such as disabilities, chronic diseases or senior citizens. • The criterion of “efficiency” was not investigated in depth due to the lack of specific financial infor- mation regarding the funds allocated for coordina- tion tasks of the Health Cluster work. However, the study attempted to answer basic questions about funding in terms of analysing the performance of the Health Cluster against the available known budgets (e.g. YHRP). • Inconsistencies in using a single language in admin- istering interviews might affect the quality of the transcripts. Thus, some critical concepts might be lost during translation, i.e. translating the two Ara- bic interviews to English. • From an accountability perspective, the people in need are the end user of the Health Cluster part- ners’ field interventions, and their opinions could have benefited the findings of this evaluation study (Hallam 1998). However, the limited study scope combined with the difficulty in accessing the field in Yemen prevented the incorporation of inter- views with the affected population within the study design. Authors’ contributions SA wrote and developed the topic, collected the data and analysed and wrote the full manuscript. MT supported the development of topic, revised and edited all sections of the manuscript, contributed to the analysis and was a major contributor in writing the manuscript. FD revised the abstract, provided inputs to the background, methods, conclusion and recommendations sections and proposed the manuscript title and journal selection and edited references. All authors read and approved the final manuscript. • The role of Health Cluster observers - Médecins Sans Frontières (MSF), ICRC, and the private sec- tor in the humanitarian coordination was not addressed because the study focused on full cluster members. Availability of data and materials Availability of data and materials Not applicable. Not applicable. • The principal investigator’s previous experience in the Cluster Approach in Yemen might have created bias during data collection and analysis. Neverthe- less, comparison with other studies on humanitar- ian health coordination and examination of results by external health experts were applied to minimise the researcher’s bias. Author details 1 Institute of Public Health, Jagiellonian University Medical College, Krakow, Poland. 2 School of Health and Related Research, The University of Sheffield, Sheffield, UK. 3 Institute of Research for International Assistance, Akkon Hoch‑ schule, Berlin, Germany. 4 Heidelberg Institute of Global Health, Heidelberg University, Heidelberg, Germany. 5 Health Economics and Social Security Department, Institute of Public Health, Jagiellonian University Medical College, ul. Skawińska 8, 31‑066 Kraków, Poland. • Limited scholarly work, especially peer-reviewed, on the Health Cluster in Yemen Received: 27 January 2021 Accepted: 28 February 2022 Funding This study was implemented in partial fulfilment of a master’s degree in Public Health (Europubhealth+) with funds from the European Commission’ Erasmus Mundus excellence scholarship. • Females might be underrepresented in the study. One contributing factor is that most cluster leads are pre- dominantly males (at the study time). Females who participated in this study represented one NNGO and one INNGO. References C S ( Accessed 27 Feb 2019 World Bank (2015) Global poverty line update. Available at: http://www.worldbank. org/en/topic/poverty/brief/global-poverty-line-faq. Accessed 19 May 2019 Michael, M. (2019) ‘Vaccines blocked as deadly cholera raged across Yemen’. Available at: https://www.apnews.com/b821a9b1811d4b4d803fffd4f e132b4e. 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https://openalex.org/W4315646451	https://victorianpopularfiction.org/wp-content/uploads/2023/01/001-Basdeo-and-Nesvett-VPFJ-4-2-1.pdf	English	null	Reappraising Penny Fiction	Victorian popular fictions	2,023	cc-by	9,812	Reappraising Penny Fiction Abstract This Introduction to the Special Volume of Victorian Popular Fictions Journal titled “Reappraising Penny Fiction” defines penny fiction, surveys its prehistory, and reconstructs its emergence in the nineteenth- century British media and globally. The article then engages with the ongoing scholarly debate about “penny dreadfuls” and theorises how misconceptions about the genre developed and were circulated by critics and scholars. Finally, the article introduces the central questions and themes of the special issue, as well as the individual articles. Victorian penny fiction has long been considered disturbing yet compelling; we hope that our volume reveals why that is so. Stephen Basdeo and Rebecca Nesvet Early in the twentieth century, A.E. Waite (1857–1942) struggled to write one of the earliest bibliographies of penny fiction – that is, the Victorian fiction published in penny-a-number periodicals and pamphlet-style penny parts and meant largely for the entertainment of working- class readers in Britain and in cities throughout the Empire. A self-appointed scholar of the occult who was fascinated by Arthur Machen and befriended Aleister Crowley, Waite decided that no arcana were more obscure than the secret canon of “penny bloods” and “dreadfuls,” as his generation called penny fiction. “It may sound inconceivable,” he wrote: … and yet is of literal fact, that for a century past at least a multifarious literature … has been circulating in England, above all in London, by its thousands and myriads, its constituents dissolving almost at their birth and leaving so few traces behind that it is comparable to a realm of hiddenness, a world almost unknown. (Waite 1997: 1) (Waite 1997: 1) It is not surprising that, as late as the 1930s, Waite described this bibliographic endeavour in terms drawn from the discourse of (imperial) exploration because, for seventy or eighty years previously, the middle-class press had represented penny fiction and its working-class readership as hidden entities. Waite aimed to become the penny press’s Mayhew or Greenwood, infiltrating it and revealing its secrets to middle-class readers. It is not surprising that, as late as the 1930s, Waite described this bibliographic endeavour in terms drawn from the discourse of (imperial) exploration because, for seventy or eighty years previously, the middle-class press had represented penny fiction and its working-class readership as hidden entities. Waite aimed to become the penny press’s Mayhew or Greenwood, infiltrating it and revealing its secrets to middle-class readers. Similar imagery already pervaded Margaret Oliphant’s “The Byways of Literature: Reading for the Million” (1858). Though giving “the masses all credit for their gift of reading” Oliphant proposed that “before we glorify ourselves over the march of intelligence, let us pause first to look into their books” (King and Plunkett 2005: 197). Critiquing the naïveté of the Society for the Diffusion of Useful Knowledge, she mentioned “that unknown intelligence,” “the working man” (198). Having “invested a sixpence” in penny literature, she decided to find out what the million read (196). Keywords y penny fiction; penny bloods; penny dreadfuls; G.W.M. Reynolds; Eugene Sue; James Malcolm Rymer; Pierce Egan; periodicals; serialisation dime novels; world literature Date of Acceptance: 31 December 2022 Date of Publication: 13 January 2023 Double Blind Peer Reviewed Date of Acceptance: 31 December 2022 Date of Publication: 13 January 2023 Double Blind Peer Reviewed Recommended Citation: Basdeo Stephen and Rebecca Nesvet. 2022. “Reappraising Penny Fiction: Introduction.” Victorian Popular Fictions, 4.2: 1-17. ISSN: 2632-4253 (online) DOI: https://doi.org/10.46911/DHBV6145 Recommended Citation: Basdeo Stephen and Rebecca Nesvet. 2022. “Reappraising Penny Fiction: Introduction.” Victorian Popular Fictions, 4.2: 1-17. ISSN: 2632-4253 (online) DOI: https://doi.org/10.46911/DHBV6145 Recommended Citation: This work is licensed under a Creative Commons Attribution 4.0 International License. Volume 4: Issue 2 (Autumn 2022) Victorian Popular Fictions 1 Andrew King identifies the respective rhetorical goals of Oliphant and Collins’s investigations of the penny fiction of 1858. Oliphant, struggling to articulate her own politics while adhering to Blackwood’s conservative outlook, finds penny fiction an update of medieval romance, inhabiting what King calls a “stereotypical and chivalrous imaginative world” in contrast with which middle-class literature does not represent progress, but decay (King 2004: 35). Her analysis is ultimately “profoundly material” (35). Collins’s “The Unknown Public” has been read as a response to Oliphant but is more likely a justification of Collins’s friend Mark Lemon’s editorial practices at the London Journal, which Lemon had taken over in 1857 and which in 1858 was facing a commercial downturn (36). In “The Unknown Public,” Collins “ventriloquis[es]” Lemon’s “desperation” (36). Stephen Basdeo and Rebecca Nesvet Wilkie Collins explored similar materials in his article “The Unknown Public” (1858), in which, unlike the voice of the Oliphant, he went (fictionally) undercover to obtain and study fifteen samples of penny fiction. He called penny fiction “these mysterious publications,” and claimed “the discovery of an Unknown Public, a public to be counted by millions; 1 ISSN: 2632-4253 (online) 1 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) the mysterious, the unfathomable, the universal public of the penny-novel journals” (208-9). Moving from the rhetoric of discovery to that of colonisation, Collins located this public “right out of the pale of literary civilisation” (209). He declared that, in the penny papers, “[a]n immense public has been discovered: the next thing to be done is, in a literary sense, to teach that public how to read” (1858, quoted in King and Plunkett 2005: 216). That, in Collins’s mind, meant how to read better material than the penny press provides them.1 However, the penny press, which mixed fiction with other genres in its pages, was by that point an important fixture in literary culture, and imbued with its own aesthetics, politics, and culture. What is Penny Fiction? Yet categorising Steele and Addison’s serial and largely fictional Spectator as a forerunner of the penny fiction of George W.M. Reynolds (1814–79), James Malcolm Rymer (1814–84), J.F. Smith (1806-90), M.E. Braddon (1835-1915) and others would probably sit uneasily with most scholars of eighteenth- and nineteenth-century literature, even if its traces are clear in penny publications that aimed at (even if they did not succeed in obtaining) some form of respectability. Andrew King for example has traced what he calls “the parergic” (“a system whereby texts are based on originals that are invested with greater symbolic capital and authority.” 2004: 27) in the mass-market penny fiction magazine, the London Journal (1845-1927), edited (1845-6) by Reynolds: even though its status was low, the London Journal explicitly emulated Addison and Steele in the late 1840s (King 2004: 57-9). Many Victorianists will associate Reynolds with the so-called “penny blood.” This leads us into a discussion of two of this issue’s key questions: what do we mean by “penny blood,” “dreadful,” and, indeed, “awful” and what is the history of those terms? We must bear in mind that not every sensational literary work that sold for a penny was a penny blood; the “blood” writer Pierce Egan the Younger’s Home Circle magazine contained several thrilling stories, yet anyone who looks at its contents – which included works by Nathaniel Hawthorne and George Otway – could not class this magazine as containing anything resembling a “blood” story (Basdeo 2020). This perhaps raises another question: to what extent does a story’s appearance in a journal such as the London Journal demarcate it as not respectable? To take an example from Reynolds again, the famous author’s Faust: A Romance of the Secret Tribunals (1845–6) first appeared in the London Journal and thereafter was sold in standalone serialised instalments as well as collected in a volume form in 1847. As a text it appears in multiple forms, each with distinct paratextual features and this suggesting different reading strategies that will make links to differing corpora of texts: in magazine form it will link most easily to other parts of the magazine; in standalone serial forms to other standalone serials (especially to what we think of as the core meaning of “penny bloods”), in volume form to other volume-form fiction (which will include the more expensive and more respectable fiction). What is Penny Fiction? In Victorian Britain, popular literature, briefly put, was a heterogenous form of mass reading entertainment which included prose fiction, poetry, ballads and satires which were cheaply printed and retailed usually for the price of one or two pennies (or less, if second-hand). Since it was cheap it was considered disposable or recyclable, unlike expensive volume form fiction. Within this very broad category of ephemeral literature were several formats of literature which included broadsides – usually a single printed page containing a poem or short story – as well as chapbooks, small books containing eight duodecimo-sized pages. These chronicled a variety of fantastic tales in prose and rhyme. In addition, there were the more recognisable – for Victorianists at least – penny magazines, and 16-page “bloods” and “dreadfuls.” Penny fiction, in our understanding, is a distinct form of popular literature and a distinctly Victorian phenomenon. Such a demarcation is necessary because any definition of popular and indeed penny fiction will always be imperfect, and what is applicable to the Victorian era may not be applicable to the literature in the centuries preceding it. The medieval era had popular literature, for instance; escrowez or schedulae – which Steven Justice translates as ‘broadsides’ – were handwritten single sheets of parchment that contained poetry, news, and satires and were pinned up in village, town squares, and on church doors (Justice 1994: 28-9). Neuburg (1964) and Shepard (1977) see the beginnings of cheap literature in the early modern broadsides that retailed at a low price and contained ballads or told stories of strange and fantastical occurrences. Poetry and ballads are a different genre to prose fiction, and in terms of genre they can be more easily separated from the serialised prose fiction that dominated in the Victorian era. Yet when it comes to cheap, fictional prose from before the Victorian era, our definition encounters further problems. For example, Joseph Addison and Richard Steele’s weekly Spectator (1711–15) retailed for the majority of its run at one penny (and, after the stamp tax was imposed, two pence), and a critique of society was offered through the eyes of fictional correspondents relating contrived stories of life in Augustan London (Mr. Spectator, Sir Roger de Coverley, Will Honeycomb, and the 2 2 ISSN: 2632-4253 (online) Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) “Lawyer of the Inner Temple”). What is Penny Fiction? Many scholars cite the genres of crime and gothic fiction as major influences upon penny fiction and, in particular, penny bloods. Many mid-century penny bloods did indeed contain murders and robberies in stories written in the style of the Newgate novel of the 1830s while gothic influences are clearly detectable in close readings and analyses. Yet these focuses – which confine penny fiction inside the neat confines of crime and gothic – arguably do a disservice to a corpus of literature which was just as varied as “mainstream” literature. For an example one may turn, once more, to the works of Pierce Egan the Younger. They sold for a penny yet there was very little in them that modern scholars, were they to read his works, would classify as proper to a penny blood. Egan’s Robin Hood and Little John (1838–40), Wat Tyler (1840–1), Adam Bell (1842), and Edward the Black Prince (1850) mark him out as a skilled historical novelist not prone to sensationalism or violence for violence’s sake. His later novel Clifton Grey (1856) was a tale of the Crimean War which offered a critique of contemporary events, while his 1860s novels serialised in the London Journal such as The Poor Girl, The Flower of the Flock, and The Wonder of Kingswood Chace would sit easily alongside any silver fork or sensation novel (see King, 2004: 126). Further complications arise when we consider the republication of expensive literary works in penny format. The publisher John Dicks did this frequently in his Library of Standard English Works later in the century (Humpherys 2016: 94). In this long-running series, the works of W.M. Thackeray and William Harrison Ainsworth stand alongside Reynolds; James Fenimore Cooper is printed in the same volume as Sue, as is Henry Fielding. When the series was published in the 1880s, Victorian readers would have encountered such mainstream works and literary classics alongside penny bloods and penny fiction. In such a series, does mainstream literature become penny literature, or does penny literature enter the mainstream? John Dicks’s sons further complicated the Victorian “canon” when the firm began publishing sixpenny “People’s Editions” of major English novelists’ works. An advertisement for this series includes the works of Dickens, Bulwer Lytton, Ainsworth, Reynolds, Egan the Younger, along with a host of other writers whose names have now been largely forgotten (“Advertisement” 1885). What is Penny Fiction? Such brief considerations might suggest that penny fiction was as much an “idea” as a genre and, second, just because something was sold for a penny does not mean that it was necessarily “sensational,” gory, or a “blood.” Further questions related not only to format and dissemination considered in this special issue include how illustration, translation and transatlantic (re)publication of penny fiction, feuilleton novels, and dime novels illuminate the history of penny fiction. For example, many scholars of “Victorian Popular Fiction” will know that the roman feuilleton (newspaper-published fiction serial) paved the way for British serial fiction and that, among feuilleton authors, Eugène Sue had a significant influence on the popularity of penny fiction in Britain (Chevasco 2003), and that transatlantic publication of popular fiction was a “two-way” phenomenon (King 2004: 9). However, as one article in this issue shows, there is an entire world of cheap fiction beyond the Francophone and Anglophone. The Portuguese-speaking world, in particular, was ripe for the reception of penny fiction with European authors such as Sue and Reynolds directly referenced by authors in Brazil (see Basdeo and Guerra’s article in this volume). Comparably, in Portugal, where Camilo Branco continued the “mysteries” novel tradition with his Misterios de Lisboa, its sequel O Livro Negro de Padre Diniz – in which the mysterious priest from Misterios de Lisboa continues his campaign of writing wrongs in Lisbon’s high society – and Misterios de Fafe. Sue and Reynolds had their own, often not very favourable, reputations in France and Britain and there was, in the public mind at least, a clear demarcation between their works which were written 3 ISSN: 2632-4253 (online) 3 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) for the “lower orders” and those of Scott, Dickens, and Thackeray. The Portuguese and Spanish worlds, however, saw no such line demarcating the literature of Britain and France. This has obvious implications for scholars who seek to incorporate discussions of decolonisation and the decentring of European literary modes and canon into their discussions: penny fiction was not insular and confined solely to the Anglosphere but had a reach much broader than was previously known. Another question that some of the articles contained in this issue seek to grapple with is the following: What links penny fiction with other literary genres and phenomena? What is Penny Fiction? There are no straightforward answers here but they are questions that are worth thinking about if scholars are ever to meaningfully reappraise penny fiction in the Victorian world. The seven essays that comprise this special issue will start to respond to some of those important questions. “Bloods and Dreadfuls” Perhaps one of the only definitive answers we can arrive at is to say that prose fiction selling at a low price was popular with the Victorian reading public. “Mainstream” novels being expensive and retailing at a declared price of 31 shillings for their three-volume form, it is no wonder that many of them were imitated by writers eking out a living for the various penny publishers and later 4 ISSN: 2632-4253 (online) 4 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) reprinted. Today, critics tend to assume that the terms “penny dreadful” and “penny blood” have always been in use. “Throughout the history of cheap popular fiction marketed for the working- class,” one scholar claims, “the term ‘Penny Dreadful’ was the blanket title for all inexpensive, serialised tales of sensation fiction sold on London’s streets in penny parts,” while “due to the excessive amount of blood, gore, and hellish woodcuts each tale contained,” these works were also “labeled Penny Bloods” (Herr 2008: 11). However, much of this “history” is myth. It took some decades after penny fiction’s 1830s inception for critics to recognise it as a distinct literary entity, separate from cheap literature, penny papers, and crime fiction. As late as 1857, an editorial on “Cheap Literature as it Effects Authors and Publishers” in The London Miscellany struggled to define the contents – fictional and otherwise – of penny papers. The editorial ultimately settled upon “literature (the cost of which is within the reach of the poorest) available to the knowledge and understanding of the least educated of those who have lately learned the art of reading” (“Cheap Literature” 1857: 376). It took even longer for penny fiction to be explicitly demonised as the “penny blood” and “penny dreadful.” To be sure, cheap literature had always posed real threats to the status quo. As the Chartist writer Thomas Frost recalled, “[t]he first locomotive was not viewed with more fear and distrust than the first elementary school and the first penny periodical” (Frost 1880: 7). Initially, a major target of this fear was political radicalism. In an 1838 Fraser’s essay, a young Thackeray accused penny papers of luring working-class men into ginshops and brothels, and also of introducing them to the “cant … of the Radical spouters,” which he labels “hatred” (Thackeray 1838: 290). “Bloods and Dreadfuls” Yet one of the most successful publishers of penny newspapers and penny fiction in other formats, Edward Lloyd, denounced violent political action and promoted liberalism, thereby helping to build a mass liberal consensus (McWilliam 2019: 209-11). The London Journal, from the 1860s onwards “perceived as part of the ‘purified penny press” (King 2004: 29) became the indicative penny fiction weekly, “a metonym for the whole genre” (141). While the penny press acclimated people to news and pleasure reading during the growth of what Ian Haywood has called a radical “counter-public sphere,” this was deeply unsettling to the gatekeepers of British culture (Haywood 2004: 2-4). The middle-class reaction to this development was to blame cheap literature, crime news, and outlaw fiction for the crimes committed by working-class people, particularly after the 1840 execution of the valet Benjamin Courvoisier for the murder of his aristocratic employer. Infamously, Courvoisier had owned a copy of William Harrison Ainsworth’s Jack Sheppard, which proved suggested a cause and effect relationship between reading and action (Haugtvedt 2021: 78-9). Jack Sheppard was not a penny publication in itself, having debuted in the more expensive Bentley’s Miscellany, but it was frequently imitated in the penny press. Examples include Thomas White’s twelve-part version (c.1840) and Edward Leman Blanchard’s inventive Jack Sheppard; or, London in the Last Century, a Romance of Reality (c.1847). As we have seen, criticism such as Collins’s “The Unknown Public” and Oliphant’s “The Byways of Literature: Reading for the Million,” both published in 1858, exploited anxieties about the growth of a working-class reading nation, just as Thackeray had done two decades earlier. Meanwhile, penny fiction itself dramatically evolved, becoming, for a variety of reasons, less lurid, more adventurous, and geared explicitly towards boy readers (Springhall 1998: 41). This development did not alleviate the moral panic. In fact, the panic increased. The passage of the 1870 Education Act, which promised to increase the size of the working-class reading public, provoked further anxiety and saw the advent of the terms “penny blood” and ISSN: 2632-4253 (online) 5 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) “penny dreadful.” As John Springhall notes, an early instance of “penny dreadful” appears in John Camden Hotten’s Slang Dictionary of 1874 (Springhall 1998: 38). Hotten defines the “penny dreadful” as “a penny publication” that “depend[s] more upon sensationalism than upon merit, artistic or literary, for success” (Hotten 1874: 250). “Bloods and Dreadfuls” Primary source databases reveal that “penny blood” and “penny dreadful” both emerged slightly earlier (around 1870) and that “penny dreadful” was consistently the more popular term. ProQuest’s British Periodicals collection contains twelve periodical pieces that include the term “penny blood.” These are dated 1869–1933, but the earliest date is an outlier, accounting for only one article, which calls Reynolds a “penny blood-and-thunder novelist” (“Mr. Charles Lever” 1869: 301), referring to the sort of “vulgar oath” that Byron employs in Childe Harold (Byron 317 [Canto 8, line 1]). ProQuest British Periodicals also incorporates 465 mentions of “penny dreadful” dating between 1870 and 1926. The earliest appears in an 1870 editorial by Antony Trollope in St. Paul’s Magazine,. Trollope purports to reveal the despair of a writer employed at several “penny dreadful papers” ([Trollope] 1870: 669). Google NGram, which generates data visualisations of word frequencies in the Google Books corpus, produces similar results. According to Google NGram, “penny blood” and “penny dreadful” both appear circa 1870, with “penny dreadful” considerably outstripping the term “penny blood” (Fig. 1). In British Periodicals and Google NGram results, both “penny blood” and “penny dreadful” appear most frequently in content from the 1890s, a decade which, as Springhall documents, saw sensational court cases in which boy defendants were identified as readers of penny fiction (Springhall 1998: 10). Fig. 1: Google NGram Viewer, 2022. Fig. 1: Google NGram Viewer, 2022. ISSN: 2632-4253 (online) 6 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) This cultural history shapes the earliest scholarly studies of Victorian penny fiction, studies that assume that this literature is gratuitously sanguinary and associated with sexual transgression. An early bibliography of popular literature including “bloods” and “dreadfuls,” Montague Summers’s Gothic Bibliography (1940), found a printer only in London’s Fortune Press, which in 1934 had been subjected to an Obscenity Act police raid that turned up erotica misattributed to the still-notorious Oscar Wilde (Ladenson 2013: 125). In 1948, E.S. Turner (born 1909), a proud collector of penny fiction and of later-Victorian boys’ adventure books, called penny periodicals “the journals” that boys’ “parents did not want them to read.” He pronounced them to be typically consumed “in the privacy of bed” and “associated with a feeling of guilt” (Turner 1975: 11-16). “Bloods and Dreadfuls” Turner boasts that “[p]robably there is no product of the human brain which has been the object of such remorseless and misconceived abuse as the boy’s thriller,” for “on no theme have magistrates, clergymen and schoolmasters talked more prejudice to the reported inch” (11). This hyperbole makes penny fiction sound very much like erotica. It is no wonder, then, that from the late Victorian era until today, critics and scholars have characterised the authors of penny fiction as mysterious men confined to the shadows. In Trollope’s 1870 St. Paul’s piece, the supposed “penny dreadful” author had been rusticated from a Cambridge college for insubordinating a tutor. He lived in “dirty lodging” and abandoned his wife and children (“An Editor’s Tales” 1870: 670). “My life, of course, is a mistake,” he concluded (670). Turner calls Frank Richards/Charles Hamilton, “the creator of Billy Bunter … something of an enigma” and “the first man to have achieved an entry in Who’s Who under an alias” (Turner 1975: 8). Turner’s contemporary Arthur Waite recycled Oliphant’s 1858 terminology and declared in his unpublished bibliography The Quest for Bloods (abandoned c.1940) that “penny bloods” occupy the “byways” of literature (Waite 1997: 1). While his self-aggrandising explorationist hyperbole is demonstrably untrue, it reinforces the idea that penny fiction is not respectable. The belief that penny fiction was produced by seedy recluses persisted, for instance in the catalogue of a 1998 British Library exhibit showcasing the Barry Ono Collection of popular fiction. “At all periods” of the Victorian era, this catalogue claims, “the writers of penny fiction have tended to lead Bohemian lives which are difficult to chart accurately,” and “only a few” of them “seem to have been, or have become, conventional respectable citizens” (James and Smith 1995: xiv). The truth of this claim depends upon the meaning of “respectable:” Reynolds was a radical but also a public figure with a well-documented life. As Basdeo observes, penny fiction writer Pierce Egan the Younger, son of Pierce Egan the author of the comic bestseller Life in London (1822), also had radical political leanings, but was hardly obscure and generally considered a pillar of society. The Critical Tradition A paradigm shift in Victorian penny fiction scholarship occurred in the mid-twentieth century, led by British scholars of the New Left tradition. In this era, Britain saw intense debates about the nature of literature, the remit of literary studies, and the value of popular culture. T.S. Eliot presented a particularly limiting theory of literary value in his Notes Toward the Definition of Culture (1948), which maintained that “culture” excludes “the less developed culture of mass society,” and that organised religion constitutes the most robust “frame-work for a culture, and protects the mass of humanity from boredom and despair” (Eliot 1948: 60, 121, 34). ISSN: 2632-4253 (online) 7 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) Echoing Arnold almost a century earlier, Eliot defended class hierarchy as a safeguard of “culture” and insisted that “if Christianity goes, the whole of our culture goes” (1948: 126). Vociferously disagreeing with every point of Eliot’s manifesto, Raymond Williams, having newly graduated from Cambridge and teaching adult education modules for Oxford University, came up with a competing, Marxist-derived, theory of culture that seeded the movement that shortly thereafter came to be known as “cultural materialism.” “Culture is ordinary,” Williams insisted: popular culture was as worthy of study as the literary canon, which, in any case, was a subjective, constantly evolving entity (Williams 1989: 1). In 1963, the historian E.P. Thompson published The Making of the English Working Class, which, though many of its points have since been qualified and refined, remains a vital survey of Chartist and other radical movements that shaped the civic world of penny fiction’s readers, writers, and publishers. Stuart Hall and Paddy Whannell expanded upon Williams in The Popular Arts (1964). Here they argued – in what would come to be known as cultural studies – that popular art, ranging from music to movies, also deserved analysis as it could illuminate past and present cultural values and processes. In short, what the people read matters as much as what the elites read, as do genres of popular entertainment and information that do not fit easily into the major categories of poetry, stage drama, and literary fiction. From the same intellectual perspective, Louis James published his monograph Fiction for the Working Man: 1830-1850 in 1963. The Critical Tradition This study established for the first time that penny fiction was a fit subject for academic literary criticism, that its authors’ and editors’ aesthetics, ethics and economics were complex and well-thought-out, and that without knowledge of penny fiction, we really cannot claim to understand the Victorian imagination or the Victorian reading community. Written with a deep understanding of social history and a great respect for the eccentric collectors of penny literature who shared their libraries and homes with him, James writes of the “penny issue” novel and the “domestic[ity]” of much penny fiction, which, due to its hitherto sensationalist reputation, seemed a “paradox” at the time (James [1963] 2017: 114, 120-1). Since James’s study, more and more critics have begun to study penny fiction, and even teach it. A later seminal work is John Springhall’s Youth, Popular Culture, and Moral Panics (1998) which examines the moral panics about popular culture’s impact on youth, beginning with “penny dreadfuls” and the “penny gaffs” (minor theatre shows) often adapted from them, and tracing the phenomenon through the 1990s racist panics about “gangsta rap.”. Individual figures in Victorian penny fiction have attracted attention. For us the most significant is G.W.M. Reynolds, a figure of astonishing radical convictions and foresight. Reynolds features in anthologies, critical editions, research monographs and even blogs and popular articles. Other widely studied figures of the period include the publisher Edward Lloyd and the novelist James Malcolm Rymer. The two penny serials that have attracted the most critical attention were both written by Rymer in the 1840s for Lloyd: Varney the Vampyre and The String of Pearls. This was facilitated by two facsimile editions of Varney that made that text more accessible than it had been since the 1850s: one in a beautiful three-volume quarto edition by Devendra P. Varma (originally 1970, reprinted 1998) and a cheap set of two paperback volumes edited by E.F. Bleiler that came out in the same year. ISSN: 2632-4253 (online) 8 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) As for Sweeney Todd in The String of Pearls, Robert Mack’s The Wonderful History of Sweeney Todd: The Life and Times of an Urban Legend (2007) is a magisterial study of the urban mythological tradition that incorporates Rymer’s invention of the Barber of Fleet-street. 2 In 2002, Smith disambiguated Rymer and Prest’s writings in a pamphlet for the London antiquarian bookseller Jarndyce, establishing rudimentary Rymer and Prest canons and clarifying that Rymer was responsible for The String of Pearls (Sweeney Todd). In 2020, Nesvet further clarified the attribution question, documenting George Augustus Sala’s 1892 introduction of the Prest attribution by way of a gossip column never intended to be read as literary history. Criticism of The String of Pearls is growing at a rate that precludes comprehensive survey, but an important contribution is Maisha Wester’s “Text as Gothic Murder Machine: The Cannibalism of Sawney Beane and Sweeney Todd.” Wester argues that Todd and the Newgate Calendar villain Beane both function as Gothic “meaning machines,” articulating popular anxieties about racial, ethnic, and geographic others as well as ambivalence about the figuratively cannibalistic nature of urban English society during what the 20th century came to call “the Hungry Forties.” Anna Gasperini’s 2019 monograph on medical and anatomical controversies in Victorian popular fiction focuses with unusual depth and breadth on Rymer, considering the impact of the 1832 Anatomy Act and going beyond The String of Pearls and Varney to consider more obscure texts. 3 See also in this journal, Nesvet (2019). The Critical Tradition Several editions of The String of Pearls (1846–7 serial version) exist, edited by Mack (2007), Dick Collins (2005, helpfully updated 2010), and Rohan McWilliam (2015).2 Scholarship on Chartism, beginning with the British Marxist classics of the late twentieth century, has enabled fascinating work on the intersections between Chartism, other forms of radicalism, and Victorian penny fiction. Troy Boone correctly identifies Chartism as a major topic of Rymer’s Varney, the Vampyre’s political vision. Like some Chartist leaders, Boone argues, Varney promotes nonviolent political agitation to working-class readers, especially children. A more recent high contribution is Rohan McWilliam’s article “Sweeney Todd and the Chartist Gothic: Politics and Print Culture in Early Victorian Britain” (2019). McWilliam usefully distinguishes the “Chartist Gothic” (his coinage) ideology of Reynolds’s romances from Lloyd’s far more conciliatory liberalism. Pointing out the middle-class heroes of The String of Pearls and its refusal to demand political change, McWilliam persuasively contends that unlike Reynolds’s Chartist Gothic works, it is no radical text. Taking this idea further, Rob Breton’s monograph The Penny Politics of Popular Fiction (2021), significantly informed by Gregory Vargo’s An Underground History of Victorian Popular Fiction (2018), persuasively contends that the palpably radicalised public sphere of the Chartist era forced penny publishers including Lloyd and Reynolds to engage with radical culture, in a diverse array of manners.3 The exciting critical renaissance in penny fiction studies is indebted to the Victorian Popular Fiction Association and the Research Society for Victorian Periodicals, both of which have expanded the field of Victorian literary studies to incorporate multimodal, non-elite penny fiction. Meanwhile, independent presses such as Victorian Secrets, Valancourt, and Edwin Everett Root are introducing penny literature to a new generation of readers. Databases such as Nineteenth Century Collections Online and ProQuest British Periodicals have made many of the primary sources newly accessible to members of institutions with the funds to subscribe to them. Meanwhile, the open access bibliographic database Price One Penny (www.priceonepenny.info) has achieved its tenth anniversary and is still being expanded. What Earhart calls the New Historicist inflected “Era of the Archive.” whereby multimodal archives of disparate materials have, from the 1990s onwards, displaced linear, text-based editions, become the 9 9 ISSN: 2632-4253 (online) Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) dominant means by which American digital humanities scholars encounter material (Earhart 2015: 38-9). The Critical Tradition One example of this kind of curated archive relevant to Victorian Popular Fictions publishes both criticism and rare images of illustrated penny fiction in their original publishing contexts: John Adcock’s personal blog Yesterday’s Papers, which commenced in 2008, draws extensively upon Adcock’s personal collection to cover penny fiction from the 1840s through the turn of the century. More recently, the blog Reynolds’s News and Miscellany by Basdeo and his collaborators explores and meticulously contextualises radical culture, journalism, and penny fiction from the 1840s-1890s. Again, this offers invaluable facsimiles of items from the author’s extensive personal library as well as public-domain source (Basdeo and Thomas 2021). Resources such as these open access private enterprises, along with the larger subscription-based archives, will facilitate further work on penny fiction – a grass roots addition to outputs from multinational publishers that needs to be done in part because the myths about penny fiction, begun in the nineteenth century, linger in critical discourse. This special issue of Victorian Popular Fictions aims to perform a small part of that much-needed reappraisal and to inspire more to come. New Readings Brooke Cameron’s article “Domestic Plots and Class Reform in Varney the Vampire” fills that gap, by reading Varney’s serial marriage plots as a critique of bourgeois models of marriage promoted in the 1840s via controversial public health campaigns. Building upon research on marital, sexual, and sanitary rhetoric in Victorian urban life, Cameron shows that Varney resists the marriage plots that the Victorian establishment tried to foist upon working-class people in fiction and real life. Cameron’s article not only questions how we read Varney’s numerous marriage plots but also complicates the history of the novel. Read in relation to Edwin Chadwick’s 1842 sanitary report, Varney offers refreshing pushback to authoritarian attempts to legislate working-class mores. This is only one of the ways in which close reading and the recontextualising of penny fiction can lead us to reconsider both popular fiction more broadly and the history of the novel itself. Sara Hackenberg participates in a similar process of reconsideration and reappraisal in her article “The Magician of Civilised Life”: The Literary Detective in Mary Elizabeth Braddon’s Early Penny Fiction.” As she points out, penny fiction is the precursor of the later detective novel. In Rymer’s Ada the Betrayed and The String of Pearls, magistrates collaborate with heroines to conduct investigations that ultimately uncover conspiracy and murder. The pseudonymous ‘Edward Ellis’s’ serial Ruth the Betrayer, or, the Female Spy, printed in 1862–3 by Reynolds’s publisher John Dicks, imagined a woman police detective long before the London Metropolitan Police ever hired such a person (Bredesen 2019). Braddon is now acknowledged as a forerunner of crime fiction, though not as consistently as she might be. Hackenberg observes Braddon’s characterisation of the nascent figure of the “detective police officer” and, reading Braddon’s corpus longitudinally, shows how she reinvents the City Mysteries genre popularised by Sue and Reynolds to “personify” as the central “mystery” the figure of the “most powerful plotter.” In the process she elevates his nemesis, the police detective. We need not wonder why in A Study in Scarlet, Sherlock Holmes, an adolescent in the 1860s, was particularly drawn to ‘sensational literature’ (Doyle 1898: 91). By the following decade, well into the era of the boys’ adventure periodical, British children had a wealth of penny fiction from which to choose their reading. Competition between publishers of penny papers that targeted children became intense. Madeline P. New Readings The seven articles that follow reappraise penny fiction in ways that both expand upon the critical tradition and challenge or refine it. To begin with, penny fiction is fiction: this is what distinguishes it from the other offerings of the Victorian penny press, including news serials and other kinds of periodic columns. The fictional nature of penny romances and tales might cause us to forget that they very often promised to reveal the real or actuelle: in other words, history or news. Sophie Raine’s “‘Founded on Fact’: Paratextual Politics in the Penny Dreadful” recentres penny fiction’s facticity. Pointing out that Rymer observed that apparent “truthfulness” makes fiction popular, Rymer documents the marketing of penny fiction via paratexts which emphasise how a text is “founded on fact,” thus transforming fiction into an apparent truth. Raine finds that what Gary Kelly refers to as the “footnote novel” – a form used by Romantic-period writers which incorporated “factual, public, and political material” (1993, 157) – resurfaces in penny fiction. Raine argues that the penny novelists used apparently scholarly references to create authorial credibility and reinforced it by establishing a meaningful relationship between the author and the reader. Moving from aesthetics to ethics, this special issue explores penny fiction’s politics as advanced by writers including James, Humpherys, McWilliam, and Breton. The same authors who strove to describe the world as it was also wished to change some aspects of it, a wish they shared with the palpably radicalised working-class audience of the Chartist era. Readings of the political contexts of penny fiction have been plentiful in recent years, helpfully establishing that penny fiction was neither escapist nor apolitical. However, no attention has been paid to the name- dropping of a major Chartist figure, William Lovett, author of the Charter, in the most famous penny serial. In “What’s in a Name? Mr. and Mrs. Lovett and The String of Pearls,” Rebecca Nesvet considers what this name’s invocation as that of the villainous yet nonviolent Mrs. Lovett might have meant to its primary author, Rymer, and its original audience. ISSN: 2632-4253 (online) 10 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Political readings of penny fiction often evade questions of gender, such as those explored by Anna Clark in The Struggle for the Breeches (2005). New Readings Gangnes examines this exigency in her article ‘Editor, Reader, and Value for Money in Young Folks’. In one of the most successful boys’ papers, Young Folks, which introduced readers to Stevenson’s Treasure Island and Kidnapped, the editorial team of James Henderson and his staff constantly revised their rhetorical strategies in order to remain appealing to his boy audience. Gangnes traces the evolution of Young Folks as its editor pursues a delicate balance of “value for money” in the eyes of children over more than a decade of the fin-de-siècle.Equally drawn to penny fiction were writers and readers from well beyond Britain Scholars such as Rebecca S. Wingo (2017), Pamela Bedore (2013), and Yu-Fang Cho (2007) have done important work on the “dime novels” of late nineteenth-century America; especially on dime fiction’s complicity with myth-building about the phantasmic “American West” and the indigenous people(s) whom myths of the West in the era of Wounded Knee marginalised and misrepresented. In “Young America: Dime Novels and Juvenile Authorship,” Laurie Langbauer in the present issue goes back to the 1830s and 1840s to demonstrate that dime novels incorporated nationalist narratives and projects that scholars have traditionally associated only with adult-literature, or, to put it another way, so-called “serious” fiction. Langbauer grounds the emergence of the dime novel in the “Young America” movement 11 ISSN: 2632-4253 (online) 11 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) galvanised by the New York publishing baron Evert Duyckinck. Although Duyckinck died before the dime novel’s heyday, his influence endured. “Wide-awake” youth – supposedly revolutionary young thinkers, revolutionary because they were young – contributed to the dime papers, which were highly interactive in ways not yet acknowledged by scholars. They invented a new mode of authorship that encapsulated what Duyckinck thought it meant to be American. This aspect of dime fiction’s origins-story shifts both that body of literature and youth authorship from the margins of American literature to the forefront. Additional instalments of Langbauer’s larger project, on youth, agency, race, nation, and nationalism in American dime novels, are scheduled to appear in The Journal of Juvenilia Studies in 2023 and 2024, so, as so much penny fiction has said, this investigation is “to be continued in our next.” Stephen Basdeo and Luiz F.A. Guerra’s article takes us to the Empire of Brazil. New Readings The nineteenth-century British and Brazilian empires were in many ways similar in that they were constitutional monarchies with a parliamentary system dominated by Liberal and Conservative parties. Both boasted a thriving literary culture comprising periodicals, newspapers, chapbooks, and books, and there was a distinction between “cheap” books and expensive ones. Unsurprisingly there was some cultural exchange between the two empires. The British had a significant presence in Rio de Janeiro, while one of Brazil’s most prominent Romantic poets, Álvares de Azevedo, looked to the likes of Lord Byron, Walter Scott, and Percy Shelley as examples of artistic brilliance and admired British statesmen such as Robert Peel for their political brilliance (de Azevedo 1849/ 1942: 406). It is in this cultural context that Basdeo and Guerra investigate the reach of Reynolds’s Mysteries of London and Sue’s Mysteres de Paris, in the form of what might be called a loose adaptation and extension of the mysteries tradition by Juana Manso titled Misterios del Plata (1852). Serialised in the women’s periodical O Jornal das Senhoras and paying homage to Sue and Reynolds in the introduction, Manso’s tale is a radical story of female agency which sees a young woman fight against the despotic regime of the Argentine dictator Juan Rosas. Basdeo and Guerra show how an understanding of Manso’s text, and a discussion of the several Latin American mysteries novels that were published around the same time, has implications for the study of Victorian popular literature as a whole. As several previously published pieces in this journal have shown, it is now time for Victorian Popular Literature to join the discussion of ‘global’ world literatures and across languages. Once more voyaging to the USA, we conclude our reappraisal of penny, dime, and other cheap fiction with a very personal account: a miniature memoir of sorts. Fans of Sweeney Todd, in any of his kaleidoscopic array of transmedia forms, will know Robert L. Mack’s edition of The String of Pearls – the first critical edition of that important work of penny fiction – and his interdisciplinary – and diabolically fun – excavation of the Sweeney Todd phenomenon, The Wonderful History of Sweeney Todd: The Life and Times of an Urban Legend (2007). In this issue’s “coda” entitled “Confronting the ‘Real’ Sweeney Todd: A Journey of Discovery,” Mack returns to his personal introduction to the legend. “Such Style, Such Flair” In the Dr. Who episode “Shada,” written in 1979 by cult novelist Douglas Adams but never broadcast in full, the Doctor and his companion Romana visit a time-traveling Cambridge don. In Professor Chronotis’s rooms at St. Cedd’s College, the adventurers search for a dangerous book. Rumoured to have the power to destroy the world, it must never be read. The Doctor and Romana frantically rifle through Chronotis’s shelves. They do not find this book, but instead they discover a copy of “Sweeney Todd.” The Doctor reflects on his childhood adoration: “He was a boyhood hero of mine,” the Doctor reflects. The demure Romana is surprised: “A great criminal, your hero?” “Oh yes,” the Doctor confesses, “But he had such style, such flair … Like me.” This space-age Jack Sheppard’s appeal to Time Lords and humans alike reveals why penny fiction has proved compelling to generations: always disreputable, its instantly recognisable aesthetic has “a flair” that we would like to think we continue to emulate. Waite saw its storytelling surviving in the movies and he was certainly correct with regard to adaptations of Reynolds in his own era; a silent film produced in 1915 – the only surviving copy of which has Dutch subtitles – was titled The Mysteries of London and related a scene from Reynolds’s Mysteries of the Court of London (Coleby 1915). In India, where Reynolds garnered great respect (Bhattacharya 2008), an adaptation of his novel Leila: The Star of Mingrelia was, on its release in 1931, the most expensive Indian movie produced at the time. Quite pertinently for the point about moving pictures as successors to serialised penny fiction, the poster for this movie advertised it as “a serial in 20 reels” over “2 chapters.” Given penny fiction’s seriality, television and podcasts are more likely successors. John Logan, screenwriter of the 2007 Hollywood film Sweeney Todd, created the Showtime and Sky television show Penny Dreadful (2014-17). This award-winning show takes place in 1890s London and features well-known characters from the nineteenth century literary canon, including Frankenstein and his Creature, a vampire, and Dracula’s Mina Harker. These characters interact with updates of other pop cultural icons such American werewolf Ethan Lawrence Talbot, borrowed from Universal Studios’ 1941 film The Wolf Man (Manea 12). None of Penny Dreadful’s principal characters derive directly from penny fiction texts, but some aspects of the penny fiction phenomenon feature prominently. New Readings Like so many Victorians, he first met Todd in the live theatre, though, in his case, he met Todd in Stephen Sondheim and Hugh Wheeler’s musical Sweeney Todd, The Demon Barber of Fleet-Street: A Musical Thriller, when it premiered at Broadway’s Uris Theatre in 1979. The musical was not an immediate hit and Mack documents the critical confusion that it met before charting how, via this particular adaptation, Sweeney Todd garnered a truly global audience for the first time and began to be reinvented as a historical figure, which he most decidedly was not. ISSN: 2632-4253 (online) 12 Volume 4: Issue 2 (Autumn 2022) Victorian Popular Fictions “Such Style, Such Flair” One example is the show’s “plunder[ing]” of London’s “popular culture archive” (Louttit 3). Another is Penny Dreadful’s continual insistence that “the social life of the city is a performance and that those around us may be harbouring mysterious inner lives” (8), which is obviously indebted to the urban mysteries genre. Also perhaps reminiscent of Reynolds’s Mysteries of London is Penny Dreadful’s exploration of “dual social realities” (Akilli and Öz 18) by contrasting elite and “impoverished and the marginalized” Londoners’ life experiences (Manea 44). Scholars observe that Penny Dreadful inhabits (Louttit 3) yet “historicises” (Manea 44) the cinematic setting “Horror London,” to which the Sweeney Todd transmedia phenomenon has contributed. In 2020, Logan and his co-producers created the show Penny Dreadful: City of Angels, a “spiritual descendant” of the original Penny Dreadful (Horton). Penny Dreadful: City of Angels takes place in 1938 Los Angeles and depicts such actual evils as racist policing and the proliferation of Fascism. Like penny fiction, Penny Dreadful found ISSN: 2632-4253 (online) 13 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) new life across the Atlantic. However, even this spinoff is riddled with tropes drawn from Victorian penny fiction. The hero, Los Angeles Police Detective Santiago Vega, the force’s first Chicano detective, struggles with his brother, a labour organiser. The antagonistic brothers’ duality recalls that of the brothers Markham and Montague in Reynolds’s The Mysteries of London. Additionally, in a situation anticipated in many examples of Victorian penny fiction, the Nazi physician Dr. Craft schemes to have his perfectly sane wife committed to a mental asylum. The success of the Penny Dreadful television franchise demonstrates that a Victorian mode of popular storytelling can be appropriated to realise any imaginable chronotrope and can resonate powerfully with audiences around the world. Clearly, penny fiction should be more central to Victorian studies than historically it has been. In any case, now that we know what the vast majority of Victorian readers enjoyed reading, we can no longer shut the door and pretend that it is an undiscoverable arcana. Sweeney Todd and his ilk are here to stay. Boone, Troy. 2004. Youth of Darkest England: Working-Class Children at the Heart of Victorian Empire. New York: Routledge. Volume 4: Issue 2 (Autumn 2022) sen, Dagni, ed. 2019. Ruth the Betrayer, or, the Female Spy. London: Valancourt Books. Breton, Rob. 2021. The Penny Politics of Popular Fiction. Manchester: Manchester University Press. Byron, George Gordon, Lord. 1977. Don Juan, edited by T.G. Steffan, E. Steffan, and W.W. Pratt. London: Penguin. “Cheap Literature, as it Effects Authors and Publishers.” 1857. The London Miscellany, 14.1: 376. Chevasco, Berry Palmer. 2003. Mysterymania: The Reception of Eugène Sue in Britain, 1838–1860. Bern: Peter Lang. Cho, Yu- Fang. 2007. “A Romance of (Miscege)Nations: Ann Sophia Stephens’ Malaeska: The Indian Wife of the White Hunter (1839, 1860).” Arizona Quarterly, 63.1: 1-25. Clark, Anna. 1997. The Struggle for the Breeches: Gender and the Making of the British Working Class. Oakland, CA: University of California Press. Coleby, A. E. dir. N.d. [1915]. The Mysteries of London. United Kingdom: Martin’s Exclusives. The British Film Institute BFI Player. https://player.bfi.org.uk/free/film/watch-mysteries-of-london-1915-online Collins, Dick, ed. 2010. Sweeney Todd (The String of Pearls). Ware: Wordsworth. Collins, Wilkie. 1858. “The Unknown Public.” Household Words, 18.439, 217-22, extracted in Victorian Print Media: a Reader, edited by Andrew King and John Plunkett, 207-216. Oxford: Oxford University Press. de Azevedo, Álvares. [1849] 1942. ‘Discurso: Recitado no dia 11 de agosto de 1849 na sessão academica, commemoradora do anniversario da criação dos cursos juridicos do Brasil’. In Obras completas de Álvares de Azevedo, ed. Homero Pires, 2 vols, 399-416. Rio de Janeiro: Companhia Editora Nacional. Doyle, Arthur Conan, Sir. [1878] 1898. A Study in Scarlet: A Novel. New York and London: Street and Smith. Earhart, Amy L. 2015. Traces of the Old, Uses of the New: The Emergence of Digital Literary Studies. Ann Arbor: University of Michigan Press. Elliott, T.S. 1948. Notes Towards the Definition of Culture. New York: Harcourt Brace. Frost, Thomas. 1880. Forty Years' Recollections: Literary and Political. London: Sampson Low, Marston, Searle, and Rivington. Gasperini, Anna. 2019. Nineteenth Century Popular Fiction, Medicine and Anatomy: The Victorian Penny Blood and the 1832 Anatomy Act. London: Palgrave. Geier, Ted. 2017. Meat Markets: A Cultural History of Bloody London. Oxford: Oxford Un l, Stuart and Paddy Whannell. 1964. The Popular Arts. London: Hutchinson International. Haugtvedt, Erica. 2021. “Class and Complex Transmedia Character in Jack Sheppard (1838-9).” Victorian Popular Fictions Journal, 3.2: 78-9. DOI: https://doi.org/10.46911/oyym7051 Haywood, Ian. 2004. The Revolution in Popular Literature: Print, Politics, and the People, 1790-1860 Cambridge: Cambridge University Press. Herr, Curt, ed. 2008. Varney, the Vampyre, or the Feast of Blood. Humpherys, Anne. 2016. “John Dicks’s Cheap Reprint Series, 1850s-1890s: Reading Advertisements.” In Media and Print Culture Consumption in Nineteenth-Century Britain: The Victorian Reading Experience, edited by Paul Raphael Rooney and Anna Gasperini, 93-110. London: Palgrave. Bibliography Adams, Douglas. 2013. Doctor Who: Shada. [DVD.] BBC Video. Adams, Douglas. 2013. Doctor Who: Shada. [DVD.] BBC Video. Adams, Douglas. 2013. Doctor Who: Shada. [DVD.] BBC Video. Adcock, John. Yesterday’s Papers. 2008–. http://john-adcock.blogspot.com/2017/08/three-centuries-of- penny-dreadfuls.html Adcock, John. Yesterday’s Papers. 2008–. http://john-adcock.blogspot.com/2017/08/three-centuries-of- penny-dreadfuls.html “Advertisement.” In Olivia Serres. 1885 [1832]. The Secret History of the Court of England. “People’s Edition.” London: John Dicks. Ainsworth, William Harrison. c.1840. The Eventful Life and Unparalleled Exploits of the Notorious Jack Sheppard, the Housebreaker. London: Thomas White. Akilli, Sinan and Seda Öz. 2016. “‘No More Let Life Divide’: Victorian Metropolitan Confluence in Penny Dreadful.” Critical Survey 28.1: 15-29. DOI: https://doi.org/10.3167/cs.2016.280103 Autrey, Max L. 1975. “A Source for Roger Chillingworth.” American Transcendental Quarterly, 26: 24-6. Basdeo, Stephen, and Jessica Elizabeth Thomas. 2021. “British Library Selects Reynolds’s News and Miscellany for Inclusion in Digital Archive.” Reynolds’s News and Miscellany: History, Literature, Poetry, and Literary Criticism, https://reynolds-news.com/2021/08/11/reynolds-news-british- library-stephen-basdeo Basdeo, Stephen. 2018-. Reynolds’s News and Miscellany: History, Literature, Poetry, and Literary Criticism. https://reynolds-news.com/ Basdeo, Stephen. 2020. “The First British Edition of Nathaniel Hawthorne’s “Feathertop” in Home Circle,” Nathaniel Hawthorne Review, 46.2: 207-12. DOI: https://doi.org/10.5325/nathhawtrevi.46.2.0207 Bhattacharya, Sucheta. 2008. “G.W.M. Reynolds: Rewritten in Nineteenth Century Bengal.” G.W.M. Reynolds: Nineteenth-Century Fiction, Politics, and the Press, edited by Anne Humpherys and Louis James, 249-60. Abingdon: Routledge. Bedore, Pamela S. 2012. Dime Novels and the Roots of American Detective Fiction. Basings S. 2012. Dime Novels and the Roots of American Detective Fiction. Basingstoke: Palgrave Bedore, Pamela S. 2012. Dime Novels and the Roots of American Detective Fiction. Basingstoke: Palgrave. Blanchard, Edward Litt Leman. c.1847. Jack Sheppard; or, London in the Last Century, a Romance of Reality. London: G. Mansell. Blanchard, Edward Litt Leman. c.1847. Jack Sheppard; or, London in the Last Century, a Romance of Reality. London: G. Mansell. Bleiler, E.F., ed. 1970. Varney, the Vampyre, or, the Feast of Blood. 2 vols. London: Dover. Boone, Troy. 2004. Youth of Darkest England: Working-Class Children at the Heart of Victorian Empire. New York: Routledge. 14 ISSN: 2632-4253 (online) Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) Camarillo: Zittaw Press. Horton, Adrian. 2020. “Penny Dreadful: City of Angels Review: Magnetic but Messy Escapism.” The Guardian 23 April. https://www.theguardian.com/tv-and-radio/2020/apr/23/penny-dreadful-city-of- angels-review-los-angeles Hotten, John Camden. 1874. The Slang Dictionary: Etymological, Historical, and Anecdotal. London: Chatto and Windus. Humpherys, Anne. 2016. “John Dicks’s Cheap Reprint Series, 1850s-1890s: Reading Advertisements.” In Media and Print Culture Consumption in Nineteenth-Century Britain: The Victorian Reading Experience, edited by Paul Raphael Rooney and Anna Gasperini, 93-110. London: Palgrave. ISSN: 2632-4253 (online) 15 Victorian Popular Fictions Volume 4: Issue 2 (Autumn 2022) Volume 4: Issue 2 (Autumn 2022) James, Elizabeth, and Helen R. 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https://openalex.org/W2560031281	http://old.scielo.br/pdf/rod/v67n4/2175-7860-rod-67-04-0871.pdf	English	null	Floral biology of Sesbania virgata: an invasive species in the Agreste of Paraíba, northeastern Brazil	Rodriguésia	2,016	cc-by	4,479	1 This work is a part of the Thesis of the first author presented at PPGA/CCA/UFPB. 2 Universidade Federal da Paraíba, Depto. Ciências Básicas e Sociais/CCHSA, Campus III, 58220-000, Bananeiras, PB, Brazil. 3 Universidade Federal da Paraíba, Depto. Fitotecnia e Ciências Ambientais/CCA, Campus II, 58397-000, Areia, PB, Brazil. 4 Universidade Federal da Paraíba, Depto. Engenharia e Meio Ambiente/CCAE, Campus IV, R. da Mangueira s.n., Centro, 58297-000, Rio Tinto, PB, Brazil. 5 Author for correspondence: venia_camelo@hotmail.com Resumo A reprodução parece ser uma parte importante da estratégia de invasão bem-sucedida de Sesbania virgata. Apesar disso, a sua biologia floral é pouco estudada. O objetivo do nosso estudo foi preencher esta lacuna no conhecimento estudando a biologia floral de uma população de S. virgata no agreste brasileiro. Sesbania virgata é um arbusto que apresenta inflorescências racemosas. Suas flores são zigomorfas, pedunculadas e hermafroditas com corola amarelo-clara, androceu diadelfo, anteras com deiscência longitudinal, e estigma inconspícuo. As flores de Sesbania virgata apresentam três diferentes tipos de pétalas: o estandarte (11,92 mm), as alas (8,27 mm) e a quilha (9,3 mm). A maior intensidade do período de floração ocorreu entre novembro e janeiro. A média de grãos de pólen por flor foi de 22.918, com viabilidade polínica de 99,93%. A antese é diurna e assincrônica. Embora S. virgata seja autocompatível, a razão pólen/óvulo sugere a presença de um sistema reprodutivo misto (xenogamia + autogamia) nesta planta. A floração contínua, a morfologia floral, e o sistema reprodutivo especializado são indispensáveis para garantir o sucesso reprodutivo desta espécie no habitat estudado. A sua floração contínua e grande população faz de S. virgata uma fonte de recursos para os visitantes florais. Portanto, esta espécie apresenta uma efetiva estratégia reprodutiva que garante seu sucesso como espécie invasora. Palavras-chave: sistema reprodutivo, polinização, Fabaceae. Rodriguésia 67(4): 871-878. 2016 http://rodriguesia.jbrj.gov.br DOI: 10.1590/2175-7860201667402 Rodriguésia 67(4): 871-878. 2016 http://rodriguesia.jbrj.gov.br Rodriguésia 67(4): 871-878. 2016 http://rodriguesia.jbrj.gov.br DOI: 10.1590/2175-7860201667402 Floral biology of Sesbania virgata: an invasive species in the Agreste of Paraíba, northeastern Brazil1 Vênia Camelo de Souza2,5, Leonaldo Alves de Andrade3, & Zelma Glebya Maciel Quirino4 Vênia Camelo de Souza2,5, Leonaldo Alves de Andrade3, & Zelma Glebya Maciel Quirino4 Abstract Although reproduction seems to play an important role in the successful invasive strategy of Sesbania virgata, its floral biology is poorly known. We aimed at filling this gap by studying the floral biology of a population of S. virgata in the Agreste of Brazil. Sesbania virgata is a shrub that bears racemose inflorescences. Its flowers are zygomorphic, pedunculate, hermaphrodite, and have a pale yellow corolla, diadelphous androecium, anthers with longitudinal dehiscence, and inconspicuous stigma. S. virgata flowers display three different types of petals: banner (11.92 mm), wing (8.27 mm), and keel (9.3 mm). The flowering peak occurred from November to January. The average number of pollen grains per flower was 22,918, with a pollen viability of 99.93%. Its anthesis is diurnal and asynchronous. Although S. virgata is autogamous, its pollen/ovule ratio suggests that this plant has a mixed reproductive system (xenogamy + autogamy). Continuous flowering, flower morphology, and specialized breeding system are essential to assure the reproductive success of this species in the studied habitat. Sesbania virgata is a food source for flower visitors, due to its continuous flowering and large population. Therefore, it has an effective reproductive strategy that assures its invasion success. Keywords:breedingsystem pollination Fabaceae Key words: breeding system, pollination, Fabaceae. 5 Author for correspondence: venia_camelo@hotmail.com Introduction species. It plays an important role as a floristic element in several vegetation types, mainly within the Atlantic Forest, where Fabaceae species stand out for their diversity and abundance (Lima 2000). Despite the richness and ecological Fabaceae is the third largest and the second most economically important family of Angiosperms (Judd et al. 2009). In Brazil, the family is represented by ca. 190 genera and 2,100 872 Souza, V.C. et al. Souza, V.C. et al. importance of Fabaceae in Brazil, only a few studies have investigated the pollination biology and reproduction of this family (see Agostini et al. 2006; Nogueira & Arruda 2006; Guedes et al. 2009; Leite & Machado 2009). This situation is especially true for the invasive species of this family. Leite & Machado 2009). Such studies comprised mainly invasive species whose breeding systems take part in the invasion strategy. One example is Raphiodon echinus, typically found in the Sertão of Pernambuco, especially in temporarily flooded areas, road margins, and abandoned crops. Data available suggest that R. echinus is autogamous, but needs pollinators to secure its reproductive success (Dias & Kill 2007). Sesbania virgata (Cav.) Pers. is commonly used in restoration programs due to its aggressiveness in terms of growth rate and ability to quickly cover up the soil. This species is native to several areas of Brazil. It occurs mainly in pioneer riparian vegetation of midwestern and southeastern regions (Carpanezzi & Fowler 1997; Braggio et al. 2002), in Brazilian Savanna (Cerrado) and Atlantic Forest areas (Braggio et al. 2002). The breeding system of each species is crucial to assure the perpetuation of its descendants and enable the colonization of new habitats and the occurrence of natural evolutionary processes. Analyzing floral biology and pollination mechanisms is very important (Silva & Pinheiro 2007), especially in the case of species with invasive potential. In this sense, knowledge of floral morphology, pollination, and breeding mechanisms will contribute to set the ground for the control and management of this species. The present study, therefore, aimed at analyzing the floral biology of S. virgata in a population located in northeastern Brazil, to help understand how reproduction integrates the species invasion strategy. The biological traits of invasive plants influence their colonization success, and studying their reproductive system is particularly important for their management (Blanco 1972; Estes et al. 1983). Study area The present study assessed a natural population of Sesbania virgata in the municipality of Areia, state of Paraíba, northeastern Brazil (06o58’04”S, 35o41’33”W, 527 m a.s.l.). The area is located in a region known as Brejo Paraibano (ca. 269,424 km2) (IBGE 2011). The climate is mild, with average annual temperatures ranging from 22 to 26oC. It has a rugged relief where open rainforests predominate (IBGE 1992), and highland swamps are also found. The occurrence of S. virgata as invasive in the Brazilian Caatinga is somewhat recent, dating back to no longer than three decades (Andrade 2006). The preferential sites for the species within this biome are riparian forests around reservoirs (Andrade 2013). This species is also considered invasive in other regions of Brazil, and has a negative effect on the structure and diversity of native plant communities in all three study sites along riparian forests of the Paraiba River. In Area II, Salgado de São Félix, state of Paraíba, S. virgata had a higher value of importance for the shrub-arboreal layer (Souza 2012). Introduction However, Pyšek & Richardson (2007) stated that the role of plant traits in biological invasion is highly stage- and habitat-specific. When invasive species are able to set flowers and fruits in human-modified habitats, even in the absence of pollinators, in-depth studies are strongly needed. Self-fertilization assures the perpetuation of those species and thus also plays a role in biologic invasion. Species description Sesbania virgata is a shrub that reaches up to 6 m in height, 25 cm in diameter at breast height (DBH), and 5 m in crown width. It has a short lifespan (no longer than eight years) and a moderate ability to compete with grasses and resprout from stump after pruning or fire. It grows spontaneously on humid soils and can be associated with Rhizobium (Carpanezzi & Fowler 1997). Its fruits are nucoid, indehiscent, their color changes from green to opaque brown during the ripening, and they show a dry texture associated with rough skin. Their average values are 5.81 mm in length, 7.82 in width, and 5.81 in thickness. The seeds (ca. Studies on other invasive plants have associated invasion success with reproductive strategy, which is consistent with Williamson & Fitter (1996). For instance, characteristics such as independence of pollinators, large fruit set, and fast fruit ripening can facilitate the permanence of another plant species, Ipomoea eriocalyx, in invaded environments. Studies on floral biology, phenology, pollination mechanisms, and breeding systems in species of the Caatinga have been carried out since 1990 (Machado & Lopes 2002; Rodriguésia 67(4): 871-878. 2016 873 Biologia floral da espécie invasora Sesbania virgata (Cav.) Pers. sampled ten individual plants for analyzing their floral biology. 4.44 per fruit) are reniform, light chestnut-brown with a smooth, glossy, and hard seed coat. Their average values are 6.87 mm in length, 4.4 mm in width, and 3.22 mm in thickness. The average weight of 1,000 seeds is 7.78 g. The germination is epigeal, and the seedling bears proto-leaves composed of 4–9 leaflets and a sub-woody, white or brown radicle (Araujo et al. 2004). For the analysis of floral morphology, we fixed the flowers in ethanol 70% and took them to the Laboratory of Plant Ecology at the Campus IV of the Federal University of Paraiba (UFPB) at Rio Tinto. The data recorded included: flower morphology, flower biometry, anthesis, breeding system, pollen viability, number of pollen grains, and flower visitors. We measured the size of the floral elements of 30 flowers (3 flowers per plant, ten plants) with a digital caliper. We collected Data collection Data collection Data collection and analysis were carried out from April to December 2010. We randomly Rodriguésia 67(4): 871-878. 2016 Figure 1 – Pollination experiments carried out in Areia, state of Paraíba, northeastern Brazil – a-b. fruit set; c. bagged flowers; d. flower marking. a b c d a a d b b c d d b c Figure 1 – Pollination experiments carried out in Areia, state of Paraíba, northeastern Brazil – a-b. fruit set; c. bagged flowers; d. flower marking. Rodriguésia 67(4): 871-878. 2016 Souza, V.C. et al. 874 inflorescences in the field. Next, we took the inflorescences to the Laboratory of Botany at the Agricultural Sciences Center, municipality of Areia, state of Paraíba, where we analyzed their flower morphology under a stereomicroscope. Rodriguésia 67(4): 871-878. 2016 Pollen viability and pollen/ovule ratio Pollen viability and pollen/ovule ratio To estimate the pollen viability, we removed the anthers of ten pre-anthesis flower buds and stained them with acetic carmine 2% (Radford et al. 1974). We considered viable the pollen grains stained in red. We performed pollen grain counting in a Neubauer chamber and determined pollen/ ovule ratio following Cruden (1977). Although the pollen/ovule ratio per se does not prove that a species is autogamous, it corroborates this hypothesis. Anthesis Results and Discussion Characteristics of the species Sesbania virgata is a shrub that bears raceme inflorescences. Its flowers are zygomorphic, pedunculate, hermaphrodite, and show a pale yellow corolla, diadelphous androecium bearing anthers with longitudinal dehiscence, and inconspicuous stigma. In S. virgata flowers, three types of petals can be easily distinguishable: banner (11.92 mm), wings (8.27 mm), and keel petals (9.3 mm). The average number of pollen grains per flower was 22,918, which have high viability: 99.93% (Tab. 1). Brito et al. (2010) studied the reproductive biology of other Fabaceae species and also reported high pollen viability: 97% in Sophora tomentosa and 99% in Crotalaria vitellina. According to Etcheverry et al. (2008), the flowers of Fabaceae species show an intricate architecture. Therefore, pollen reception can only be achieved by highly specialized mechanisms. Although the population studied shows a continuous flowering pattern (Newstrom et al. 1994), a flowering peak occurred in the dry season, from November 2010 to January 2011. We assessed the species’ breeding system through pollination experiments (Fig.1) carried out in the field. We marked 20 individual plants and covered 23 inflorescences bearing pre-anthesis flower buds with voile bags to test for spontaneous self-pollination and fruit set. We left additional 23 inflorescences under natural conditions (control group), with flowers exposed to flower visitors. In both groups, we assessed pollination success through fruit yield. We monitored anthesis for five consecutive days. We marked pre-anthesis flower buds and recorded information on anthesis beginning and duration. We carried out non-standardized observations of flower visitors during anthesis, and photographed the flowers whenever possible. To compare the number of fruits formed between treatments, we calculated a t test in BioEstat 5.0. Besides carrying male gametes, the pollen grains are also a protein source for flower visitors and paramount for the reproductive success of plants (Faegri & van der Pijl 1979). Sesbania virgata shows, on average, 22,918 pollen grains per flower, which makes the pollen one of the most important nutritional resources offered to pollinators. Anthesis The anthesis is diurnal and begins with the banner petal slowly unbending at ca. 10:00. R d i é i 67(4) 871 878 2016 Table 1 – Floral traits of Sesbania virgata in a population studied in Areia, state of Paraíba, northeastern Brazil. Traits Measurements Length (mm) 12.7 (15,29 ± 10,25) Width (mm) 3.0 (7,94 ± 1,72) Sepals (mm) 5.1 (5,96 ± 3,56) Peduncle (mm) 3.8 (5,11 ± 2,36) Stigma (mm) 8.8 (10,64 ± 7,09) Pollen/ovule ratio 4,583.6 Pollen viability (%) 99.93% Pollen grains (mean value) 22,918 Floral traits of Sesbania virgata in a population studied in Areia, state of Paraíba, northeastern Brazil. Rodriguésia 67(4): 871-878. 2016 875 Biologia floral da espécie invasora Sesbania virgata (Cav.) Pers. success was 55.67% in the control (pollination free from visitors) and 27.34% in the treatment of spontaneous self-pollination. Aguiar et al. (2012) reported a mixed reproductive system in Oeceoclades maculate composed of cross- pollination and autogamy. Similarly to our study species, this system is probably related to its success in colonizing new and isolated habitats. This process is asynchronous: most flowers are completely open by 12:00, but some flowers begin anthesis only at 14:00. The increase in temperature probably favors anthesis, as its peak occurred around 12:00. The flowers remained open until the following day. Flower opening occurs from the base to the apex of the inflorescence. There are flowers and flower buds at different development stages on the same inflorescence. The extended flower opening favors pollinator activity throughout the day. This strategy may benefit cross-pollination, as it makes the pollen available as a resource in different plants and flowers simultaneously for flower visitors. g The population studied showed a continuous flowering pattern. Therefore, a given number of flowers per day on each plant, in a large amount at the peak of the phenophase, could restrict the movement of pollinators. Indeed, we observed few pollinators visiting several flowers per plant, as well as several different plants. Some pollinators were bees (Xylocopa sp.), and we collected them. We bagged flowers after the first visit of pollinators to reduce the pollen flow and avoid additional visits of other pollinators. However, those flowers set fruits. According to Stephenson (1981), this could reduce the pollen flow among plants, which would favor self-pollination. The asynchronous pattern seems to be common in some Fabaceae species. Anthesis Usually, the anthesis is diurnal and lasts only one day (Primack 1985), but some species may show differences in flower duration, as observed in Mucuna sp. flowers (Agostini 2004). Guedes et al. (2009) classified the anthesis of Canavalia brasiliensis Mart. ex Benth (Fabaceae) as diurnal and asynchronous. Souza et al. (2012) classified the anthesis of Senna cana (Fabaceae) as diurnal and very slow. According to the same author, the reproductive success of S. cana and its subsequent establishment result not only from the morpho-structural characters of the flower, but also from the vegetative phenology and reproductive strategies. Autogamy is a reproductive characteristic of invasive and pioneer species that occupy clearings and forest edges (Williamson 1996; Holsinger 2000). In the present study, S. virgata individuals occurred in areas similar to clearings, i.e., high-light environments. Repeated cycles of colonization and low density may favor this kind of reproduction to assure the perpetuation of the species (Barret et al. 2008). Although several Fabaceae species are autogamous (Arroyo 1981), there is evidence that cross-fertilization also takes place (Borges 2006). References Aguiar, J.M.R.B.V.; Pansarin, L.M.; Ackerman, J.D. & Pansarin, E.R. 2012. Biotic vs abiotic pollination in Oeceoclades maculata (Lindl,) Lindl. (Orchidaceae: Epidendroideae). Plant Species Biology 27: 86-95. Agostini, K. 2004. Ecologia da polinização de Mucuna sp. nov. (Fabaceae) no litoral norte de São Paulo, Brasil. Tese de Doutorado. Universidade Estadual de Campinas, Campinas. 97p. Agostini, K.; Sazima, M. & Sazima, I. 2006. Bird pollination of explosive flowers while foraging for nectar and caterpillars. Biotropica 38: 674-678. Andrade, L.A. 2006. Espécies exóticas invasoras no nordeste do Brasil: impactos nos ecossistemas locais. In: Mariath, J.E.A. & Santos, R.P. (org.). Os avanços da botânica no início do século XXI: morfologia, fisiologia, taxonomia, ecologia e genética. Sociedade Botânica do Brasil, Porto Alegre. 752p. Autogamy would work as a safety mechanism to assure reproduction when pollinators are scarce (Endress 1994). Hence, autogamy might contribute to increasing seed set and the species’ fitness. Andrade, L.A.; Fabricante, J.R. & Alves, A.D.S. 2008. Algaroba [Prosopis juliflora (Sw.) DC.]: Impactos sobre a fitodiversidade e estratégias de colonização em área invadida na Paraíba. Natureza & Conservação 6: 61-67. Results indicate that, despite the high pollination efficiency, populations of this rewarding orchid species (Epipactis palustris) were strongly pollen limited. On the other hand, costs of early inbreeding depression were high, which may prevent rapid evolution of complete selfing (Jacquemyn & Brys 2015). Andrade, L.A.; Fabricante, J.R. & Oliveira, F.X. 2009. Invasão biológica por Prosopis juliflora (Sw.) DC. Impactos sobre a diversidade e a estrutura do componente arbustivo-arbóreo da caatinga no estado do Rio Grande do Norte, Brasil. Acta Botanica Brasiliense, 23: 935-943. According to Raven (1996), the appearance of the hermaphrodite flower was an important evolutionary development in Angiosperms. The presence of carpels and stamens in the same flower allows the pollinator to remove and deposit pollen simultaneously. This process increases the efficiency of the pollinator at each visit and, thus, gives a selective advantage to the plant. Andrade, L.A. de. 2013. Plantas invasoras: espécies vegetais exóticas invasoras da Caatinga e ecossistemas associados. UFPB/CCA, Areia. 100p. Araújo, E.C.; Mendonça, A.V.R.; Barroso, D.G.; Lamônica, K.R. & Silva, R.F. 2004. Caracterização morfológica de frutos, sementes e plântulas de Sesbania virgata (CAV.) PERS. Revista Brasileira de Sementes 26: 105-110. Arroyo, M.T.K. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill, R.M. & Raven, P.H. (eds.). Advances in legume systematics: part 2. Royal Botanic Garden, Kew. Pp. 723-769. Conclusions Sesbania virgata is autogamous, which is an important mechanism to assure colonization. The reproductive success and establishment of the species is a result of its reproductive strategy, as well as of its production of flowers/pollen, mainly during the dry season. This strategy is advantageous for the studied species in the marshes (“Brejo”) of Paraíba, where the climate is favorable and with no heavy rains that might harm flowers. Baker, H.G. 1974. The evolution of weeds. Annual Review of Ecology and Systematics 5: 1-24. Barret, S.C.H.; Colautti, R.I. & Eckert, C.G. 2008. Plant reproductive systems and evolution during biological invasion. Molecular Ecology 17: 373- 383. Blanco, H.G. 1972. A importância dos estudos ecológicos nos programas de controle das plantas daninhas. O Biológico 38: 343-350. Borges, H.B.N. 2006. Biologia reprodutiva de Centrosema pubescens Benth. (Fabaceae). Boletim do Museu Paraense Emilio Goeldi Ciências Naturais. Belém. Disponível em <http://scielo.iec.pa.gov. br/scielo.php?script=sci_arttext&pid=S1981- 81142006000100003&lng=pt&nrm=iso>. Acesso em 3 fevereiro 2016. Breeding system Sesbania virgata is autogamous, as fruits were formed in both treatments: control and spontaneous self-pollination (Tab. 2). This result provides evidence that S. virgata does not depend on pollinators to transfer pollen grains from the anthers to the stigmatic surface. The pollen grains were already available when the flower bud was closed. This strategy assures the maintenance of genetic variability, which is necessary for the permanence of the species in the habitat in the face of environmental pressures that are common in tropical areas. The mesquite tree (Prosopis juliflora), a legume (Fabaceae) of the subfamily Mimosoidae, is another invasive species with a reproductive biology similar to that of S. virgata. It has invaded extensive river margins and degraded areas, which resulted in high population density (Andrade et al. 2009; Leão et al. 2011). There was a significant difference in fruit set between the control and the treatment of spontaneous self-pollination (t = 3.0720, p = 0.0018). This result suggests that pollinators increased fruit set in this species. The reproductive Rodriguésia 67(4): 871-878. 2016 Table 2 – Pollination and fruit set in flowers of Sesbania virgata (in each treatment) in Areia, state of Paraíba, northeastern Brazil. Treatments Flowers/Fruits (Fr/Fl) Success % Spontaneous self-pollination 12.69/3.47 27.34 Open pollination (control) 12.95/7.21 55.67 Rodriguésia 67(4): 871-878. 2016 Rodriguésia 67(4): 871-878. 2016 Souza, V.C. et al. Souza, V.C. et al. 876 In spite of the fruit set observed in autogamy experiments, the pollen/ovule ratio also suggests S. virgata as a preferentially xenogamous plant. However, in self-pollination experiments, we observed the formation of fruits. Therefore, this classification may not be suitable for S. virgata, as spontaneous self-pollination also occurs. According to Baker (1974), a certain balance between self-compatibility and cross-pollination is beneficial to weeds. The author states that once a seed is dispersed to a distant place, the formation of a new population will depend on the self-pollination capacity of the species. Sesbania virgata is autogamous, which seems to be a good strategy combined with the ability of this species to invade degraded areas, such as the study site. Acknowledgements We thank the students from the Laboratory of Plant Ecology, especially Francisco Thiago, Ariosto, and Beatriz Symara, for their help during the experiments. Rodriguésia 67(4): 871-878. 2016 877 Biologia floral da espécie invasora Sesbania virgata (Cav.) Pers. seed set in a rewarding orchid. American Journal of Botany 102: 67-72. Braggio, M.M.; Lima, M.E.L.; Veasey, E.A. & Haraguchi, M. 2002. Atividades farmacológicas das folhas de Sesbania virgata (CAV.) PERS. Revista Arquivos do Instituto Biológico 69: 49-53. Judd, W.S.; Campbell, C.S.; Kellogg, E.A.; Stevens, P.F. & Donoghue, M.J. 2009. Sistemática vegetal: um enfoque filogenético. Artmed, Porto Alegre. 632p. Brito, V.L.G.; Pinheiro, M. & Sazima, M. 2010. Sophora tomentosa e Crotalaria vitellina (Fabaceae): biologia reprodutiva e interações com abelhas na restinga de Ubatuba, São Paulo. Biota Neotropica 10: 185-192. Leão, T.C.C.; Almeida, W.R.; Dechoum, M. & Ziller, S.R. 2011. Espécies exóticas invasoras no nordeste do Brasil. In: Leão, T.C.C.; Almeida, W.R.; Dechoum, M. & Ziller, S.R. (org.). Contextualização, manejo e políticas públicas. CEPAN/Instituto Hórus, Recife. Pp. 21-76. Carneiro, A.M. & Irgang, B.E. 2005.Origem e distribuição geográfica das espécies ruderais da Vila de Santo Amaro, General Câmara, Rio Grande do Sul. IHERINGIA, Série Botânica 60: 175-188. Cruden, R.W. 1977. Pollen-ovule ratios: a conservative indicator of breeding systems in flowering plants. Evolution 31: 32-46. Carneiro, A.M. & Irgang, B.E. 2005.Origem e distribuição geográfica das espécies ruderais da Vila de Santo Amaro, General Câmara, Rio Grande do Sul. Leite, A.V. & Machado, I.C. 2009. Biologia reprodutiva da catingueira (Caesalpinia pyramidalis Tul., Leguminosae-Caesalpinoideae), uma espécie endêmica da Caatinga. Revista Brasileira de Botânica 32: 79-88. IHERINGIA, Série Botânica 60: 175-188. Cruden, R.W. 1977. Pollen-ovule ratios: a conservative indicator of breeding systems in flowering plants. Evolution 31: 32-46. Carpanezzi, A.A. & Fowler, J.P.A. 1997. Quebra da dormência tegumentar de sementes de Sesbania virgata (Cav.) Pers. EMBRAPA. Comunicado Técnico, 14. EMBRAPA CNPF, Colombo. Pp. 1-2. Lima, H.C. 2000. Leguminosas arbóreas da Mata Atlântica: uma análise da riqueza, padrões de distribuição geográfica e similiaridades florísticas em remanescentes florestais do Estado do Rio de Janeiro. Tese de Doutorado. Universidade Federal do Rio de Janeiro, Rio de Janeiro. 151p. Dias, C.T.V. & Kiill, L.H.P. 2007. Ecologia da polinização de Raphiodon echinus (Nees e Mart.) Schauer (Lamiaceae) em Petrolina, PE, Brasil. Acta Botanica Brasilica 21: 977-982. Machado, I.C. & Lopes, A.V. 2002. A polinização em ecossistemas de Pernambuco: uma revisão do estado atual do conhecimento. In: Tabarelli, M. & Silva, J.M.C. (eds.). Acknowledgements Diagnóstico da biodiversidade de Pernambuco. Editora Massangana, Recife. 722p. Endress, P.K. 1994. Diversity and evolutionary biology of tropical flowers. Cambridge University Press, Cambridge. 407p. Estes, J.R.; Amos, B.B. & Sullivan, J.R. 1983. Pollination from two perspectives: the agricultural and biological sciences. In: Jones, C.E. & Little, R.J. (eds.). Handbook of experimental pollination biology. Scientific and Academic Editions, New York. 558p. Newstrom, L.E.; Frankie, G.W. & Baker, H.G. 1994a. A new classification for plant phenology based on flowering patterns in lowland tropical rain forest trees at La Selva, Costa Rica. Biotropica 26: 141- 159. Nogueira, E.M.L. & Arruda, V.L.V. 2006. Fenologia reprodutiva, polinização e sistema reprodutivo de Sophora tomentosa L. (Leguminosae - Papilionoideae) em restinga da praia da Joaquina, Florianópolis, sul do Brasil. Revista Biotemas 19: 29-36.l Etcheverry, A.V.; Alemán, M.M. & Fleming, T.F. 2008. Flower morphology, pollination biology and mating system of the complex flower of Vigna Caracalla (Fabaceae: Papilionoideae). Annals of Botany, Oxford Journals 102: 305-316. Primack, R.B. 1985. Longevity of individual flowers. Annual Review of Ecology and Systematics 16: 15-37. Faegri, K. & van der Pijl, L. 1979. The principles of pollination ecology. Pergamon Press, Oxford. 244p. Guedes, R.S.; Quirino, Z.G.M. & Gonçalves, E.P. 2009. Fenologia reprodutiva e biologia da polinização de Canavalia brasiliensis Mart. ex Benth. (Fabaceae). Revista Biotemas 22: 27-37. Pyšek, P. & Richardson, D.M. 2007. Traits associated with invasiveness in alien plants: where do we stand? Biological invasions. Nentwig, W. Springer- Verlag Berlin Heidelberg, New York. Pp. 97-122. Holsinger, K.E. 2000. Reproductive systems and evolution in vascular plants. Proceedings of the National Academy of Sciences, USA 97: 7037-7042.i Radford, A.E.; Dickison, W.C.; Massey, J.R. & Bell, C.R. 1974. Vascular plant systematics. Harper & Row Publishers, New York. 891p. Instituto Brasileiro de Geografia e Estatística. 2011. Cidades. Disponível em <http://www.ibge.gov.br/ cidadesat/painel/painel.php?codmun=250990>. Acesso em 7 dezembro 2011.i Raven, P.H.; Evert, R.F. & Eichhorn, S.E. 1996. Biologia vegetal. Guanabara Koogan, Rio de Janeiro. 728p.l Silva, A.L.G.S. & Pinheiro, M.C.B. 2007. Biologia floral e da polinização de quatro espécies de Eugenia L. (Myrtaceae). Acta Botanica Brasilica 21: 235-247. Instituto Brasileiro de Geografia e Estatística. 1992. Manual técnico da vegetação brasileira. Série manuais técnicos em geociências. Vol. 1. Fundação Instituto Brasileiro de Geografia e Estatística - IBGE, Rio de Janeiro. 92p. Souza, I.M.; Coutinho, K. & Funch, L.S. 2012.Estratégias fenológicas de Senna cana (Nees & Mart.) H.S. Irwin & Barneby (Fabaceae: Caesalpinioideae) como mecanismo eficiente para atração de polinizadores. Artigo recebido em 15/07/2015. Aceito para publicação em 11/05/2016. Acknowledgements Acta Botanica Brasilica 26: 435-443. Jacquemyn, H. & Brys, R. 2015. Pollen limitation and the contribution of autonomous selfing to fruit and Rodriguésia 67(4): 871-878. 2016 878 Souza, V.C. et al. Souza, V.C. et al. Sukopp, H. & Trepl, L. 1987. Extinction and naturalization of plant species as related to ecosystem structure and function. Ecological Studies 61: 245-276. Souza, V.C. 2012. Invasão biológica por Sesbania virgata (CAV) Pers. na Paraíba, Brasil: Biologia reprodutiva, ecofisiologia de sementes e estrutura populacional. Tese de Doutorado. Universidade Federal da Paraíba, Areia. 199p. Williamsom, M. 1996. Biological invasions. Chapman e Hall, London. 256p. Stephenson, A.G. 1981. Flower and fruit abortion: proximate causes and ultimate functions. Annual Review of Ecology and Systematics 12: 253-279. Williamson, M.H. & Fitter, A. 1996. The characters of successful invaders. Biological Conservation 78: 163-170. Rodriguésia 67(4): 871-878. 2016
https://openalex.org/W4323566436	https://ijsr.internationaljournallabs.com/index.php/ijsr/article/download/750/650	English	null	The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code	Journal of Social Research	2,023	cc-by-sa	3,964	P-ISSN: 2827-9832 E-ISSN: 2828-335x http://ijsr.internationaljournallabs.com/index.php/ijsr P-ISSN: 2827-9832 E-ISSN: 2828-335x http://ijsr.internationaljournallabs.com/index.php/ijsr THE LEGAL FORCE OF THE COOPERATION AGREEMENT LETTER IN A COOPERATION AGREEMENT IS REVIEWED ACCORDING TO ARTICLE 1320 OF THE CIVIL CODE Irma Shintia Kumaralo, Risdalina, Indra Kumalasari M Faculty of Law, Universitas Labuanbatu irmashintiak@gmail.com, risdalinasiregar@gmail.com, indrakumalsarim@gmail.com Irma Shintia Kumaralo, Risdalina, Indra Kumalasari M Faculty of Law, Universitas Labuanbatu irmashintiak@gmail.com, risdalinasiregar@gmail.com, indrakumalsarim@gmail.com In every activity that is an agreement, of course, there is an agreement that has been made. Whether it is in business, or in certain special matters. This study aims to find out and analyze the regulation of cooperation agreement letters in the civil code (hyperdata). As well as knowing and analyzing the Principles and Determining Factors in Making a Draft Contract (Cooperation Agreement). This research belongs to the normative type of research. So it can be known that agreements are specifically regulated in the Civil Code, Book III, Chapter II on "Agreements Born of Contracts or Agreements" and Chapter V to Chapter XVIII which regulates the principles of legal principles and legal norms of treaties in general, as well as legal norms of treaties that have special characteristics better known as named agreements. In addition, basically, the contract that the parties conclude applies as a law to those who make it. The factors that must be considered by the parties are the legal authority of the parties, taxation, over legal rights, agricultural issues, choice of law, dispute resolution, termination of contracts, and the form of standard agreements agreed upon Keywords: letter of agreement, cooperation, engagement INTRODUCTION Article 1266 of the Civil Code can be cited as follows: Article 1266 of the Civil Code can be cited as follows: "The void condition is deemed to always be included in a reciprocal agreement if one of the parties does not fulfill its obligations. "The void condition is deemed to always be included in a reciprocal agreement if one of the parties does not fulfill its obligations. In such a case consent is not null and void, but annulment must be requested to the Court (Santosa, 2015). This request must also be made, even if the void condition regarding non- fulfillment of the obligation is stated in the consent (Dalimunthe, 2017). In such a case consent is not null and void, but annulment must be requested to the Court (Santosa, 2015). This request must also be made, even if the void condition regarding non- fulfillment of the obligation is stated in the consent (Dalimunthe, 2017). “If the void condition is not stated in the agreement, then the Judge in view of the circumstances, at the request of the defendant, is free to give a period of time to fulfill the obligation, but that period shall not be more than one month." Article 1267 of the Civil Code can be cited as follows: Article 1267 of the Civil Code can be cited as follows: "The party against whom the engagement is not fulfilled may vote; forcing the other party to comply with the agreement, if it is still possible, or demanding the cancellation of the agreement, with reimbursement of costs, losses, and interest." "The party against whom the engagement is not fulfilled may vote; forcing the other party to comply with the agreement, if it is still possible, or demanding the cancellation of the agreement, with reimbursement of costs, losses, and interest." The waiver of Articles 1266 and 1267 is so that in the event of default or non-fulfillment of the contents of the agreement by one of the parties, then: The waiver of Articles 1266 and 1267 is so that in the event of default or non-fulfillment of the contents of the agreement by one of the parties, then: The cancellation of an agreement does not need to go through the process of a void application to the court but can be based on the agreement of the parties themselves (Lambsdorff, 2002). INTRODUCTION The cancellation of an agreement does not need to go through the process of a void application to the court but can be based on the agreement of the parties themselves (Lambsdorff, 2002). A party whose agreement is not fulfilled may force the other party to fulfill the contents of the agreement or demand the cancellation of the agreement to the court by charging reimbursement of costs, losses, and interest (Chang & Ive, 2007). INTRODUCTION In every activity that is agreement, of course, there is an agreement that has been made (Harahap, 1982). Whether it is in business, or in certain or special matters (Kolk, 2016). Articles 1320, 1266, and 1267 of the Civil Code describe the legal terms of an agreement and the waiver of articles in the occurrence of default. Articles 1320, 1266, and 1267 of the Civil Code describe the legal terms of an agreement and the waiver of articles in the occurrence of default. In the explanation of Article 1230 of the Civil Code (Civil Code) can be found the legal terms of an agreement, in general, can be known as follows: In the explanation of Article 1230 of the Civil Code (Civil Code) can be found the legal terms of an agreement, in general, can be known as follows: The four legal terms of an agreement include: 1. An agreement that binds both parties. 2. Proficiency in making an engagement. 3. A particular subject matter. 4. A cause that is not forbidden. 4. A cause that is not forbidden. The four valid juridical requirements of an agreement contract are as follows: • Objective valid terms based on article 1320 of the Civil Code. 1. Specific object/Subject. 2. Allowed/justified/legalized causation. • Subjective legal terms under article 1320 of the Civil Code. 1. The existence of the agreement and will. 2. Authority to do. 2. Authority to do. 917 The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code • Common legal terms outside article 1320 of the Civil Code. 1. The contract must be performed in good faith. 2. The contract must not conflict with applicable customs. 3. The contract must be performed on the basis of the principle of propriety. 4. The contract must not violate the public interest. • Special legal terms. 1. Written terms for a particular contract. 1. Written terms for a particular contract. 2. The terms of the notarial deed for a particular contract. 3. The terms of the deed of an official other than a notary for a particular contract. 4. The condition of permission from the authorized official for a particular contract. RESULTS AND DISCUSSION Regulation of Cooperation Agreement Letter in the Civil Code (KuhPerdata) The Civil Code is a law that is a source of formal law as well as a source of material law for treaty law in force in Indonesia. The Agreement is specifically regulated in the Civil Code, Book III, Chapter II on "Agreements Born of Contracts or Agreements" and Chapters V through Chapter XVIII which regulate the legal principles and legal norms of treaties in general. As well as the legal norms of treaties that have special characteristics that are better known as named agreements (Harefa & Tuhana, 2016). Based on Article 1233 of the Civil Code regarding covenants, explains that: "An engagement was born because of an agreement or because of a law." (Civil Code). Based on Article 1233 of the Civil Code regarding covenants, explains that: "An engagement was born because of an agreement or because of a law." (Civil Code). Furthermore, Article 1333 of the Civil Code states that: "An agreement is an act in which one or more persons bind themselves to another or more persons." Furthermore, Article 1333 of the Civil Code states that: "An agreement is an act in which one or more persons bind themselves to another or more persons." Based on provisions in Article 1320 of the Civil Code, it states that there are 4 (four) conditions for the validity of an agreement, namely: Based on provisions in Article 1320 of the Civil Code, it states that there are 4 (four) conditions for the validity of an agreement, namely: 1. There is a word of the agreement for those who bind themselves; 2. The ability of the parties to make an agreement; 2. The ability of the parties to make an agreement; 3. A certain thing; and 4. A cause (causa) that is lawful. The first and second terms are called subjective terms because they pertain to the subject of the agreement. Meanwhile, the third and fourth terms related to the object of the agreement are called objective conditions. The difference between the two terms is also associated with the null and void issue (nieteg or null and ab initio) and the vernietigbaar=voidable of an agreement. The difference between the two terms is also associated with the null and void issue (nieteg or null and ab initio) and the vernietigbaar=voidable of an agreement. METHOD The research method used in this study is a normative legal research method. Normative legal research is legal research carried out by examining library materials or secondary data (Soekanto, 2007). According to Marzuki (2017), normative legal research is a process to find a rule of law, legal principles, and legal doctrines to answer the legal issues faced. In this type of legal research, often the law is conceptualized as what is written in legislation or law is conceptualized as a rule or norm that is a benchmark for human behavior that is considered appropriate (Asikin, 2004). 2, 3., February 2023 918 The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code RESULTS AND DISCUSSION If the objective conditions in the agreement are not met then the Agreement is null and void or the agreement is void, the law considers the agreement to have never existed. If the objective conditions in the agreement are not met then the Agreement is null and void or the agreement is void, the law considers the agreement to have never existed. If the subjective conditions are not met then the Agreement may be canceled or as long as the agreement has not been or is not canceled by the court, then the agreement in question continues to be in force. The four conditions for the validity of the treaty are the basis for the birth of a treaty. In the law of covenants, we know five important principles that are at once the essence of the law of covenants (Sinaga, 2018). The five principles are: In the law of covenants, we know five important principles that are at once the essence of the law of covenants (Sinaga, 2018). The five principles are: 1. The principle of freedom to enter into agreements 4. Principles of good faith Article 1338 paragraph (3) of the Civil Code states that, "All agreements shall be executed in good faith". This principle states that the parties, namely the creditor and the debtor, must carry out the substance of the contract based on the trust or firm belief or good will of the parties (Mackaay, 2011). 2. The principle of consensual The principle of consumerism can be traced in the formulation of Article 1320 paragraph (1) of the Civil Code. In this article, it is determined that one of the conditions for the validity of the agreement is the agreement of both parties. The agreement is valid if an agreement has been reached on the subject matter and no more formalities are needed (Hayek, 2002). In this article, it is determined that one of the conditions for the validity of the agreement is the agreement of both parties. The agreement is valid if an agreement has been reached on the subject matter and no more formalities are needed (Hayek, 2002). However, various provisions of the law stipulate that for the validity of the agreement must be made in writing or is required to be made by a deed made by an authorized official (for example, a deed of establishment of a Limited Liability Company). The principle of consensual known in the Civil Code relates to the form of agreements. 3. The principle of pacta sunt servanda The principle of consensual known in the Civil Code relates to the form of agreements. 3. The principle of pacta sunt servanda This principle is translated as the principle of legal certainty summarized in the formulation of Article 1338 paragraph (1) of the Civil Code, "All agreements made validly apply as laws". The principle of pacta sunt servanda states the judge or third party must respect the substance of the agreement entered into by the parties, as befits a statute. This principle is translated as the principle of legal certainty summarized in the formulation of Article 1338 paragraph (1) of the Civil Code, "All agreements made validly apply as laws". h l f d h j d hi d h This principle is translated as the principle of legal certainty summarized in the formulation of Article 1338 paragraph (1) of the Civil Code, "All agreements made validly apply as laws". The principle of pacta sunt servanda states the judge or third party must respect the substance of the agreement entered into by the parties as befits a statute The principle of pacta sunt servanda states the judge or third party must respect the substance of the agreement entered into by the parties, as befits a statute. They may not intervene or intervene in the substance of the agreement concluded by the parties. 1. The principle of freedom to enter into agreements 1. The principle of freedom to enter into agreements Freedom of entering into treaties is one of the principles in the common law prevailing in the world (Anand, 2011). Freedom of entering into treaties is one of the principles in the common law prevailing in the world (Anand, 2011). This principle gives every citizen the freedom to enter into an agreement on anything, provided that it does not conflict with laws and regulations, propriety and public order. This principle gives every citizen the freedom to enter into an agreement on anything, provided that it does not conflict with laws and regulations, propriety and public order. Article 1338 paragraph (1) of the Civil Code states, "All agreements made lawfully apply as laws to those who make them". The principle of freedom of entering into a treaty is a principle that gives freedom to the parties to the agreement to: a. Make or not make an agreement; b. Enter into an agreement with anyone; 2, 3., February 2023 919 The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code c. Determine the content of the agreement, its implementation and terms; d. Determine the form of the agreement i.e. written or unwritten; e. Accept or deviate from the provisions of the legislation of a rational nature. All agreements or the entire contents of the agreement, provided that they are qualified, apply to their makers with the same force as the law. The parties to the agreement are free to enter into an agreement with any content in an agreement by taking into account the applicable legal limitations, including the limitations of moral norms living in society. 5. The principle of personality It is the principle that determines that a person who will perform and or make a covenant is for individual purposes only (van Dyke, 1977). This can be seen in Article 1315 and Article 1340 of the Civil Code. It is the principle that determines that a person who will perform and or make a covenant is for individual purposes only (van Dyke, 1977). This can be seen in Article 1315 and Article 1340 of the Civil Code. Article 1315 is formulated, "Generally no man may bind himself on his own behalf or ask for the establishment of a promise, except for himself". Article 1315 is formulated, "Generally no man may bind himself on his own behalf or ask for the establishment of a promise, except for himself". Article 1315 relates to the formulation of Article 1340 of the Civil Code, "Agreements are valid only between the parties who make them" (http://titintrianash.blogspot.com/2015/06/normal-0-false-false-false-in-x-none-x.html. Titin Triana. Legal Certainty of Oral Treaties.). Article 1315 relates to the formulation of Article 1340 of the Civil Code, "Agreements are valid only between the parties who make them" (http://titintrianash.blogspot.com/2015/06/normal-0-false-false-false-in-x-none-x.html. Titin Triana. Legal Certainty of Oral Treaties.). In addition to the five principles above, there are still some basic things that can be used as guidelines in making covenants. This provision is universal and morally liable. 2, 3., February 2023 920 The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code Some of these basic principles are: Some of these basic principles are: a. The principle of trust; b. The principle of legal equality; c. The principle of balance; d. The principle of legal certainty; e. Moral principles; f. The principle of propriety; g. The principle of habit; and h. The principle of protection. CONCLUSION Based on the above explanation, it can be concluded that the agreement is specifically regulated in the Civil Code, Book III, Chapter II on "Agreements Born of Contracts or Agreements" and Chapter V to Chapter XVIII. Which regulates the principles of legal principles and legal norms of treaties in general, as well as the norms of treaty law that have special characteristics better known as named treaties. In addition, basically, the contract that the parties conclude applies as a law to those who make it. Therefore, drafting a contract requires the thoroughness and thoroughness of the parties. The factors that must be considered by the parties to enter into and make the contract are the legal authority of the parties, taxation, over legal rights, agricultural issues, choice of law, dispute resolution, termination of the contract, and the form of the agreed standard agreement. The Legal Force of the Cooperation Agreement Letter in a Cooperation Agreement is Reviewed According to Article 1320 of the Civil Code In this case, the author takes one example of a cooperation agreement contract by PT. Clipan Finance Indonesia Tbk Labuhanbatu branch of Barton car showroom in Labuhanbatu Region. In the cooperation agreement Letter Number 01 / PKS-SR / CFI-843 / VII / 2022 article 1 concerning Rights and Obligations in point 1 between PT. Clipan Finance Indonesia Tbk Labuhanbatu Branch Against Barton Mobil Showroom said that PT. Clipan Finance Indonesia Tbk Labuhanbatu Branch will conduct research and analysis of customer capabilities in accordance with the creditworthiness requirements set by PT. Clipan Finance Indonesia Tbk. Clipan Finance Indonesia Tbk Labuhanbatu Branch. Principles and Determining Factors in Making a Draft Contract (Cooperation Agreement) Principles and Determining Factors in Making a Draft Contract (Cooperation Agreement) What is meant by the principles in contract design is the basis or principles that must be considered in designing a contract (Sangiovanni-Vincentelli et al., 2012). Every contract designer who will design a contract, whether it is a contract that has been known in the Civil Code or that lives and develops in society, must pay attention to the principles in designing the contract. Erman Rajaguguk stated that there are ten basic principles that must be considered in contracts that are commonly used in Indonesia and deserve the attention of international trade contract designers. The ten things include: (1) Use of the term, (2) The principle of freedom of contract, (3) The principle of bidding and acceptance, (4) Good faith, (4) Good faith, (5) Risk switching, (6) Indemnity, (7) Emergency (8) Reasons for termination, (9) Choice of law, and Dispute resolution. Factors To Be Considered in The Drafting Of The Contract Basically the contract that the parties make applies as a law to those who make it. Factors To Be Considered in The Drafting Of The Contract Basically the contract that the parties make applies as a law to those who make it. Thus, the contract concluded by the parties equates its binding force with the Act. Therefore, designing a contract requires thoroughness and accuracy from the parties, both the creditor and debtor, the investor and the rights of the state concerned, and the contract designer and notary. 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https://openalex.org/W2073714350	https://hal.archives-ouvertes.fr/hal-03502039/file/bg-8-743-2011.pdf	English	null	Short-term dynamics of microplankton abundance and diversity in NW Mediterranean Sea during late summer conditions (DYNAPROC 2 cruise; 2004)	Biogeosciences	2,011	cc-by	15,431	Short-term dynamics of microplankton abundance and diversity in NW Mediterranean Sea during late summer conditions (DYNAPROC 2 cruise; 2004) S. Lasternas, A. Tunin-Ley, F. Ibanez, V. Andersen, M. -D. Pizay, R. Lemee To cite this version: S. Lasternas, A. Tunin-Ley, F. Ibanez, V. Andersen, M. -D. Pizay, et al.. Short-term dynamics of microplankton abundance and diversity in NW Mediterranean Sea during late summer conditions (DYNAPROC 2 cruise; 2004). Biogeosciences, 2011, 8 (3), pp.743-761. 10.5194/bg-8-743-2011. hal-03502039 Distributed under a Creative Commons Attribution 4.0 International License Short-term dynamics of microplankton abundance and diversity in NW Mediterranean Sea during late summer conditions (DYNAPROC 2 cruise; 2004) sternas1,2,, A. Tunin-Ley1,2, F. Iba˜nez1,2, V. Andersen1,2, M.-D. Pizay1,2, and R. Lem´ee1,2 S. Lasternas1,2,, A. Tunin-Ley1,2, F. Iba˜nez1,2, V. Andersen1,2, M.-D. Pizay1,2, and R. Lem´ee1,2 1Universit´e Pierre et Marie Curie-Paris6, Laboratoire d’Oc´eanographie de Villefranche, 06230 Villefranche-sur-Mer, France 2CNRS, Marine Microbial Ecology and Biogeochemical Group, Laboratoire d’Oc´eanographie de Villefranche UMR 7093, 06230 Villefranche-sur-Mer, France present address: IMEDEA (CSIC-UIB), Instituto Mediterr´aneo de Estudios Avanzados, Departm Research, 07190 Esporles (Mallorca), Spain present address: IMEDEA (CSIC-UIB), Instituto Mediterr´aneo de Estudios Avanzados, Department of Global Change Research, 07190 Esporles (Mallorca), Spain present address: IMEDEA (CSIC-UIB), Instituto Mediterr´aneo de Estudios Avanzados, Department of Global Change R h 07190 E l (M ll ) S i Received: 23 September 2008 – Published in Biogeosciences Discuss.: 22 December 2008 Revised: 31 January 2011 – Accepted: 19 February 2011 – Published: 22 March 2011 Abstract. Changes in marine phytoplankton communities at short-time scales have rarely been examined. As a part of the DYNAPROC 2 cruise (14 September–17 October 2004), conducted in the NW Mediterranean Sea, we daily sampled and determined taxonomic composition and abundance of the microphytoplankton and the dominant microzooplank- tonic groups from both net and bottle sampling, during 4 cy- cles of 5 days. Hydrological conditions were characterised by the dominance of a stratiﬁed water column and nutrient- depleted conditions. However the stratiﬁcation index re- vealed a destabilization of the water column from the be- ginning of the second cycle, related to a wind stress event accompanied with coastal water intrusion. This conducted to an increase of taxonomic richness and a general decrease of evenness, depicting the dominance of species with the best ﬁtness. We also emphasised on the great interest of taxo- nomic studies, as able to provide valuable information on biogeochemical-important groups of species, potential wa- ter masses indicators and trophic aspects of the community that are ignored or largely underestimated with other kind of phytoplankton studies, such as pigment analysis. HAL Id: hal-03502039 https://hal.science/hal-03502039v1 Submitted on 24 Dec 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ doi:10.5194/bg-8-743-2011 © Author(s) 2011. CC Attribution 3.0 License. Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ doi:10.5194/bg-8-743-2011 © Author(s) 2011. CC Attribution 3.0 License. Biogeosciences 2.2 Field sampling analysed samples taken during 4 periods of 5 days from the cruise DYNAPROC 2 (DYNAmic of rapid PROCesses in water column that control vertical ﬂux of matter) per- formed in the NW Mediterranean from September to Novem- ber 2004. This expected transition period from summertime oligotrophic stratiﬁed system to autumnal system was con- sidered favourable and pertinent because of higher frequen- cies of wind events. It was completing the DYNAPROC 1 cruise conducted in May 1995 in order to document the ef- fects of episodic wind events on the dynamics of the ecosys- tem during a post-bloom period, characterised by the transi- tion from a mesotrophic to an oligotrophic system (Andersen and Prieur, 2000). We also analyzed the intake of taxonom- ical studies in biogeochemical cycle’s studies, mass indica- tors or trophic aspects. This was feasible since DYNAPROC 2 was a multidisciplinary cruise that documented many hy- drological parameters and considerable stocks and rates mea- surements (Andersen et al., 2009). analysed samples taken during 4 periods of 5 days from the cruise DYNAPROC 2 (DYNAmic of rapid PROCesses in water column that control vertical ﬂux of matter) per- formed in the NW Mediterranean from September to Novem- ber 2004. This expected transition period from summertime oligotrophic stratiﬁed system to autumnal system was con- sidered favourable and pertinent because of higher frequen- cies of wind events. It was completing the DYNAPROC 1 cruise conducted in May 1995 in order to document the ef- fects of episodic wind events on the dynamics of the ecosys- tem during a post-bloom period, characterised by the transi- tion from a mesotrophic to an oligotrophic system (Andersen and Prieur, 2000). We also analyzed the intake of taxonom- ical studies in biogeochemical cycle’s studies, mass indica- tors or trophic aspects. This was feasible since DYNAPROC 2 was a multidisciplinary cruise that documented many hy- drological parameters and considerable stocks and rates mea- surements (Andersen et al., 2009). Two complementary sampling methods were used to sample the main microplankton groups, in order to obtain the most exhaustive and accurate description of the species possible, with emphasis put on phytoplanktonic groups. Hydrologi- cal bottle sampling allowed collecting small-sized and abun- dant microphytoplanktonic species. This method was also appropriate to preserve delicate or colonial organisms. 2.1 Cruise track The DYNAPROC 2 cruise was conducted in 2004 from 14 September to 17 October (Julian days 261 to 292) on board R/V Thalassa, at the vicinity of the NW Mediterranean French JGOFS Station DYFAMED (43◦25′ N, 7◦52′ E), ap- proximately 45 km South of Cape Ferrat, France, where max- imal depth is 2350 m. It was divided into two legs (here after leg 1 and leg 2) that consisted of two cycles of ﬁve days each (cycles 1 to 4). In order to describe the hydrological environ- ment, a grid of 16 stations, centred on a ﬁxed station (“central point”), was occupied at least four times during the cruise. All net and water sampling were done near the central point during the whole cruise. Many hydrological parameters are then available since the whole project consisted in evaluat- ing the DYNAmic of rapid PROCesses in water column that control vertical ﬂux of matter. In order to quantify the sta- bility of the water column, an index of the intensity of the stratiﬁcation was calculated as suggested by Bowden (1983) as: 8 = g/H ∫0 −H(ρm–ρ) zdz, where 8 is an index of poten- tial energy anomaly (J m−3), H is the water column height (m), ρ is the density at any depth z, and ρm is the mean den- sity of the water column. This index estimates the deﬁcit in potential energy due to a density gradient. A highly mixed water column will present small values of 8. Water density was derived from temperature-salinity measurements. Inte- grated values of 8 were obtained for the 0–90 m layer for all stations. More explanations on cruise development and hy- drological/meteorological evolutions could be found in An- dersen et al. (2009). 2.2 Field sampling Net sampling was the most appropriate to efﬁciently collect less abundant species (which were often the biggest) since the net ﬁltered a great volume of seawater. Dominant groups of the microzooplankton were also enumerated. 3 Hydrological bottle sampling Samples were collected every 10 m from 90 m to the sur- face with hydrological NISKIN bottles coupled with a Seabird CTD (Conductivity-Temperature-Depth) instrument that recorded continuously hydrological parameters (temper- ature, salinity, density and in situ ﬂuorescence). Aliquots of 250 ml of seawater from each depth, ﬁxed with acid lugol’s solution (ﬁnal concentration, 2% v/v; Throndsen, 1978) were stored in dark cold room (4 ◦C) until microscopic analysis. Samples were collected every day of each cycle (∼12.00 a.m., ITU), yielding a total of 200 samples. Aliquots were analysed using the Uterm¨ohl method with sedimenta- tion of 100 ml seawater in settling chambers. Counts and observations were done with an inverted light microscope (Zeiss©Axiovert 35), at 200x and 400x magniﬁcation. For each sample, a minimum of 250 living cells was counted. Instead of analysing strictly microphytoplankton species, which are formally deﬁned as ranging from 20 to 200µm, we preferred counting every species belonging to the 3 groups typical of the microphytoplankton: diatoms, dinoﬂagellates and silicoﬂagellates. For instance, some small species of the genus Gymnodinium, with a mean size lower than 10µm, were counted. Coccolithophorids were not counted, since acid lugol’s solution alters calciﬁed structures. Enumeration of microzooplanktonic groups (tintinnids and naked ciliates) was also carried out. Naked ciliates were separated in 3 size classes (equivalent diameter <30µm, between 30 and 50µm, >50µm). 1 Introduction Marine phytoplankton constitutes the base of the pelagic food-web and is responsible for about half of the net annual primary production on Earth (Field et al., 1998; Falkowski et al., 2004). Current studies on monitoring marine phyto- plankton are mainly based on satellite-derived values from remote sensing (Kyewalyanga et al., 2007) or deal with the pigment diversity (Marty et al., 2002), rather than examining changes in diversity or species composition. This is surpris- ing since it is generally believed that ecosystems functioning, stability and efﬁciency are linked to diversity (e.g., Loreau et al., 2001). Moreover, the few existing studies on marine phytoplankton communities assessed seasonal to pluriannual periods (Venrick et al., 1997; Caroppo et al., 1999; G´omez and Gorsky, 2003). Short-time scale studies of the phyto- plankton diversity dynamics (i.e. day to day or more often) have concerned lakes (Padis´ak et al., 1988; Padis´ak, 1993; Pinckney et al., 1998) or were based on pigment analyses or automated submerged ﬂow cytometer in seawater systems (Bidigare and Ondrusek, 1996; Thyssen et al., 2008). Short term dynamics of marine phytoplankton abundance and di- versity are then poorly documented even if phytoplankton could be considered as a good indicator for quick environ- mental changes (see review in Thyssen et al., 2008). In order to study the impact of natural disturbances (as wind events and changes in the mixing depth) on marine mi- crophytoplankton abundance and taxonomic diversity as well as related dynamics of main microzooplanktonic groups, we Correspondence to: R. Lem´ee (lemee@obs-vlfr.fr) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 744 4 Net sampling In addition to the hydrological bottle sampling, we also made daily net sampling during each cycle using a phytoplankton net (53µm mesh size, 54 cm diameter and 280 cm length). A vertical haul was performed from 90 m depth to the surface at the same time of the day (∼10.00 a.m., ITU) to collect large microplankton. The volume ﬁltered by the net was calculated as equivalent to the surface at the opening of the net multi- plied by the height of sampled water column. Due to bad Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ www.biogeosciences.net/8/743/2011/ 745 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity where pi = ni/N, ni = number of individuals of one taxon and N = total number of individuals. Evenness was estimated as Pielou’s index J′ = H′/log2 S. The net samples were di- vided into three replicates before counting. This allowed to apply the nonparametric jackknife 1 method (Manly, 1991), which gives estimations of the biodiversity parameters after correcting the bias induced by the sampling effort and the sampling size (Carpentier and Lepˆetre, 1999). For richness, jackknife 1 = SO+(r1(n-1)/n), where SO is the observed tax- onomic richness, n the number of replicates and r1 the num- ber of taxa occurring in one single replicate. For diversity and evenness, jackknife 1 = 68i/n, with 8i = nSt-(n-1)Sti-1, where St is the estimation of the indicator for the n replicates and Sti-1 the estimation of the indicator for the n-1 repli- cates. These estimations were calculated on a minimal basis of 100 counted cells per replicate, which represents a mini- mal total of 300 cells per sample. For diversity calculations, Magurran (2004) recommended to use abundances ranging from 200 to 500 cells per samples. The jackknife 1 method was not applied on bottle samples, for which no replicates were available. In order to identify the important genus or taxa within dinoﬂagellates and within genus Ceratium from net samples, a dominance index was also calculated as δ = 100(n1+n2)/N, where n1 and n2 are the abundances of the two most important taxa and N the total cell concentration (Hulburt, 1963). weather, there were no net samples for Julian days 285 and 289. The samples were split once or twice using a Motoda splitter (Motoda, 1959) and preserved with acid lugol’s solu- tion (Throndsen, 1978) to 2% ﬁnal concentration. 4 Net sampling The cells were enumerated in 1mL calibrated slides using a light mi- croscope (Alphaphot 2-YS2, Nikon Instruments, Badhoeve- dorp, Netherlands). More than 250 living cells were counted per samples. Dinoﬂagellates were identiﬁed at the genus level. We focussed more particularly on genus Ceratium Schrank since it is specious in the Mediterranean and often dominates among large armoured dinoﬂagellates, thus con- stituting a good model for biodiversity studies (Tunin-Ley et al., 2007). Determination of taxa belonging to genus Cer- atium was carried out at intraspeciﬁc level. Although they do not belong to phytoplankton, the total radiolarians were also enumerated in net samples, without detailed taxonomic identiﬁcation, because they exhibited signiﬁcant abundances. 4.1.1 Taxonomic identiﬁcation The species identiﬁcation of cells within the 3 selected groups was based on the following references: Tr´egouboff and Rose (1957a, b), Massuti and Margalef (1960), Dodge (1982), Sournia (1986), Ricard (1987), Balech (1988), Hasle and Syversten (1996), Steidinger and Tangen (1997) and Horner (2002). In net samples, Ceratium cells were iden- tiﬁed at the infraspeciﬁc level, using the monograph and the parasystematic nomenclature published by Sournia (1967). Recent molecular and morphological analysis assigned all marine Ceratium species to Neoceratium gen. nov. (G´omez et al., 2010). This new genus name was not used in this work since this naming is still in discussion among special- ists (Calado and Huisman, 2010). Moreover, it is rarely used by scientists working on ecology or diversity of microphy- toplankton. Some limits with regard to identiﬁcation con- strained our results. Thus, for small-sized cells, resolution of light microscopy and ﬁxation with acid lugol made some determinations difﬁcult. For instance, some Gymnodiniales (dinoﬂagellates) determination was completed to the genus level. However, different species were distinguished via size, form and ornamentation. In the same way, species name were not ascribed to some Pseudo-Nitzschia, which were dis- criminated as several species belonging to Pseudo-nitzchia delicatissima complex or Pseudo-nitzchia seriata complex (Hasle and Syversten, 1996). 4.2 Data analysis The variability of the main planktonic groups was analysed using a multivariate analysis producing a synthesis of co- occurring factors. The Correspondence Analysis (CA) aims to describe the total inertia of a multidimensional set of data in a sample of fewer dimensions (or axes) that is the best summary of the information contained in the data (Benzecri, 1973). The CA is applied to contingency tables and uses a Chi square metric. The different calculations of this method are detailed in Legendre and Legendre (2000). This ordina- tion method was applied to our matrix describing the plank- tonic groups in bottle samples (samples in rows and plank- tonic groups in columns), in order to underline the kinetics of plankton composition over time. The composite mean depths’ and the composite mean day’s coordinates, calcu- lated as the average of the samples of the same depth, or the average of the samples of the same day, were used as illus- trative qualitative variables. Spearman’s rank coefﬁcients (Siegel and Castellan 1988) were used in order to explore the correlations between plank- tonic groups diversity and the stratiﬁcation index, as estima- tion of a disturbance. 5.1.1 Meteorological data Wind regime was characterised by the alternation of strong wind events and calm periods. The leg 1 was accompanied with two signiﬁcant wind events (Fig. 1a), one at the begin- ning of cycle 1 (day 261 to day 262), which lasted 48 h, and a latter one during cycle 2 (day 269) with a highest intensity yet shorter in time. Both corresponded to NE winds, whereas winds are usually blowing from W or SW in the area at this period (Raybaud et al., 2008). By contrast, the end of leg 2 was dominated by persistent and strong NE and SW winds resulting in a decrease of stratiﬁcation (Fig. 4a). In addition, an episode with signiﬁcant precipitations occurred just be- fore the campaign started. Water temperature (°C) 0 (c) Depth (m) Salinity 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 −90 −80 −70 −60 −50 −40 −30 −20 −10 0 38.25 38.3 38.35 38.4 38.45 (c) 5.1.2 Hydrological and biological parameters (CTD acquisition) The hydrological structure along the main part of the cruise revealed a typical late summer situation with a stratiﬁed wa- ter column, whereas a transition from summer period to au- tumn structure was expected (i.e. destratiﬁcation). Thus, the thermocline ranged from 20 to 40 m depth (Fig. 1b), with sur- face temperatures above 20 ◦C. Salinity dynamics were char- acterised by the intrusion of Low Salinity Water (LSW) with likely coastal characteristics (< 38.3) during leg 1 (day 265 to day 274) just under the thermocline (Fig. 1c). This phe- nomenon, detailed by Andersen et al. (2009) and Raybaud et al. (2008), was also observed to a lesser degree during cycle 4 (day 283 to day 286). Each time, this resulted in a light shal- lowing of the thermocline, as a consequence of the dilatation of the less dense water mass. Along with a deepening of the temperature and the halocline, the destratiﬁcation seemed to be initiated at the end of the cruise. The signiﬁcant decrease of the stratiﬁcation index observed at this period (Fig. 4a) de- picted the disturbance that occurred in response to the regime of persistent and strong wind at this time. At the begin- ning of cycle 1, the ﬂuorescence measurements displayed a chlorophyll-a peak about 70–80 m depth, followed by an- other distinctive one about 50 m depth (Fig. 1d). All along the second part of the cruise, the Deep Chlorophyll Maxi- mum (DCM) was located just under the thermocline, follow- ing its shift to a deeper depth. Consequently, the in-vivo ﬂu- orescence evolution showed up tendency to get deeper at the end of campaign. Depth (m) Relative fluorescence 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 −90 −80 −70 −60 −50 −40 −30 −20 −10 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Julian days (d) Fig. 1. Hydrological conditions during DYNAPROC 2 cruise, (a) Wind strength, (b) Water temperature, (c) Salinity, (d) Relative ﬂuorescence. most diverse group was dinoﬂagellate, with 92 species (32 armoured and 60 unarmoured), followed by 55 species of di- atoms (35 centrics and 25 pennates). We found only 2 species of silicoﬂagellates. Additionally we encountered Leptocylin- drus mediterraneus cells (centric diatom), but they were inte- grated neither in abundance nor in diversity calculation since they were not alive. 5.1.2 Hydrological and biological parameters (CTD acquisition) Near 58% of taxa were determined to the species level, 98% to genus level; the rest was composed of indeterminate armoured dinoﬂagellates. Maximum abun- dances for each taxa over the whole cruise period and the whole water column were indicated in Table 1. The maxi- mum abundance of total microphytoplankton was often just under the thermocline and reached more than 40 000 ind l−1 at the beginning of cycle 4 (Fig. 2a). Throughout the study, diatoms were mostly in the lower depths (Fig. 2b), with abun- dance ranging from 200 to 800 cell l−1 in surface and from 1000 up to 5000 cell l−1 below the thermocline. Temporally, two distinct peaks of abundance were observed. The ﬁrst one (from 4000 to 5000 cell l−1) occurred at 80 m depth at the beginning of the ﬁrst cycle (Julian day 262) and was 5 Results 5.1 Physical environment 5.1.1 Meteorological data 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 0 10 20 30 40 50 Wind strength (Kts) Depth (m) Water temperature (°C) 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 −90 −80 −70 −60 −50 −40 −30 −20 −10 0 14 15 16 17 18 19 20 Depth (m) Salinity 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 −90 −80 −70 −60 −50 −40 −30 −20 −10 0 38.25 38.3 38.35 38.4 38.45 Depth (m) Relative fluorescence 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 −90 −80 −70 −60 −50 −40 −30 −20 −10 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Julian days (a) (b) (c) (d) Fig. 1. Hydrological conditions during DYNAPROC 2 cruise, (a) Wind strength, (b) Water temperature, (c) Salinity, (d) Relative ﬂuorescence. 5 Results 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 0 10 20 30 40 50 Wind strength (Kts) Depth (m) Water temperature (°C) 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 −90 −80 −70 −60 −50 −40 −30 −20 −10 0 14 15 16 17 18 19 20 Salinity (a) (b) (c) 4.1.2 Diversity indexes Biodiversity parameters were calculated for the main plank- tonic groups in bottle samples on one hand, and in net sam- ples on the other hand. The taxonomic richness S corre- sponds to the number of taxa in each group. The diversity was calculated using Shannon’s index H′ = −Ppi log2 pi, Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 746 Biogeosciences, 8, 743–761, 2011 5.2.1 Abundances Summing over the whole cruise, 151 species of microphy- toplankton were found in hydrological bottle samples. The Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 747 Table 1. List of microphytoplanktonic species encountered in hydrological bottle samples during DYNAPROC 2 cruise, wit abundance recorded in samples in cells per liter. Species Authorities Max. abundance Bacillariophyceae Achnanthes sp. a Bory 60 Bacteriastrum delicatulum Cleve 100 Bacteriastrum furcatum Shadbolt 60 Cerataulina dentata Hasle 180 Cerataulina pelagica (Cleve) Hendey 20 Chaetoceros afﬁnis Lauder 60 Chaetoceros decipiens Cleve 60 Chaetoceros didymus Ehrenberg 80 Chaetoceros laciniosus Sch¨utt 400 Chaetoceros peruvianus Brightwell 80 Chaetoceros sp. a Ehrenberg 40 Chaetoceros sp. b Ehrenberg 240 Climacosphenia sp. a Ehrenberg 120 Corethron sp. Castracane 3240 Coscinodiscus sp. a Ehrenberg 120 Coscinodiscus sp. b Ehrenberg 20 Cylindrotheca closterium (Ehrenberg) Reimann & Lewin 180 Dactyliosolen phuketensis (Sundstr¨om) Hasle 600 Entomoneis sp. a Ehrenberg 20 Gossleriella tropica Sh¨utt 40 Guinardia striata (Stolterfoth) Hasle 200 Haslea wawrikae (Husedt) Simonsen 140 Hemiaulus hauckii Grunow ex Van Heurck 10 Hemidiscus cuneiformis Wallich 20 Lauderia annulata Cleve 150 Leptocylindrus danicus Cleve 140 Leptocylindrus minimus Gran 240 Lyrella sp. a Karajeva 20 Navicula sp. a Bory 50 Navicula sp. b Bory 40 Navicula sp. c Bory 40 Navicula sp. d Bory 2800 Nitzschia bicapitata Cleve 670 Planktonielle sol (Wallich) Sch¨utt 30 Pleurosigma sp. a Smith 20 Proboscia alata (Brightwell) Sundstr¨om 160 Pseudo-nitzschia sp. a (Nitzschia delicatissima complex) Peragallo 760 Pseudo-nitzschia sp. b (Nitzschia delicatissima complex) Peragallo 1300 Pseudo-nitzschia sp. c (Nitzschia seriata complex) Peragallo 800 Pseudo-nitzschia sp. d (Nitzschia seriata complex) Peragallo 900 Pseudo-nitzschia sp. e (Nitzschia seriata complex) Peragallo 360 Pseudo-nitzschia sp. f (Nitzschia seriata complex) Peragallo 380 Pseudo-nitzschia sp. g (Nitzschia seriata complex) Peragallo 100 Rhizosolenia decipiens Sundstr¨om 60 Rhizosolenia hebetata f. semispina (Hensen) Gran 40 Synedra pulchella K¨utzing 160 Synedra sp. a Ehrenberg 20 Thalassionema frauenfeldii (Grunow) Hallegraeff 280 Table 1. List of microphytoplanktonic species encountered in hydrological bottle samples during DYNAPROC 2 cruise, with maximal abundance recorded in samples in cells per liter. Table 1. List of microphytoplanktonic species encountered in hydrological bottle samples during DYNAPROC 2 cruise, with maximal abundance recorded in samples in cells per liter. Table 1. List of microphytoplanktonic species encountered in hydrological bottle samples during DYNAPROC 2 cruise, with maximal abundance recorded in samples in cells per liter. www.biogeosciences.net/8/743/2011/ 5.2.1 Abundances anktonic species encountered in hydrological bottle samples during DYNAPROC 2 cruise, with maximal in cells per liter. Max. abundance Bacillariophyceae Achnanthes sp. a Bory 60 Bacteriastrum delicatulum Cleve 100 Bacteriastrum furcatum Shadbolt 60 Cerataulina dentata Hasle 180 Cerataulina pelagica (Cleve) Hendey 20 Chaetoceros afﬁnis Lauder 60 Chaetoceros decipiens Cleve 60 Chaetoceros didymus Ehrenberg 80 Chaetoceros laciniosus Sch¨utt 400 Chaetoceros peruvianus Brightwell 80 Chaetoceros sp. a Ehrenberg 40 Chaetoceros sp. b Ehrenberg 240 Climacosphenia sp. a Ehrenberg 120 Corethron sp. Castracane 3240 Coscinodiscus sp. a Ehrenberg 120 Coscinodiscus sp. b Ehrenberg 20 Cylindrotheca closterium (Ehrenberg) Reimann & Lewin 180 Dactyliosolen phuketensis (Sundstr¨om) Hasle 600 Entomoneis sp. a Ehrenberg 20 Gossleriella tropica Sh¨utt 40 Guinardia striata (Stolterfoth) Hasle 200 Haslea wawrikae (Husedt) Simonsen 140 Hemiaulus hauckii Grunow ex Van Heurck 10 Hemidiscus cuneiformis Wallich 20 Lauderia annulata Cleve 150 Leptocylindrus danicus Cleve 140 Leptocylindrus minimus Gran 240 Lyrella sp. a Karajeva 20 Navicula sp. a Bory 50 Navicula sp. b Bory 40 Navicula sp. c Bory 40 Navicula sp. d Bory 2800 Nitzschia bicapitata Cleve 670 Planktonielle sol (Wallich) Sch¨utt 30 Pleurosigma sp. a Smith 20 Proboscia alata (Brightwell) Sundstr¨om 160 Pseudo-nitzschia sp. a (Nitzschia delicatissima complex) Peragallo 760 Pseudo-nitzschia sp. b (Nitzschia delicatissima complex) Peragallo 1300 Pseudo-nitzschia sp. c (Nitzschia seriata complex) Peragallo 800 Pseudo-nitzschia sp. d (Nitzschia seriata complex) Peragallo 900 Pseudo-nitzschia sp. e (Nitzschia seriata complex) Peragallo 360 Pseudo-nitzschia sp. f (Nitzschia seriata complex) Peragallo 380 Pseudo-nitzschia sp. g (Nitzschia seriata complex) Peragallo 100 Rhizosolenia decipiens Sundstr¨om 60 Rhizosolenia hebetata f. semispina (Hensen) Gran 40 Synedra pulchella K¨utzing 160 Synedra sp. a Ehrenberg 20 Thalassionema frauenfeldii (Grunow) Hallegraeff 280 Thalassionema nitzschioides (Grunow) Mereschkowsky 580 Thalassionema sp. a Grunow ex Mereschkowsky 740 Thalassiosira sp. a Cleve 260 Thalassiosira sp. b Cleve 240 Thalassiosira sp. c Cleve 380 Thalassiosira sp. d Cleve 1200 Toxarium undulatum Bailey 20 Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Table 1. Continued. Spiraulax kofoidii Graham 40 Tryblionella compressa (Bailey) Poulin 280 Undeterminated armoured Dinoﬂagellate sp. a 40 Undeterminated armoured Dinoﬂagellate sp. b 20 Undeterminated armoured Dinoﬂagellate sp. c 20 Naked species Amphidinium sp. a Clapar`ede & Lachmann 4000 Amphidinium sp. b Clapar`ede & Lachmann 4020 Asterodinium gracile Sournia 20 Cochlodinium sp. a Sch¨utt 360 Dissodinium pseudolunula Swift ex Elbr¨achter & Drebes 20 Gymnodinium sp. a Stein 20000 Gymnodinium sp. b Stein 800 Gymnodinium sp. c Stein 5600 Gymnodinium sp. d Stein 8000 Gymnodinium sp. e Stein 160 Gymnodinium sp. f Stein 2400 Gymnodinium sp. g Stein 20 Gymnodinium sp. h Stein 60 Gymnodinium sp. i Stein 2400 Gymnodinium sp. j Stein 16800 Gymnodinium sp. k Stein 20 Gymnodinium sp. l Stein 60 Gyrodinium falcatum Kofoid & Swezy 20 Gyrodinium sp. a Kofoid & Swezy 7200 Gyrodinium sp. b Kofoid & Swezy 8800 Gyrodinium sp. c Kofoid & Swezy 2800 Gyrodinium sp. d Kofoid & Swezy 3500 Gyrodinium sp. e Kofoid & Swezy 1340 Gyrodinium sp. f Kofoid & Swezy 1400 Gyrodinium sp. g Kofoid & Swezy 2000 Gyrodinium sp. h Kofoid & Swezy 1200 Gyrodinium sp. i Kofoid & Swezy 20 Kofoidinium velelloides Pavillard 100 Petalodinium porcelio Cachon & Cachon 60 Polykrikos kofoidii Chatton 80 Pronoctiluca pelagica Fabre-Domergue 160 Scaphodinium mirabile Margalef 20 Torodinium teredo (Pouchet) Kofoid & Swezy 140 Dictyochophyceae Dictyocha ﬁbula Ehrenberg 420 Dictyocha speculum Ehrenberg 750 In contrast to the diatoms, dinoﬂagellates showed a pref- erence for the upper layers during the whole cruise (Fig. 2c). The group of naked dinoﬂagellates, mainly represented by small-sized species Gyrodinium spp. and Gymnodinium spp., dominated in both legs. Abundance increased from 5000 to 20 000 ind l−1 during leg 1 to 10 000 to 35 000 ind l−1 during cyles 3 and 4. The observed armoured species Scrippsiella sp. was probably associated with the intrusions of less saline water (Fig. 3b). Silicoﬂagellates (Dictyochophyceae) abun- dance was lowest in comparison with the two other groups mainly the centric species Corethron sp. probably in senes- cent phase since intracellular part was sometimes affected. The second peak occurred at 50 m depth during cycle 3 (Ju- lian day 278) and was mainly composed of pennate species represented by the complexes Pseudo-nitzchia delicatissima spp. and Pseudo-nitzchia seriata spp. In samples of cycles 1 and 2, we also found the centric species Leptocylindrus mediterraneus and its epiphyte (Fig. 3a), the heterotrophic ﬂagellate Rhizomonas setigera. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 748 Table 1. Continued. Dinophyceae Armoured species Alexandrium sp. a Halim 1200 Alexandrium sp. b Halim 800 Amylax triachanta (J¨orgensen) Sournia 20 Ceratium declinatum (Karsten) J¨orgensen 20 Ceratium furca (Ehrenberg) Clapar`ede & Lachmann 20 Ceratium fusus (Ehrenberg) Dujardin 20 Ceratium hexacanthum Gourret 20 Ceratium horridum (Cleve) Gran 20 Ceratium limulus Gourret 10 Ceratium lineatum (Ehrenberg) Cleve 20 Ceratium pentagonum Gourret 50 Ceratium platycorne Daday 20 Ceratocorys sp. a Stein 40 Dinophysis acuminata Clarap`ede & Lachmann 20 Dinophysis acuta Ehrenberg 20 Dinophysis dens Pavillard 10 Dinophysis hastata Stein 20 Dinophysis pulchella (Lebour) Balech 60 Dinophysis punctata J¨orgensen 40 Dinophysis sphaerica Stein 40 Diplopsalis sp. a Bergh 2400 Gonyaulax digitale (Pouchet) Kofoid 20 Gonyaulax polygramma Stein 20 Gonyaulax sp. a Diesing 20 Gonyaulax spinifera (Clapar`ede & Lachmann) Diesing 40 Heterocapsa rotundata (Lohmann) Hansen 1800 Heterodinium leiorhynchum (Murray & Whitting) Kofoid 80 Heterodinium sp. a Kofoid 20 Heterodinium sp. b Kofoid 20 Heterodinium sp. b Kofoid 20 Katodinium glaucum (Lebour) Loeblich III 4400 Lingulodinium polyedrum (Stein) Dodge 40 Oblea sp. a Balech ex Loeblich & Loeblich 70 Oxytoxum constrictum (Stein) B¨utschli 20 Oxytoxum laticeps Schiller 40 Oxytoxum longiceps Schiller 40 Oxytoxum milneri Murray & Whitting 20 Oxytoxum reticulatum (Stein) Sch¨utt 40 Oxytoxum scolopax Stein 40 Oxytoxum tesselatum Stein 50 Pavillardinium sp. a De Toni 40 Phalacroma rapa Jorgensen 10 Phalacroma rotundatum (Clapar`ede & Lachmann) Kofoid & Michener 60 Phalacroma sp. a Stein 20 Podolampas palmipes Stein 40 Podolampas spinifera Okamura 60 Prorocentrum gracile Sch¨utt 510 Prorocentrum scutellum Schr¨oder 20 Prorocentrum triestinum Schiller 300 Protoperidinium cassum Balech (Balech) 300 Protoperidinium divergens (Ehrenberg) Balech 20 Protoperidinium ovatum Pouchet 20 Protoperidinium pyriforme (Paulsen) Balech 20 Protoperidinium sp. a Bergh 800 Schuettiella mitra (Sch¨utt) Balech 460 Scrippsiella sp. a (Stein) Loeblich III 12800 www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 749 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 750 Table 2. List of dinoﬂagellates genera and Ceratium taxa encountered in net samples during DYNAPROC 2 cruise, with maximal abu recorded in samples in cells per liter. Dinoﬂagellates Max. abundance Genus Ceratium Max. abundanc Acanthogonyaulax (Kofoid) Graham 6 C. arietinum Cleve var. arietinum 10 Amphidiniopsis Woloszynska 3 C. arietinum var. gracilentum (Jørgensen) Sournia 3 Amphisolenia Stein 8 C. arietinum Cleve “arietinum-gracilentum” 71 Centrodinium Kofoid 11 C. azoricum Cleve 7 Ceratium Schrank 3734 C. candelabrum (Ehrenberg) Stein var. candelabrum 61 Ceratocorys Stein 105 C. candelabrum var. depressum (Pouchet) Jørgensen 70 Corythodinium Loeblich Jr. & Loeblich III 134 C. candelabrum (Ehrenberg) Stein “candelabrum-depressum” 30 Dinophysis Ehrenberg 237 C. candelabrum(Ehrenberg) Stein“candelabrum-depressum” 24 Dissodinium Klebs in Pascher 36 C. candelabrum (Ehrenberg) Stein “depressum-candelabrum” 35 Goniodoma Stein 1420 C. carriense Gourret var. carriense 16 Gonyaulax Diesing 465 C. carriense var. volans (Cleve) Jørgensen 18 Heterocapsa Stein 26 C. carriense Gourret “carriense-volans” 3 Heterodinium Kofoid 30 C. concilians Jørgensen 237 Kofoidinium Pavillard 81 C. contrarium (Gourret) Pavillard 71 Micracanthodinium Deﬂandre 3 C. declinatum f. brachiatum Jørgensen 145 Noctiluca Suriray 21 C. declinatum f. normale Jørgensen 145 Ornithocercus Stein 16 C. declinatum var. majus Jørgensen 30 Oxytoxum Stein 60 C. euarcuatum Jørgensen 26 Palaeophalacroma Schiller 7 C. extensum (Gourret) Cleve 65 Phalacroma Stein 346 C. falcatiforme Jørgensen 35 Podolampas Stein 289 C. falcatum (Kofoid) Jørgensen 16 Prorocentrum Ehrenberg 17 C. furca var. eugrammum (Ehrenberg) Schiller 59 Protoperidinium Bergh 342 C. furca (Ehrenberg) Clarap`ede & Lachmann “furca-eugrammum” 4 Pyrophacus Stein 42 C. furca (Ehrenberg) Clarap`ede & Lachmann “furca-eugrammum” 59 Pyrocystis Murray ex Haeckel 29 C. furca (Ehrenberg) Clarap`ede & Lachmann “eugrammum-furca” 14 Spatulodinium Cachon & Cachon 53 C. fusus (Ehrenberg) Dujardin 149 Spiraulax Kofoid 79 C. gibberum var. dispar (Pouchet) Sournia 19 C. gravidum Gourret 7 No genera: 27 C. hexacanthum f. hiemale Pavillard 4 C. hexacanthum Gourret var. hexacanthum 5 C. horridum var. buceros (Zacharias) Sournia 13 C. horridum (Cleve) Gran “horridum-buceros” 14 C. horridum (Cleve) Gran “buceros-horridum” 298 C. inﬂatum (Kofoid) Jørgensen 4 C. limulus (Gourret ex Pouchet) Gourret 219 C. longirostrum Gourret 30 C. longissimum (Schr¨oder) Kofoid 4 C. macroceros (Ehrenberg) Vanh¨offen var. macroceros 32 C. macroceros (Ehrenberg) Vanh¨offen “macroceros-gallicum” 24 C. massiliense f. armatum (Karsten) Jørgensen 5 C. massiliense (Gourret) Jørgensen var. massiliense 11 C. massiliense var. protuberans (Karsten) Jørgensen 32 C. minutum Jørgensen 11 C. www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity The k d h d f b d h Total microphytoplankton Diatoms 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 150 250 500 1000 2000 3000 4000 Dinoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 5000 10000 15000 20000 25000 30000 35000 40000 25000 10000 25000 Silicoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 50 100 200 300 400 500 600 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 D 5000 10000 15000 20000 (b) (c) (d) Cell.l-1 Cell.l-1 Cell.l-1 dance of microphytoplankton during DYNAPROC 2 cruise, from hydrological bottle sampling (a) Total microphytoplankton, phyceae, (c) Dinophyceae, (d) Dictyochophyceae. Julian days Diatoms 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 150 250 500 1000 2000 3000 4000 (b) Cell.l-1 (b) 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 Depth (m) 150 250 500 1000 2000 3000 Dinoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 5000 10000 15000 20000 25000 30000 35000 40000 25000 10000 25000 Silicoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 50 100 200 300 400 500 600 (c) (d) Cell.l-1 Cell.l-1 Fig. 2. Abundance of microphytoplankton during DYNAPROC 2 cruise, from hydrological bottle sampling (a) Total microphyto (b) Bacillariophyceae, (c) Dinophyceae, (d) Dictyochophyceae. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 751 Total microphytoplankton 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 5000 10000 15000 20000 25000 30000 35000 40000 (a) Cell.l-1 Diatoms 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 150 250 500 1000 2000 3000 4000 Dinoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 5000 10000 15000 20000 25000 30000 35000 40000 25000 10000 25000 Silicoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 50 100 200 300 400 500 600 Total microphytoplankton 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 5000 10000 15000 20000 25000 30000 35000 40000 (a) (b) (c) (d) Cell.l-1 Cell.l-1 Cell.l-1 Cell.l-1 Fig. 2. Abundance of microphytoplankton during DYNAPROC 2 cruise, from hydrological bottle sampling (a) Total mic (b) Bacillariophyceae, (c) Dinophyceae, (d) Dictyochophyceae. (Fig. 2d). Two species were found, Dictyocha ﬁbula and Dictyocha speculum, the ﬁrst one being the most abundant. Sili ﬂ ll h d k d i l i i dance were identiﬁable (days 264–265 and 27 from 400 cell l−1 up to 600 cell l−1). S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity paradoxides Cleve 3 C. pavillardii Jørgensen 12 C. pentagonum f. turgidum (Jørgensen) Jørgensen 80 C. pentagonum var. tenerum Jørgensen 316 C. pentagonum Gourret “robustum-tenerum” 62 C. pentagonum Gourret “robustum-tenerum” 1587 C. platycorne Daday var.platycorne 96 C. platycorne var.compressum (Gran) Jørgensen 267 C. platycorne Daday “platycorne-compressum” 56 C. platycorne Daday “compressum-platycorne” 99 C. ranipes Cleve 22 C. schroeteri Schr¨oder 3 C. setaceum Jørgensen 24 C. symmetricum Pavillard var. symmetricum 113 C. symmetricum var. coarctatum (Pavillard) Graham & Bronikovsky 151 C. symmetricum var. orthoceras (Jørgensen) Graham & Bronikovsky 17 C. trichoceros (Ehrenberg) Kofoid 6 C. tripos var. atlanticum (Ostenfeld) Paulsen 83 C. tripos var. pulchellum (Schr¨oder) Lopez 97 C. tripos (M¨uller) Nitzsch “atlanticum-pulchellum” 9 C. tripos (M¨uller) Nitzsch “atlanticum-pulchellum” 35 No species: 34 No infraspeciﬁc taxa: 64 Table 2. List of dinoﬂagellates genera and Ceratium taxa encountered in net samples during DYNAPROC 2 cruise, with maximal abundance recorded in samples in cells per liter. Table 2. List of dinoﬂagellates genera and Ceratium taxa encountered in net samples during DYNAPROC 2 cruise, with maximal abundance recorded in samples in cells per liter. www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Julian days Dinoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 5000 10000 15000 20000 25000 30000 35000 40000 25000 10000 25000 (c) Cell.l-1 (c) Julian days Silicoflagellates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 50 100 200 300 400 500 600 (d) Cell.l-1 (d) Fig. 2. Abundance of microphytoplankton during DYNAPROC 2 cruise, from hydrological bottle sampling (a) Total microphytoplankton, (b) Bacillariophyceae, (c) Dinophyceae, (d) Dictyochophyceae. (Fig. 2d). Two species were found, Dictyocha ﬁbula and Dictyocha speculum, the ﬁrst one being the most abundant. Silicoﬂagellates showed marked spatio-temporal variations. Abundance varied from less than 50 cell l−1 to more than 600 cell l−1. During the 2 ﬁrst cycles, two maxima of abun- dance were identiﬁable (days 264–265 and 270 with a range from 400 cell l−1 up to 600 cell l−1). The end of cycle 2 marked the decrease of abundance that appeared then really weak during the two last cycles. Finally, the evolution of naked ciliates abundances (Fig. 3c) showed a slight increase www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Scrippsiella sp. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 0 200 1000 2500 5000 7500 10000 7500 Leptocylindrus mediterraneus 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 0 100 200 400 600 800 1000 Total naked ciliates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Julian days -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 0 250 500 1000 1500 2000 (a) (b) (c) Cell.l-1 Cell.l-1 Cell.l-1 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 0 5000 10000 15000 Abundances (cell.m-3) Microplankton in net samples Total Radiolarians Ceratium species Other Dinoflagellates (d) of ecologically important planktonic organisms during DYNAPROC 2 cruise, from hydrological bottle sampling (a) The us mediterraneus, (b) The dinoﬂagellate Scrippsiella sp., (c) Naked ciliates, and from net sampling (d) Dinoﬂagellates m infraspeciﬁc taxa. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 752 Leptocylindrus mediterraneus 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 0 100 200 400 600 800 1000 (a) Cell.l-1 Leptocylindrus mediterraneus Scrippsiella sp. 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 0 200 1000 2500 5000 7500 10000 7500 (b) Cell.l-1 (b) Scrippsiella sp. Total naked ciliates 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 -90 -80 -70 -60 -50 -40 -30 -20 -10 0 Depth (m) 0 250 500 1000 1500 2000 (c) Cell.l-1 (c) Julian days 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 0 5000 10000 15000 Abundances (cell.m-3) Microplankton in net samples Total Radiolarians Ceratium species Other Dinoflagellates (d) (d) Microplankton in net samples Fig. 3. Abundance of ecologically important planktonic organisms during DYNAPROC 2 cruise, from hydrological bottle sampling (a) The diatom Leptocylindrus mediterraneus, (b) The dinoﬂagellate Scrippsiella sp., (c) Naked ciliates, and from net sampling (d) Dinoﬂagellates genera and Ceratium infraspeciﬁc taxa. (from 828 to 5344 cell m−3), while Ceratium spp. repre- sented about 50% (from 834 to 3734 cell m−3) of the total dinoﬂagellates. Ceratium became largely dominant within dinoﬂagellates at the end of the cruise, while the total mi- croplankton abundance decreased. The maximum abun- dances of each taxa from net sampling are indicated in Ta- ble 2. from 500 to 1250 cell l−1 during the ﬁrst cycles leading to maximal value of 2000 cell l−1 in cycles 3 and 4. In each sample, the great majority (more than 90%) of ciliates was of small size (less than 30µm). Microplankton abundance in net samples, corresponding to radiolarians and large armoured dinoﬂagellates, decreased throughout the study, from over 8000 cells per m3 at the be- ginning to about 3000 cells per m3 at the end of the cruise (Fig. 3d). Overall, radiolarians were the dominant group www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 753 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 50 100 150 200 250 Index of stratification 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 0 50 100 Number of taxa Taxonomic Richness Ceratium Infraspecific taxa Dinoflagellates Total Microplankton Centric Diatoms Pennate Diatoms Armoured Dinoflagellates Naked Dinoflagellates 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 0 2 4 6 Diversity Shannon’s Index 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 0 0.5 1 Evenness Pielou’s Index Net sampling Bottle sampling (a) (b) (c) (d) Fig. 4. Evolution of the stratiﬁcation index in relation to the biodiversity descriptors of the main groups of the microphytoplankton during DYNAPROC 2 cruise, from hydrological bottle and net sampling (a) Stratiﬁcation index, (b) Taxonomic richness, (c) Diversity Shannon’s index, (d) Evenness Pielou’s index. 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 50 100 150 200 250 Index of stratification 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 0 50 100 Number of taxa Taxonomic Richness Ceratium Infraspecific taxa Dinoflagellates Total Microplankton Centric Diatoms Pennate Diatoms Armoured Dinoflagellates Naked Dinoflagellates 6 Diversity Shannon’s Index Net sampling Bottle sampling (a) (b) (c) 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 50 100 150 200 250 Index of stratification (a) Index of stratification Index of stratification Net sampling 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 0 2 4 6 Evenness Pielou’s Index (d) 260 262 264 266 268 270 272 274 276 278 280 282 284 286 288 0 0.5 1 Fig. 4. Evolution of the stratiﬁcation index in relation to the biodiversity descriptors of the main groups of the microphytoplankton during DYNAPROC 2 cruise, from hydrological bottle and net sampling (a) Stratiﬁcation index, (b) Taxonomic richness, (c) Diversity Shannon’s index, (d) Evenness Pielou’s index. Table 3. Spearman rank correlations between the stratiﬁcation index (8) and taxonomic richness, diversity Shannon index and evenness Pielou index of the microplankton components. ∗Correlation is signiﬁcant at the 0.05 level; g ; ∗∗∗Correlation is signiﬁcant at the 0.001 level. ∗∗Correlation is signiﬁcant at the 0.01 level; www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Taxonomic richness S Diversity Shannon index H′ Evenness Pielou Index J′ Microphytoplankton −0.82 0.78* 0.84* Diatoms −0.73* −0.47* −0.02 Centric diatoms −0.53* 0.55* 0.76*** Pennate diatoms −0.72* −0.58* 0.31 Dinoﬂagellates −0.77 0.81* 0.71*** Armoured dinoﬂagellates −0.57* 0.49* 0.33 Naked dinoﬂagellates −0.85* 0.40 0.75* www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 754 40 50 60 70 80 90 100 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Gonyaulax Ceratium / Gonyaulax Ceratium / Gonyaulax Ceratium / Podolampas Ceratium / Gonyaulax Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Gonyaulax Ceratium / Gonyaulax Dominance index % (Dinoflagellates genera) Julian day 40 50 60 70 80 90 100 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. horridum var. buceros C. pentagonum var. tenerum / C. symmetricum var. orthoceras C. pentagonum var. tenerum / C. limulus C. pentagonum var. tenerum / C. concilians C. pentagonum var. tenerum / C. horridum var. buceros C. pentagonum var. tenerum / C. declinatum f. brachiatum C. pentagonum var. tenerum / C. declinatum f. normale C. pentagonum var. tenerum / C. declinatum f. normale C. pentagonum var. tenerum / C. declinatum f. normale C. pentagonum var. tenerum / C. concilians C. pentagonum var. tenerum / C. horridum var. buceros C. pentagonum var. tenerum / C. platycorne "platycorne-compressum" C. pentagonum var. tenerum / C. pentagonum "robustum>tenerum" Dominance index % (Ceratium infraspecific taxa) Julian day (a) (b) volution of the dominance index in net samples (a) Dinoﬂagellates genera dominance index, (b) Ceratium taxa dominance index. 5.2.2 Diversity patterns showed the same feature. Yet, there was a trend of decreas- ing diversity of armoured species within dinoﬂagellates and of the diversity of centric species within diatoms (Fig. 4c). Actually, the few variations in diversity resulted in opposite trends in richness and evenness indexes. Indeed, the even- ness index sensibly decreased for total microphytoplankton (Fig. 4d), and was mainly driven by a pattern of decreasing dinoﬂagellates evenness, both displayed by armoured species and by naked species to a lesser extent. In contrast, the evenness of total diatoms remained at a high level without any clear trend of variation. Evenness of pennate diatoms was constant whereas it slowly decreased for centric species (Fig. 4d). Thus, while overall diversity appeared to be quite invariant, important variations occurred among speciﬁc tax- onomic groups. Exploring the relationship between biodi- versity and the stratiﬁcation index, we were able to highlight a trend: among each microplanktonic communities, the tax- onomic richness was negatively related to the stratiﬁcation index (Table 3). We observed higher diversity indexes of di- noﬂagellates and centric diatoms associated to stratiﬁed wa- ters (higher 8) while total diatoms and particularly pennate group diversity was negatively correlated to the stratiﬁcation index (Spearman coefﬁcient Rs = −0.58, P < 0.05; Table 3). The total richness of the microphytoplankton from hydrolog- ical bottle samples varied from 47 to 100 species (Fig. 4b). The total richness slightly increased during the ﬁrst cycles, reached a maximum during the cycle 3 and then decreased during cycle 4 although values stayed superior to the rich- ness observed in leg 1. Diatoms and dinoﬂagellates respec- tive richness followed the same pattern than total microphy- toplankton, but the increase was more marked for dinoﬂag- ellates richness. Number of diatoms species ranged from 17 to 39, with nearly equal portions of pennates (10 to 21 species) and centrics (7 to 18 species) (Fig. 4b). Dinoﬂag- ellate species richness ranged from 28 to 63 species, with a similar contribution of armoured species (14 to 32 species) and naked species (12 to 31 species). In net samples, while a total of 27 dinoﬂagellates genera was described (Table 2), the generic richness in dinoﬂagel- lates was about 20 genera along the study (Fig. 4b). S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 40 50 60 70 80 90 100 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Gonyaulax Ceratium / Gonyaulax Ceratium / Gonyaulax Ceratium / Podolampas Ceratium / Gonyaulax Ceratium / Goniodoma Ceratium / Goniodoma Ceratium / Gonyaulax Ceratium / Gonyaulax Dominance index % (Dinoflagellates genera) Julian day (a) Dominance index % (Dinoflagellates genera) 40 50 60 70 80 90 100 261 263 265 267 269 271 273 275 277 279 281 283 285 287 289 C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. pentagonum "robustum-tenerum" C. pentagonum var. tenerum / C. horridum var. buceros C. pentagonum var. tenerum / C. symmetricum var. orthoceras C. pentagonum var. tenerum / C. limulus C. pentagonum var. tenerum / C. concilians C. pentagonum var. tenerum / C. horridum var. buceros C. pentagonum var. tenerum / C. declinatum f. brachiatum C. pentagonum var. tenerum / C. declinatum f. normale C. pentagonum var. tenerum / C. declinatum f. normale C. pentagonum var. tenerum / C. declinatum f. normale C. pentagonum var. tenerum / C. concilians C. pentagonum var. tenerum / C. horridum var. buceros C. pentagonum var. tenerum / C. platycorne "platycorne-compressum" C. pentagonum var. tenerum / C. pentagonum "robustum>tenerum" Dominance index % (Ceratium infraspecific taxa) Julian day (b) Fig. 5. Evolution of the dominance index in net samples (a) Dinoﬂagellates genera dominance index, (b) Ceratium taxa dominance index. Dominance index % (Ceratium infraspecific taxa) ex in net samples (a) Dinoﬂagellates genera dominance index, (b) Ceratium taxa dominance index. Fig. 5. Evolution of the dominance index in net samples (a) Dinoﬂagellates genera dominance index, (b) Ceratium taxa dominance index. Biogeosciences, 8, 743–761, 2011 5.2.2 Diversity patterns -2 -1.5 -1 -0.5 0 0.5 1 -1.5 -1 -0.5 0 0.5 1 1.5 261 262 263 264 265 269 270 271 272 273 278 279 280 281 282 284 285 286 287 288 289 10m 20m 30m 40m 5m 50m 60m 70m 80m 90m Centric Diatoms Pennate Diatoms Armoured Dinoflagellates Naked Dinoflagellates Dictyochophyceae Ciliates<30 µm Ciliates 30-50 µm Ciliates>50 µm Leg 1 (Julian days) Leg 2 (Julian days) Depth Z Axis 1 (54.8%) Axis 2 (24.3%) In term of dominance, the genus Ceratium was prominent all along the cruise within the dinoﬂagellates, and was asso- ciated to the genus Goniodoma during the ﬁrst leg, then alter- natively to the genera Goniodoma, Gonyaulax and Podolam- pas (Fig. 5a). The association Ceratium/Podolampas at the middle of cycle 3 corresponded to a minimum of the domi- nance index (about 50%) whereas during the last cycle, the dominance of the association Ceratium/Gonyaulax exceeded 90%. Fig. 6. Spatial and temporal dynamics of the microplankton from hydrological bottle sampling during DYNAPROC 2 cruise. The Correspondence Analysis was applied on the main microplanktonic groups. Sampling depths and Julian days were added as illustrative variables (the coordinates were calculated as the average of samples of the same days or of the same depth). Within the genus Ceratium, the dominance index tended to increase throughout the study from 44% to a maximum of 73% at the end, and was globally higher during the 2 last cycles (Fig. 5b). One single taxon, C. pentagonum var. tenerum, corresponding to the thermophilic variety of the species C. pentagonum, also dominated all along the cruise. It was associated to C. pentagonum “robustum- tenerum”, C. horridum var. buceros, C. symmetricum var. orthoceras, C. limulus and C. concilians during the cycles 1 and 2, and to C. declinatum f. brachiatum, C. declina- tum f. normale, C. concilians, C. horridum var. buceros, C. platycorne “platycorne-compressum” and C. pentagonum “robustum>tenerum” during the cycles 3 and 4. These 10 prominent taxa represented a few part of the 64 infraspe- ciﬁc taxa which were encountered in the genus Ceratium (Ta- ble 2). lar vertical distribution: the mixotrophic group with ciliates and naked dinoﬂagellates was located above the thermocline in the surface layer; the armoured dinoﬂagellates were as- sociated with depths close to 50 m, i.e. 5.2.2 Diversity patterns Within the genus Ceratium, the number of infraspeciﬁc taxa in- creased from 43 to 56 taxa during the cycle 1 and then de- creased after day 269 to reach a value of 28 taxa. The diversity of microphytoplankton in bottle samples was about 3–4 bit ind−1 and was globally constant (Fig. 4c). Diversity indexes for total dinoﬂagellates and total diatoms www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 755 In net samples, the evenness varied few during the cycles 1 and 2 for dinoﬂagellates, but became more variable during the latter cycles with a ﬁnal decrease that followed a trend to increase. Within the genus Ceratium, infraspeciﬁc diversity slowly decreased and then consequently increased at the end of the cruise (Fig. 4c), whereas evenness strongly increased during the cycle 2 and then decreased as for dinoﬂagellates genera at the very end of the last cycle (Fig. 4d). Regarding to the relationship between diversity in net samples and the index of stratiﬁcation, the correlations found were not signif- icant. -2 -1.5 -1 -0.5 0 0.5 1 -1.5 -1 -0.5 0 0.5 1 1.5 261 262 263 264 265 269 270 271 272 273 278 279 280 281 282 284 285 286 287 288 289 10m 20m 30m 40m 5m 50m 60m 70m 80m 90m Centric Diatoms Pennate Diatoms Armoured Dinoflagellates Naked Dinoflagellates Dictyochophyceae Ciliates<30 µm Ciliates 30-50 µm Ciliates>50 µm Leg 1 (Julian days) Leg 2 (Julian days) Depth Z Axis 1 (54.8%) Axis 2 (24.3%) Fig. 6. Spatial and temporal dynamics of the microplankton from hydrological bottle sampling during DYNAPROC 2 cruise. The Correspondence Analysis was applied on the main microplanktonic groups. Sampling depths and Julian days were added as illustrative variables (the coordinates were calculated as the average of samples of the same days or of the same depth). 5.2.2 Diversity patterns close to the mean depth of the thermocline and the chlorophyll maximum; Dic- tyochophyceae and more particularly diatoms were charac- teristic of the deepest layers. In addition, there was an oppo- sition on axis 2 between the days of the cycles 1 and 2, as- sociated with diatoms, Dictyochophyceae and armoured di- noﬂagellates, and the days of the cycles 3 and 4 associated with ciliates, naked dinoﬂagellates and pennate diatoms. 6 Discussion The Correspondences Analysis, applied to the abundances of the main planktonic groups, yielded an interesting syn- thesis of the spatio-temporal patterns of the microplankton (Fig. 6). The planktonic groups were discriminated by their different coordinates on the 2 ﬁrst factors, explaining respec- tively 54.8% and 24.3% of the total inertia. Thus, siliceous organisms, corresponding to centric and pennate diatoms and silicoﬂagellates, had negative coordinates on axis 1, while ar- moured dinoﬂagellates and the mainly mixotrophic groups, composed of ciliates and naked dinoﬂagellates, had coor- dinates close to 0 on this axis. The axis 2 discriminated centric diatoms, Dictyochophyceae and armoured dinoﬂag- ellates (negative coordinates) from pennate diatoms and the mainly heterotrophic group (positive coordinates). Projec- tion of sampling days and sampling depths as illustrative variables indicated that the planktonic groups had particu- 6.1 Dynamics of microplankton Within radiolarians, the polycystines occur in the upper strata of the water column as they may bear algal symbionts, with maximal abundances ranging from 25 to 50 m depth in extrapolar areas (Ander- son et al., 2000). Observed densities were remarkably higher than those usually observed, with an order of magnitude of tens to hundreds individuals per cubic meter. During the ﬁrst DYNAPROC cruise (May 1995), abundances of radiolarians ranged from 200 to more than 1000 ind m−3 with maximal abundances comprised between 50 and 70 m (Andersen et al., 2001), that represents slightly lower abundances than those reported for the present cruise. Radiolarians may have sig- niﬁcantly contributed to primary production since it has been established their association with photosynthetic symbionts (Foster et al., 2006). The naked ciliates represented an im- portant part of the total microplankton, especially during the leg 2 (day 269 to day 273) and their abundances were consis- tent with values described in the Villefranche bay at the same period (G´omez and Gorsky, 2003). In addition to the overall dominance of small-sized di- noﬂagellates during the study we observed a temporal shift found during leg 2 (Marty et al., 2008) may partly explain the decline of siliceous organisms. Moreover, the develop- ment in the deep layer of diatoms, species that are able to respond very fast to nutrient pulses (Cloern and Dufford, 2005), was clearly associated to local increases in silicate concentrations (Marty et al., 2008). The disappearance of the silicoﬂagellates could then result then from the compe- tition with diatoms. As the ﬁrst observed population of di- atoms dominated by Corethron sp. was senescent, the com- petition for silica was weak, likely allowing the develop- ment of the silicoﬂagellates, whereas the competition for silica may have increased towards the middle of the cam- paign with the signiﬁcant development of mainly pennate di- atoms. Predation pressure also differed along the study, with a change in zooplankton species composition (Raybaud et al., 2008). Both diatoms (which were represented by sev- eral large-sized species), and silicoﬂagellates (which are po- tentially protected from herbivorous copepod predation due to their large spins) were well represented during leg 1 (Ju- lian day 259 to 274). Their contribution decreased during leg 2, corresponding with the presence of other predator species with more generalist and non-selective diets like Pteropods (Raybaud et al., 2008). layer (Marty et al., 2008). 6.1 Dynamics of microplankton Although a ﬁrst wind episode initiated the destratiﬁcation, the settlement of a mixing sys- tem was only obvious at the end of the campaign in re- sponse to persistent winds during the cycle 4. The domi- nance of small-sized microplankton species such as ciliates, mixotrophic and heterotrophic dinoﬂagellates (mainly Gyro- dinium spp. and Gymnodinium spp.) and radiolarians also corresponded to a typical summer situation. Dinoﬂagellates usually dominate the microphytoplankton during the strati- ﬁed period in the NW Mediterranean (G´omez, 2003; G´omez and Gorsky, 2003), since they do prefer conditions of low turbulence and warm temperatures, while low nutrients con- centrations in the surface layer and stratiﬁcation limit the de- velopment of diatoms and silicoﬂagellates. The signiﬁcant contribution of naked dinoﬂagellates probably resulted from a better adaptation to an oligotrophic environment, since sev- eral species are known to have phagotrophic activity (Stei- dinger and Tangen, 1997; Stoecker, 1999). Within less abun- dant armoured dinoﬂagellates, the prominence of Ceratium species, which characterised the net samples, has been al- ready reported in the Ligurian Sea (Halim, 1960; G´omez and Gorsky, 2003). The present abundances are of the same mag- nitude, even slightly lower, than those observed in the coastal waters of Villefranche bay at the same time (Tunin-Ley et al., 2007). Although the majority of Ceratium species possesses chloroplasts, the cells can contain vacuoles, which could result from phagotrophic behaviour (Chang and Carpenter, 1994). Radiolarians also signiﬁcantly develop in late sum- mer and autumn (Lange et al., 1997). Within radiolarians, the polycystines occur in the upper strata of the water column as they may bear algal symbionts, with maximal abundances ranging from 25 to 50 m depth in extrapolar areas (Ander- son et al., 2000). Observed densities were remarkably higher than those usually observed, with an order of magnitude of tens to hundreds individuals per cubic meter. During the ﬁrst DYNAPROC cruise (May 1995), abundances of radiolarians ranged from 200 to more than 1000 ind m−3 with maximal abundances comprised between 50 and 70 m (Andersen et al., 2001), that represents slightly lower abundances than those reported for the present cruise. Radiolarians may have sig- niﬁcantly contributed to primary production since it has been established their association with photosynthetic symbionts (Foster et al., 2006). 6.1 Dynamics of microplankton The naked ciliates represented an im- portant part of the total microplankton, especially during the leg 2 (day 269 to day 273) and their abundances were consis- tent with values described in the Villefranche bay at the same period (G´omez and Gorsky, 2003). I dditi t th ll d i f ll i d di y The trophic importance of microzooplankton is well rec- ognized; nevertheless, this group is often exclusively com- posed of a ciliate “box” in food web studies and models. We assumed that the contribution of hetero- and mixotrophic di- noﬂagellates is largely underestimated. Since Lessard and Swift (1985, 1986), we know that about half of dinoﬂagel- lates species in marine plankton do not possess chloroplasts and likely consume other plankton cells. A review of Sherr and Sherr (2007) showed that dinoﬂagellate biomass could sometimes be higher than ciliate biomass while small naked dinoﬂagellates may account for a large fraction (up to 90%) of the total heterotrophic dinoﬂagellates biomass. This last point was conﬁrmed in Paciﬁc Ocean by Masquelier and Vaulot (2007) who found, using epiﬂuorescence microscopy, that about 80% of heterotrophic dinoﬂagellates were cells smaller than 20 µm and noted that typical Uterm¨ohl method largely underestimated small dinoﬂagellates abundances. A large number of dinoﬂagellates species recorded during our study were mixotrophic or heterotrophic. For instance, all Protoperidinium species are known to lack photosynthetic pigments. Numerous cells of the order Gymnodiniales were observed, but our preserving and counting methods proba- bly largely underestimated their abundance (Masquelier and Vaulot, 2007). Given the abundance and variety of ciliates and heterotrophic dinoﬂagellates, the ecosystem of our study had a heterotrophic microplankton pool that feed on a wide range of prey, from bacteria to centric diatoms (Sherr and Sherr, 2007). The heterotrophic microplankton likely had another important role, that of a signiﬁcant food resource for mesozooplankton (Perez et al., 1997; Susuki et al., 1999; Liu et al., 2005). This has to be taken into consideration when studying the whole ecosystem functioning. In addition to the overall dominance of small-sized di- noﬂagellates during the study, we observed a temporal shift in microplankton composition from a system characterised by the presence of autotrophic organisms and by the sig- niﬁcant contribution of siliceous species (leg 1: day 261 to day 273) to a regeneration-dominated system with mainly mixotrophic or heterotrophic organisms during leg 2 (day 276 to 291). 6.1 Dynamics of microplankton In the NW Mediterranean Sea, the summer period usually starts in May–June with the development of the thermo- cline that lasts until mid-September or late October, when storms frequency is high. Along with a reinjection of nu- trients from depth toward upper layers, as a result of mix- ing effect of the storms and wind events, the development of a high pressure system following the storm period leads to calm and sunny weather conditions, known as St Martin’s summer, which triggers the autumnal phytoplankton bloom in the NW Mediterranean (Duarte et al., 1999). Despite the speciﬁc choice in timing for the DYNAPROC 2 cruise, the conditions that we encountered seemed to be characteristic of late summer stratiﬁcation, with a well-established ther- mocline and a situation of nutrient depletion in the surface Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 756 layer (Marty et al., 2008). Although a ﬁrst wind episode initiated the destratiﬁcation, the settlement of a mixing sys- tem was only obvious at the end of the campaign in re- sponse to persistent winds during the cycle 4. The domi- nance of small-sized microplankton species such as ciliates, mixotrophic and heterotrophic dinoﬂagellates (mainly Gyro- dinium spp. and Gymnodinium spp.) and radiolarians also corresponded to a typical summer situation. Dinoﬂagellates usually dominate the microphytoplankton during the strati- ﬁed period in the NW Mediterranean (G´omez, 2003; G´omez and Gorsky, 2003), since they do prefer conditions of low turbulence and warm temperatures, while low nutrients con- centrations in the surface layer and stratiﬁcation limit the de- velopment of diatoms and silicoﬂagellates. The signiﬁcant contribution of naked dinoﬂagellates probably resulted from a better adaptation to an oligotrophic environment, since sev- eral species are known to have phagotrophic activity (Stei- dinger and Tangen, 1997; Stoecker, 1999). Within less abun- dant armoured dinoﬂagellates, the prominence of Ceratium species, which characterised the net samples, has been al- ready reported in the Ligurian Sea (Halim, 1960; G´omez and Gorsky, 2003). The present abundances are of the same mag- nitude, even slightly lower, than those observed in the coastal waters of Villefranche bay at the same time (Tunin-Ley et al., 2007). Although the majority of Ceratium species possesses chloroplasts, the cells can contain vacuoles, which could result from phagotrophic behaviour (Chang and Carpenter, 1994). Radiolarians also signiﬁcantly develop in late sum- mer and autumn (Lange et al., 1997). 6.2 Biodiversity and physical perturbations Indeed, this aplastial protist is characterised by a symbiosis with a nitrogen-ﬁxing cyanobacterium (Buck and Bentham, 1998). The dynamics of L. mediterraneus may be important with regard to nitrogen budgets. Its increase in abundance noted during the second part of cruise may have helped to sustain the primary production. The changes described in the microplankton community should be related to the changes observed in the environment. This potentially raises two questions. First, did the physical perturbations that occurred (i.e. wind stress and low salinity water upcoming) affect the biodiversity of microplankton? Second, do our data support the Intermediate Disturbance Hypothesis (IDH)? Indeed, according to Connell (1978), an intermediate level of disturbance leads to a peak of diver- sity. In the absence of disturbance, the competitive exclusion principle (Hardin, 1960) would apply, the less-ﬁt species be- ing eliminated, resulting in low diversity. In contrast, highly intense or frequent disturbance can restrict the community to a few pioneer species, yielding low diversity. This theory, ﬁrstly developed for terrestrial plants, has been largely tested on phytoplankton, in part as an attempt to solve the “para- dox of the plankton” (Hutchinson, 1961), that points out the incoherence between the remarkable high richness observed in phytoplankton and the few resources which theoretically prevents the coexistence of numerous species, because of the principle of competitive exclusion. According to previous laboratory and enclosure experiments as well as ﬁeld studies, mainly focussing on freshwater ecosystems, the IDH appears to be applicable to phytoplankton (Padis´ak, 1994; Sommer, 1995; Fl¨oder and Sommer, 1999; Hambright and Zohary, 2000; Elliott et al., 2001; Weithoff et al., 2001; Shea et al., 2004). But the weakness of this theory is that it considers the response to an unmeasured event (Reynolds, 1995). While it appears effectively difﬁcult to deﬁne what a disturbance is and how intense it is, it has been proposed to consider a dis- turbance an event that alters the niche opportunities available to the species in a system (Shea et al., 2004). In our study, the stratiﬁcation index, being the resultant of physical perturbations (wind regime, intrusion of low salin- ity water), can be considered as a proxy of disturbance. Thus, exploring putative correlations between physical dis- turbances and biodiversity of phytoplankton from hydrolog- ical bottles, we observed that the decrease of the evenness and the increase of the speciﬁc richness were associated to the decrease of the stratiﬁcation index. 6.2 Biodiversity and physical perturbations The dynamics of marine phytoplankton have been as- sessed over short scales using pigment analysis (Vidussi et al., 2000). HPLC technique based on the pigment signa- ture of major phytoplankton groups constitutes an efﬁcient tool to have an overview of relative contributions and major shifts. This technique has been used successfully during DY- NAPROC 2 (Marty et al., 2008). Additionally, converting pigments to carbon equivalent could be convenient for link- ing the overall distribution of phytoplankton to the dynamic of organic matter. Yet, with this approach, groups of species that have no speciﬁc pigment signature are not identiﬁed, as well as groups without any photosynthetic pigments (i.e. het- erotrophic dinoﬂagellates). Furthermore, species composi- tion cannot be considered. Compared to pigment diversity analysis our direct microscopic observations supply comple- mentary information of biogeochemical and ecological in- terests. Thus, silicoﬂagellates characterised by a complex pigment-content composed of acylfucoxanthines (19′-BF et 19′-HF), fuco-, diadino-, diato-xanthine and also chloro- phylle a, c, and c3 (Daugbjerg and Henriksen, 2001) can not be monitored using a single pigment determination method but are easily distinguished microscopically. During DY- NAPROC 2, the dynamics of silicate compounds does not only concern diatoms; the siliceous silicoﬂagellates, even though less abundant, participated in both silicate and carbon ﬂuxes. The abundance of Dictyocha spp. encountered dur- ing the ﬁrst period of the cruise, argues for a non-negligible contribution in the silicate cycle and the existence of compe- tition with diatoms for the nutrient resources, especially sil- icate. This aspect is highlighted during the ﬁrst low salinity event that may have constituted a physiological stress with a stronger impact on diatoms. Meanwhile silicate was avail- able; it precisely corresponded with the highest abundances of silicoﬂagellates. Assuming that turbulences increased the velocity of the phytoplankton cells sinking (Ruiz et al., 2004) and that this velocity also depends on the cell shape, the spe- ciﬁc modiﬁcation that occurred within the community prob- ably inﬂuenced geochemical processes. In addition to sili- cate ﬂuxes, our study also provides information on the ni- trogen cycle. The peculiar consortium constituted by frus- tule of Leptocylindrus mediterraneus and the protozoan Rhi- zomonas setigera (Pavillard) Patterson, is involved in ni- trogen (N2) ﬁxation. As the individuals observed in olig- otrophic Paciﬁc Ocean (G´omez, 2007), R. setigera was al- ways found restricted to the central section of the frustules. 6.1 Dynamics of microplankton The slight decrease in silicate concentration Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ 757 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity Biogeosciences, 8, 743–761, 2011 6.2 Biodiversity and physical perturbations Despite the intru- sion of less saline water and the on-going destratiﬁcation that initiated during the ﬁrst leg, the diversity of the total mi- croplankton from bottle samples was quite constant along the study but the richness increased. Although the hori- zontal advection was weak, the less saline water mass has brought new species, as illustrating by the development of the coastal Scrippsiella sp., or additional individuals of some already present species, modifying the relative abundances of the species and consequently the diversity. We can postulate that perturbations were weak and progressive along the study, leading to a consistent total microplankton richness accom- panied with coexistence of species at the end of the cruise when the destratiﬁcation was likely established. Weak and frequent disturbances (several moderate wind events) would www.biogeosciences.net/8/743/2011/ www.biogeosciences.net/8/743/2011/ Biogeosciences, 8, 743–761, 2011 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity 758 have favoured and sustained diversity (here, namely species richness). mens of Scaphodinium mirabile were found in sub-surface (10 m depth, Julian day 280 and 2 m depth, Julian day 285). This species, considered as thermophylic (Stoyanova 1999; G´omez and Furaya, 2004), has already been found in NW Mediterranean Sea (Margalef, 1963; L´eger, 1971; G´omez and Gorsky, 2003). We encountered 18 specimens of Petalo- dinium porcelio, between 10 and 90 m depth (Julian days 279 to 289). It is the ﬁrst time that this species has been recorded in Mediterranean Sea since its description in 1969 (Cachon and Cachon, 1969). The two other cells of P. porcelio were reported by Stoyanova (1999) in the Black Sea and by G´omez and Furuya (2005) in the Paciﬁc Ocean. The most relative abundant morphologically-modiﬁed dinoﬂagellate was Ko- foidinium velelloides, which belongs to the family Kofoidini- aceae Taylor (Sournia, 1986). Seventy-ﬁve specimens were found, from surface to 90 m depth, throughout the study pe- riod. This species has a complex life cycle and we assumed that specimens of immature stage resembling Gymnodinium belong to this species, due to the morphology of the large velum at mature sampled stages. The species of the genus is not easy to identify because of the high morphological variability during the life cycle and the difﬁculties to delimit the species from preserved specimens (G´omez and Furaya, 2007). 6.3 Indicator and particular species Species-speciﬁc analysis also permits special attention to in- dicator species. The dinoﬂagellate Scrippsiella sp. is associ- ated with the less saline water incoming during the ﬁrst and the second leg of the cruise. As the species belonging to this genus are considered as neritic (Hasle and Syvertsen, 1996), we thus provide evidence for the coastal origin of the water with this biological marker. The species of this genus was not determined, even if it was morphologically close to Scripp- siella trochoidea, since Montresor et al. (2003) have sug- gested the existence of cryptic species in the Mediterranean Sea. Moreover, several cells of the naked dinoﬂagellate As- terodinium gracile, with ﬁve radiating elongated extensions as presented in Fig. 2a in G´omez and Claustre (2003), were sampled in our study (one cell at 50 m and the other at 60 m depth, Julian day 286). This species was initially reported in Indian Ocean (Sournia, 1986), later in the eastern Mediter- ranean Sea (Abboud-Abi Saab, 1989) and G´omez and Claus- tre (2003) found this species in the Tyrrhenian basin consid- ering it as a possible biological indicator of warming. To our knowledge, it is the ﬁrst time that A. gracile was found in the Ligurian Sea and this could indicate an extension of the spatial distribution of this species. Nevertheless, some publications suggest that the genus Asterodinium may corre- spond to life stages of a highly versatile single species re- grouping taxa of the genera Brachydinium, Karenia and Mi- croceratium (G´omez et al., 2005; G´omez, 2006). 6.2 Biodiversity and physical perturbations As for genus Asterodinium (see above), those rare species need more investigation about description as well as biology and ecology. On the opposite, in net samples, the biodiversity followed a different evolution, with a remarkable increase in Ceratium species dominance within dinoﬂagellates. Even within the genus Ceratium, which is known to be an interesting model for biodiversity studies (Tunin-Ley et al., 2007), the evenness tended to decrease at the very end of the cruise, reﬂecting an increase in the dominance of a couple of taxa. This reverse evolution may illustrate the lower abilities of some dinoﬂag- ellates, with low growth rates as in Ceratium taxa, to adapt to changes occurring at short-time scale. We can postulate that those non-opportunistic species can not get beneﬁt from the gradual disturbance which favoured the diversity of other groups. Biogeosciences, 8, 743–761, 2011 References Dodge, J. D.: Marine Dinoﬂagellates of the British Isles, Her Majesty’s Stationery Ofﬁce, England, 303 pp, 1982. 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Our results highlighted the value of such data to complete pigments analysis; the knowl- edge of microplanktonic short-term abundance and diversity evolution supplied complementary information of biogeo- chemical, biological and ecological interests. It allowed a better understanding of the interactions between autotrophs and nutrients as well as trophic relations with zooplankton. Finally, indicators species could also conﬁrm coastal water intrusion and potential warming of Mediterranean Sea. This demonstrates that traditional taxonomic studies are deﬁnitely not obsolete, with regard to the recent advances in automatic digitization and classiﬁcation (Benﬁeld et al., 2007) that will considerably decrease the time allocated to microplankton identiﬁcation in further studies. Despite their large size, some dinoﬂagellate species char- acterized by non-typical or much modiﬁed morphologies are often underestimated or absent from most of microphyto- plankton analysis. According to G´omez and Furuya (2004), this could be explained by a speciﬁc and not easily accessible literature and difﬁculty in the identiﬁcation, due to folding in ﬁxed cells. Careful analysis of our samples revealed sev- eral modiﬁed dinoﬂagellates. They mainly belonged to the family Leptodiscaceae Taylor (order Noctilucales Heackel). The species are strongly antero-posteriorly ﬂattened and dis- play a wing-like extension, called the velum. Two speci- Acknowledgements. We thank the chief scientist, V. Andersen, for organising the DYNAPROC 2 cruise, and the crew of the R/V Thalassa for ship operations. We are also grateful to John Dolan for his advices and kind re-lecture. This study was part of the PECHE project; ﬁnancial support was provided by the I.N.S.U.-C.N.R.S. through the PROOF program (JGOFS-France) and by the project “La Notion d’Esp`ece dans le Phytoplancton” in the framework of the national program EC2CO “Ecosph`ere Biogeosciences, 8, 743–761, 2011 www.biogeosciences.net/8/743/2011/ 759 S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversity S. Lasternas et al.: Short-term dynamics of microplankton abundance and diversit Cachon, J. and Cachon, M.: Contribution `a l’´etude des Noctilucidae Saville-Kent. Evolution, morphologie, cytologie, syst´ematique. II. Les leptodiscinae Cachon J. et M., Protistologica, 5, 11–33, 1969. Continentale et Cˆoti`ere”. The publication of this article is ﬁnanced by CNRS-INSU. The publication of this article is ﬁnanced by CNRS-INSU. 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https://openalex.org/W1488239349	http://sedici.unlp.edu.ar/bitstream/handle/10915/31762/Documento_completo__.pdf?sequence=1, http://sedici.unlp.edu.ar/bitstream/handle/10915/3433/Documento_completo.pdf?sequence=1, http://sedici.unlp.edu.ar/bitstream/handle/10915/3537/Documento_completo.pdf?sequence=1, https://www.econstor.eu/bitstream/10419/189558/1/doc_cedlas03.pdf, http://cedlas.econo.unlp.edu.ar/archivos_upload/doc_cedlas3.pdf	es		Comercio y desigualdad salarial en Argentina: un enfoque de Equilibrio General Computado	null	2,019	cc-by	14,649	Maestría en Economía Facultad de Ciencias Económicas Universidad Nacional de La Plata TESIS DE MAESTRIA ALUMNO Martín Cicowiez TITULO Comercio y Desigualdad Salarial en Argentina: Un enfoque de Equilibrio General Computado DIRECTOR Chisari Omar FECHA DE DEFENSA 12/7/2001 COMERCIO Y DESIGUALDAD SALARIAL EN ARGENTINA: UN ENFOQUE DE EQUILIBRIO GENERAL COMPUTADO MARTIN CICOWIEZ TESIS DE MAESTRIA MAESTRIA EN ECONOMIA UNIVERSIDAD NACIONAL DE LA PLATA DIRECTOR DE TESIS OMAR O. CHISARI LA PLATA, 27 DE NOVIEMBRE DE 2001 COMERCIO Y DESIGUALDAD SALARIAL EN ARGENTINA: UN ENFOQUE DE EQUILIBRIO GENERAL COMPUTADO MARTIN CICOWIEZ 26-11-2001 RESUMEN En este trabajo, se utiliza un modelo de Equilibrio General Computado (CGE) para analizar el efecto de la apertura comercial sobre el salario de los trabajadores calificados versus el salario de los trabajadores no calificados en la Argentina. El resultado que se obtiene es que en el caso de Argentina, el comercio sólo explica una pequeña porción (alrededor de 3%) del incremento en la desigualdad salarial. Adicionalmente, se presenta una metodología que puede emplearse para realizar ejercicios de descomposición con un modelo de CGE. I. INTRODUCCION Durante las décadas del ochenta y noventa la desigualdad salarial entre trabajadores calificados y no calificados aumentó considerablemente en varios países1. Este aumento ha sido bien documentado para los países de la OECD, en especial Estados Unidos y Gran Bretaña. En el caso de la Argentina, se registra un aumento de la desigualdad salarial durante la década del noventa (Gasparini et al., 2001). En la literatura económica se mencionan cuatro explicaciones principales para el aumento en la desigualdad salarial: el aumento del comercio internacional de bienes intensivos en mano de obra no calificada (en especial, bienes producidos en países con bajos salarios), el cambio tecnológico sesgado a favor de los trabajadores con mayor calificación, la disminución de la importancia de las instituciones que limitan el mercado (caída del salario mínimo y disminución del poder sindical) y el cambio en la estructura productiva (desindustrialización). De estas cuatro explicaciones, las que más atención han recibido en la literatura son el incremento del comercio y el cambio tecnológico. En cuanto a la importancia relativa de estos dos factores, la mayoría de los autores señalan al cambio tecnológico como causa principal 1 Para un resumen de la literatura sobre este tema, puede consultarse Burtless (1995) y Richardson (1995). -1- del aumento de la desigualdad salarial2. Estos autores señalan que sólo el cambio tecnológico sesgado contra los trabajadores no calificados es consistente con un aumento del diferencial salarial y un aumento simultáneo en la intensidad de uso del trabajo calificado. Esta literatura emplea una gran variedad de métodos econométricos. En este trabajo se emplea un modelo de Equilibrio General Computado (CGE) para determinar cuánto del cambio en el diferencial salarial entre trabajadores calificados y no calificados observado durante la década del noventa en Argentina puede ser explicado por la apertura comercial ocurrida durante el mismo período. Se elige la década del noventa porque es durante esos años que el aumento del diferencial salarial entre trabajadores con distinta calificación se ve acompañado de un proceso de apertura comercial importante. Adicionalmente, siguiendo a la literatura, se supone que el otro factor que explica el aumento de la desigualdad salarial es el cambio tecnológico y se calcula el cambio tecnológico que, combinado con la apertura comercial, hace que el modelo replique el aumento de la desigualdad salarial ocurrido durante la década del noventa en Argentina. La aplicación de un modelo de CGE al estudio de este problema se basa en la propuesta de Abrego y Whalley (1999 y 2000). Se emplean dos modelos alternativos para realizar los ejercicios de simulación. El primero es un modelo similar al empleado por Abrego y Whalley y el segundo es un modelo de CGE estático estándar más general. Aunque se trata de modelos muy simples, constituyen una primera aproximación al problema en el marco de un modelo de CGE calibrado con información para Argentina. El resto del trabajo se organiza de la siguiente manera. La Sección II presenta evidencia empírica para la Argentina sobre la evolución de la desigualdad salarial y de la política comercial durante la década del noventa. La Sección III describe la metodología del CGE. En la Sección IV se describen los modelos empleados. La Sección V presenta la información utilizada para hacer operacional (calibrar) el modelo de CGE. La Sección VI presenta los resultados obtenidos. Finalmente, la Sección VII concluye. 2 Dos excepciones a esta conclusión son Wood (1995 y 1997) y Feenstra y Hanson (1996 y 1999). -2- II. EVIDENCIA EMPIRICA PARA ARGENTINA II.1. EVOLUCION DEL DIFERENCIAL SALARIAL En este trabajo se toma como medida del diferencial salarial entre trabajadores con distinta calificación el cociente entre el salario promedio de los trabajadores calificados y el salario promedio de los trabajadores no calificados3. En el caso de la Argentina, la evidencia muestra una caída de este diferencial salarial desde fines de la década del ochenta hasta comienzos de la década del noventa y un aumento a partir de entonces y hasta el año 1998. La fuente de información utilizada para calcular este cociente es la Encuesta Permanente de Hogares (EPH) para el Gran Buenos Aires elaborada por el INDEC4. Entre 1992 y 1998 este cociente aumentó alrededor de 10%5. El Gráfico 1 permite apreciar este comportamiento del diferencial salarial. Gráfico 1: Evolución del diferencial salarial 1987-1998 2.00 1.90 1.80 1.70 1998-1 1997-1 1996-1 1995-1 1994-1 1993-1 1992-1 1991-1 1990-1 1.60 Hacia finales de la década del ochenta la Argentina comienza un proceso de apertura comercial. El arancel promedio pasó de 30,8% en 1988 a 14,1% en 1997. Además, en el mismo período, el valor de las exportaciones 3 (importaciones) se multiplicó, Se considera que un trabajador calificado es aquel que tiene educación secundaria completa, universitaria incompleta o universitaria completa. Los resultados no cambian considerablemente si se considera la clasificación de calificados y no calificados que utiliza el INDEC desde 1993. Sin embargo, cuando se utiliza esta clasificación alternativa no se observa la aceleración del aumento en el diferencial salarial a partir de 1997 que si se aprecia en el Gráfico 1. 4 El Gran Buenos Aires es un aglomerado urbano con una población de 12 millones de habitantes; alrededor de un tercio de la población total del país. 5 Este comportamiento del diferencial salarial es consistente con el de otras medidas de desigualdad durante el mismo período (Bebczuk y Gasparini, 2001). -3- aproximadamente, por 2,5 (4,5) indicando un aumento considerable del nivel de integración comercial de Argentina con el resto del mundo. Dos trabajos que estudian el efecto del aumento del comercio internacional sobre la desigualdad salarial entre trabajadores calificados y no calificados en Argentina son Galiani y Sanguinetti (2000) y Porto (2000). El resultado que obtiene el primero es que el aumento del comercio internacional sólo explica alrededor del 10% del incremento de la desigualdad salarial. El segundo, en cambio, obtiene que -bajo ciertos supuestos- la apertura comercial podría explicar hasta un 50% del aumento de la desigualdad salarial. Los trabajos citados emplean metodologías distintas. II.2. LA POLITICA COMERCIAL ARGENTINA DURANTE LOS NOVENTA En términos generales, la Argentina inició un proceso de apertura comercial de manera unilateral hacia finales de la década del ochenta a través de la eliminación de aranceles y cuotas. Sin embargo, esta primera etapa del proceso de apertura no gozó de la credibilidad necesaria para que tuviera efectos sobre la asignación de recursos. Es sólo a partir de 1992 que se genera un proceso de apertura e integración que tuvo consecuencias asignativas importantes (Cristini, 1999). La apertura comercial de los noventa se llevó a cabo a través de una baja de aranceles y de la eliminación de restricciones cuantitativas. Además de la apertura comercial unilateral, en el año 1991 la Argentina comenzó un proceso de integración regional con la creación del Mercosur. Durante los noventa, los países del Mercosur eliminaron la mayor parte de las barreras al comercio entre ellos y adoptaron un arancel externo común y lograron establecer una unión aduanera “casi perfecta”6. Además de los cambios en la política comercial, en la década del noventa se produjeron otros cambios importantes en la política económica argentina: ordenamiento fiscal, ordenamiento monetario, privatizaciones, apertura financiera, desregulación de la actividad privada. La 6 Para una descripción del proceso de apertura argentino comenzado hacia finales de los ochenta puede consultarse Berrettoni y Cicowiez (2001). -4- inflación se redujo hasta niveles internacionales y el ingreso per cápita aumento más de 50% durante la década7. Como ya se mencionó, el cambio tecnológico es el factor que más atención ha recibido en la literatura como causante del aumento de la desigualdad salarial. Aunque es difícil encontrar evidencia directa sobre este factor, existen razones para creer que ha sido importante en la Argentina durante el período considerado. En este sentido, durante la década del noventa se produjo un importante proceso de incorporación de tecnología a través de la privatización de empresas proveedoras de servicios públicos y de la importación de bienes de capital y de insumos intermedios. III. METODOLOGIA La metodología utilizada en este trabajo es la del Equilibrio General Computado (CGE). En esta sección se presentan las principales características de los modelos de CGE. Un modelo de equilibrio general tiene en cuenta que un cambio exógeno (de política o de otro tipo) que afecta directamente a una parte de la economía puede tener repercusiones a través de toda la economía. En consecuencia, debido a que capta el impacto de la reasignación de recursos entre los distintos sectores de una economía, este tipo de modelos es una herramienta ideal para identificar ganadores y perdedores luego de un cambio de política. Un modelo de CGE walrasiano hace operacional la estructura de equilibrio general de Walras. Las características de un modelo de equilibrio general walrasiano son las que se mencionan a continuación. El número de consumidores existentes en el modelo debe estar especificado. Cada uno de ellos tiene una dotación inicial de bienes y un conjunto de preferencias que resultan en funciones de demanda para cada uno de los bienes. Las demandas de mercado son las sumas de las demandas individuales. Estas demandas de mercado dependen de todos los precios, son continuas, no negativas, homogéneas de grado cero (no hay ilusión monetaria) y satisfacen la ley de Walras (a cualquier conjunto de precios el valor del gasto de los consumidores se iguala con su ingreso). Del lado de la producción, la tecnología se describe mediante funciones de producción con rendimientos constantes (o no crecientes) a escala. Los productores maximizan sus beneficios. La homogeneidad de grado cero de las funciones de 7 Para un resumen de los cambios ocurridos en la economía argentina en este período puede consultarse Bebczuk y Gasparini (2001). -5- demanda y la homogeneidad de grado uno en precios de los beneficios (si los precios se duplican se duplican los beneficios monetarios) implican que sólo los precios relativos son relevantes en este modelo; el nivel absoluto de precios no tiene ningún impacto sobre los resultados del modelo. El equilibrio de un modelo como este se caracteriza por un conjunto de precios y niveles de producción que igualan la oferta y la demanda para todos los bienes (Shoven y Whalley, 1984). Entonces, los modelos de CGE son la contraparte numérica de los modelos de equilibrio general walrasianos tipo Arrow-Debreu (1954) y están basados en el comportamiento optimizador de los agentes económicos (los consumidores maximizan su utilidad y los productores maximizan sus beneficios). Las aplicaciones numéricas del equilibrio general se iniciaron con el trabajo de Harberger (1962) sobre incidencia tributaria en el contexto de un modelo numérico de dos sectores. El trabajo de Scarf (1969) hizo posible la determinación del equilibrio de un sistema walrasiano. La utilización de modelos de CGE recibió un impulso fundamental del trabajo pionero de Shoven y Whalley (1972, 1984, 1992). Más recientemente, contribuciones como las del GTAP (Hertel, 1997) y Rutherford (1999) han contribuido al desarrollo y utilización de esta metodología. El objetivo de los trabajos que emplean modelos de CGE es analizar los efectos cuantitativos de cambios exógenos sobre la asignación óptima de recursos, la eficiencia y el bienestar. Dos de los campos de mayor aplicación han sido las finanzas públicas (por ejemplo, la evaluación de sistemas tributarios alternativos) y el comercio internacional (en especial, la evaluación de distintos acuerdos comerciales). Actualmente, los modelos de CGE son ampliamente utilizados para el análisis cuantitativo de políticas económicas. La idea básica detrás de un modelo de CGE es simple. Se construye un modelo matemático de una economía y se recopila información para un período de tiempo dado. Luego, las características de la economía en ese período de tiempo son utilizadas para resolver el modelo numéricamente. Este último paso se realiza utilizando una computadora. Un modelo de CGE tiene una estructura transparente y consistente con la teoría económica y es una herramienta ideal para evaluar políticas alternativas. En un análisis de equilibrio general se modela toda la economía. En cambio, en un análisis de equilibrio parcial se analiza la situación de un mercado en particular suponiendo constantes las condiciones en el resto de los mercados de la economía. -6- Resumiendo, un modelo de CGE como el que se emplea en este trabajo es una representación en computadora de una economía y posee las siguientes características: i. Hay varios agentes económicos que interactúan. ii. El comportamiento individual está basado en la optimización microeconómica. (Dos problemas típicos para ilustrar este punto son la maximización de la utilidad de los consumidores y la maximización de beneficios de las firmas). iii. La mayoría de las interacciones entre los agentes se realizan a través de mercados y precios. iv. Son modelos típicamente desagregados, con varios agentes y mercados. v. Los datos empleados en su construcción son pocos cuando se los compara con el número de parámetros de comportamiento y tecnológicos del modelo. Normalmente, la información para un modelo de CGE corresponde a un “equilibrio general” observado o caso base y a un conjunto de estimaciones independientes de elasticidades de oferta y demanda. Típicamente, un modelo de CGE se construye de manera tal que replique las transacciones observadas en el caso base. vi. Típicamente, las asignaciones de equilibrio no pueden caracterizarse fácilmente como la solución a un único problema de optimización (el de un planificador central). vii. La formulación de este tipo de modelos tiene como objetivo (implícito o explícito) el análisis de políticas económicas. Los pasos “normales” a seguir en un estudio de CGE son: i. Diseño general. Con base en el problema de política a tratar y en la disponibilidad de datos, se determinan las dimensiones básicas del análisis: número de regiones, de consumidores, de sectores productivos, de factores, etc. ii. Diseño de los problemas de elección individuales. Especificar para cada agente del modelo (familias, empresas y gobierno) el problema de elección que deberá enfrentar. Estos problemas podrán ser simples (por ejemplo, un gobierno que sólo transfiere lo recaudado por impuestos a los consumidores) o complicados (por ejemplo, un -7- consumidor que debe asignar su ingreso entre distintos bienes de manera tal de maximizar su utilidad respetando su restricción presupuestaria). iii. Funciones de oferta y demanda. Resolver los problemas de elección individuales del modelo. Típicamente, se trata de solucionar problemas de optimización restringida (maximización de la utilidad de los consumidores y minimización del costo de las firmas). iv. Desarrollar un sistema de notación. Se debe tener especial cuidado en las correspondencias entre mercados y precios. Selección de sets y nombres para los precios de los bienes y factores. Suele ser conveniente el empleo de más de un precio para un mismo bien si ese bien está sujeto al pago de impuestos. v. Calibración. Se trata de inferir el valor de los parámetros de las ecuaciones de comportamiento de manera tal que el caso base (el equilibrio observado) sea una solución del modelo. En este procedimiento, se combinan las ecuaciones de comportamiento con las elecciones de los agentes en el caso base y con valores para las elasticidades (parámetros libres). vi. Programación del modelo. Codificar el sistema de ecuaciones no lineales que representa el modelo. En este estudio, para la solución del sistema de ecuaciones no lineales que representa el modelo se utiliza el software GAMS (General Algebraic Modeling System) documentado en Brooke et al. (1996). vii. Replicar el caso base. Debido a que los parámetros del modelo fueron obtenidos mediante un proceso de calibración, si se asignan los valores del caso base a los parámetros y variables exógenas, el modelo debería dar el caso base como solución. Este paso es útil para confirmar la correcta codificación del modelo. viii. Realizar simulaciones. Finalmente, con el modelo correctamente codificado y calibrado se modifica el valor de alguna variable exógena o parámetro, se recalcula el equilibrio y se analizan los resultados a partir de la comparación con el caso base. La metodología que se emplea en los modelos de CGE para analizar los efectos económicos de políticas comerciales alternativas es la realización de experimentos contrafactuales o simulaciones. Se pregunta al modelo que hubiese pasado en el año base si hubiese sido -8- implementada la política comercial de interés y el resto de las políticas domésticas (fiscal y monetaria) y las condiciones externas (el comportamiento de los precios mundiales) permanecieran igual. Por lo tanto, este tipo de análisis enfatiza los efectos de la política comercial aislándola de otros factores. Debido a que no se incorporan en el análisis los cambios esperados en esos otros factores, no se trata de una predicción. Finalmente, un modelo de CGE estático no incorpora características dinámicas. Las características mencionadas deben ser tenidas en cuenta cuando se interpretan los resultados de un modelo de CGE. IV. MODELOS En esta sección se presentan los modelos utilizados en los ejercicios de simulación. En primer lugar se presenta el modelo más simple y luego el modelo más general. IV.1. MODELO SIMPLE La teoría más frecuentemente citada para explicar el nexo entre el comercio y los salarios es el modelo de Heckscher-Ohlin (HO) de comercio internacional8. Este modelo explica los patrones de comercio internacional a partir de las diferencias relativas en la dotación de factores de producción de los socios comerciales. El modelo predice que entre dos países, A y B, que comparten la misma tecnología, el país A exportará bienes que sean producidos con relativamente más del factor de producción que es relativamente abundante en el país A e importará bienes producidos con relativamente más del factor de producción que es relativamente abundante en el país B. Del modelo de HO se derivan dos teoremas acerca del efecto del comercio sobre el precio de los factores productivos: (i) El teorema de igualación del precio de los factores afirma que bajo los supuestos del modelo de HO y un régimen de libre comercio, el precio de los factores de producción será el mismo en ambos socios comerciales y (ii) El teorema de Stolper-Samuelson afirma que un incremento en el precio doméstico de un bien debido a un incremento del arancel o a una mayor protección aumentará 8 Los supuestos de la versión más sencilla de este modelo son los siguientes: (i) hay dos países, (ii) cada país produce dos bienes, (iii) la producción de cada bien requiere de dos factores de producción, (iv) cada país tiene una dotación fija de cada uno de los factores, (v) los factores son perfectamente móviles entre sectores y perfectamente inmóviles entre países, (vi) los dos países emplean la misma tecnología de producción, (vii) para todos los precios relevantes de los factores, uno de los bienes es siempre relativamente intensivo en el uso de uno de los factores, (viii) uno de los países es relativamente abundante en uno de los factores y (ix) los costos de transporte son nulos. -9- el precio real del factor de producción utilizado relativamente con mayor intensidad en la producción de ese bien. En nuestro caso, si Argentina es un país relativamente abundante en mano de obra calificada (Cristini, 1999), se esperaría un incremento del salario relativo de este tipo de trabajo luego de la apertura comercial. Sin embargo, es importante tener en cuenta que Argentina es relativamente más abundante en tierra y que es un exportador neto de bienes intensivos en recursos naturales y un importador neto de bienes intensivos en trabajo calificado y tecnología (Berlinski, 1998). Sin embargo, como demuestran Abrego y Whalley (1999), el modelo de HO sólo puede ser resuelto (numéricamente) en presencia de shocks (por ejemplo, caída de las tasas arancelarias) relativamente pequeños. Cuando se emplean las formas funcionales usuales y las dotaciones factoriales están fijas, la frontera de producción es casi lineal (Johnson, 1966). Por lo tanto, incluso shocks pequeños son acompañados de especialización en la producción9. En consecuencia, el primer modelo que se utiliza para determinar el efecto de la apertura comercial sobre la desigualdad salarial es una extensión del modelo de HO en el que se suprime el supuesto de bienes homogéneos. El modelo HO es un caso particular del que se utiliza a continuación10. ESTRUCTURA DEL MODELO SIMPLE El modelo es de una economía abierta y pequeña, que toma como dados los precios de sus exportaciones y de sus importaciones11. Sin embargo, se supone que los precios mundiales no dominan el sistema de precios doméstico. Existen tres bienes, dos factores productivos y un consumidor representativo dueño de toda la dotación factorial del país que se supone fija. Dos de los bienes se producen en el país doméstico y el tercero se importa desde el resto del mundo. El país doméstico produce un bien exportable y un bien doméstico no transable que es 9 Se podría utilizar una variante del modelo con factores específicos para eliminar el problema de la especialización. Sin embargo, en un modelo de este tipo los shocks de precios son soportados, en su mayor parte, por los factores específicos más que por los factores móviles (trabajo calificado y no calificado). 10 Como se hará evidente más adelante, esto ocurre cuando la elasticidad de sustitución (Armington) entre el bien doméstico y el importado es igual a infinito. 11 En el modelo de HO de dos países, dos bienes y dos factores, la abundancia relativa de factores determina el patrón de comercio. El modelo que se utiliza aquí contiene sólo un país pequeño que toma como dados los -10- sustituto imperfecto del bien importado. A continuación se detalla la estructura de este modelo. Para el lector interesado, en las notas al pie de página, se presenta la formulación algebraica de las formas funcionales empleadas. Lado del consumo La utilidad del consumidor representativo depende del consumo de tres bienes: el bien exportable E, el bien doméstico no transable, D, y el bien importado, M. Se supone que el consumidor representativo maximiza su utilidad en dos etapas. En la primera elige entre consumir el bien exportable y un bien compuesto, A, formado por el bien doméstico y el bien importado. En la segunda etapa determina la composición óptima del bien compuesto. Es decir, determina cuanto de bien doméstico y cuanto de bien importado consume. Las preferencias se representan con una función de utilidad tipo Cobb-Douglas (CD)12 para la primera etapa y tipo CES13 (Elasticidad de Sustitución Constante) para la segunda. Cuando el consumidor distingue entre bienes nacionales y bienes importados (por ejemplo, un auto nacional es distinto de un auto japonés) se dice que tiene preferencias tipo Armington (1969). Matemáticamente, en el nivel superior de la función de utilidad el consumidor representativo resuelve el siguiente problema: ( max U = CD A d , E d ) s. a. I = PE E d + PA A d donde U es una función de utilidad tipo Cobb-Douglas, Ad es la cantidad demandada de bien compuesto A, Ed es la cantidad demandada de bien E, PE es el precio de E, PA es el precio del bien compuesto A y I es el ingreso del consumidor representativo y está dado por precios de sus exportaciones e importaciones. El patrón de comercio del equilibrio inicial está determinado por la ventaja comparativa propia del país doméstico, no la abundancia relativa de factores. 12 Algebraicamente, la función de utilidad tipo Cobb-Douglas puede escribirse como ( ) ( ) (E ) CD A d , E d = A d αA d αE donde αΑ y αΕ son parámetros de distribución. 13 Algebraicamente, la función tipo CES puede escribirse como ⎡ CES M d , D d = γ ⎢ β M M ⎣ ( ) ε −1 d ε ( ) ε −1 d ε ( ) ⎤⎥ + βD D ⎦ ε ε −1 donde γ es un parámetro de escala, β es un parámetro de distribución y ε es la elasticidad de sustitución entre el bien importado y el bien doméstico. -11- I = w L L + wH H + R + B donde L es la dotación (exógena) de trabajo no calificado, H es la dotación (exógena) de trabajo calificado, R es la transferencia que recibe del gobierno, B es la transferencia que recibe desde el resto del mundo que se supone fija e igual al déficit comercial, wL es el salario del trabajo no calificado y wH es el salario del trabajo calificado. Resolviendo las Condiciones de Primer Orden (CPO) del problema anterior, se obtienen las demandas de bien A, Ad, y de bien E, Ed. Como se mencionó más arriba, el bien compuesto es un bien agregado tipo Armington. El consumidor debe determinar cuál es la composición óptima (cuánto de bien importado y cuánto de bien doméstico consume) que le permite minimizar el gasto total en el bien compuesto. Analíticamente, el consumidor representativo resuelve el siguiente problema: min PA A d = PM (1 + τ ) M d + PD D d ( s. a. A d = CES M d , D d ) donde el bien compuesto A se modela con una función tipo CES de la cantidad demandada de bien M, Md, y de la cantidad demandada de bien D, Dd, PM es el precio del bien M, PD es el precio del bien D y τ es la tasa del arancel a las importaciones. El precio PA se obtiene de la condición de igualdad entre el gasto total en A y la suma del gasto en M y en D. De las CPO del problema anterior surgen las funciones de demanda Md y Dd. Lado de la producción En este primer modelo se producen sólo dos bienes: el exportable E y el doméstico no transable D. En la producción sólo se utiliza trabajo no calificado L y trabajo calificado H. La tecnología es de Rendimientos Constantes a Escala y se modela con funciones de producción tipo CES14 en los dos sectores. El problema de minimización de costos que resuelve el productor de bien D es 14 Algebraicamente, la función de producción tipo CES puede escribirse como -12- min wL LD + wH H D s. a. D s = CES (LD , H D , π ) donde Ds es la oferta de bien D, LD es el empleo de L en la producción de bien D, HD es el empleo de H en la producción de bien D y π es el cambio tecnológico. Resolviendo las CPO, se obtienen las demandas de factores para la producción de D, LD y HD. La producción del bien E se modela de manera similar a la producción del bien D. El productor resuelve el siguiente problema de minimización de costos: min wL LE + wH H E s. a. E s = CES (LE , H E , π ) donde la notación utilizada es similar al caso anterior. Al igual que en el caso anterior, de la resolución de este problema se obtienen las demandas de factores en la producción de bien E, LE y HE. Gobierno En este modelo, el gobierno sólo recauda lo producido por el arancel y lo devuelve al consumidor como una transferencia de suma fija. La restricción presupuestaria del gobierno es, entonces, τ PM M d = R donde R es la recaudación total del arancel. Ω Ω −1 Ω −1 Ω −1 ⎡ ⎤ Ω CES (L, H ) = φ ⎢πδL + (1 − πδ )H Ω ⎥ ⎣ ⎦ donde φ es un parámetro de escala, δ es un parámetro de distribución, Ω es la elasticidad de sustitución entre el trabajo no calificado y el trabajo calificado y π es el cambio tecnológico. -13- Condiciones de equilibrio El equilibrio de este modelo se caracteriza por las siguientes condiciones: equilibrio en los mercados de factores, LD + LE = L y H D + H E = H , equilibrio en el mercado del bien D, ( ) D d = D s , equilibrio del sector externo, PM M d = PE E s − E d + B y cero beneficios en la producción, PE E s − wL LE − wH H E = 0 y PD D s − wL LD − wH H D = 0 . Los precios PM y PE son exógenos y el precio PD es endógeno (a diferencia de un modelo donde D y M son bienes homogéneos o sustitutos perfectos en el consumo). IV.2. MODELO GENERAL En esta sección se presenta un modelo más general que se emplea para analizar los efectos del comercio sobre la desigualdad salarial en Argentina. Se trata de un modelo estático del tipo De Melo y Robinson (1989). La estructura de este modelo es estándar en la literatura que utiliza modelos de CGE. A diferencia del modelo anterior, el Modelo General incorpora diez sectores productivos15 y tres factores primarios. La existencia de diez bienes hace posible incorporar las diferencias en el cambio que experimentaron las distintas tasas arancelarias en el período que se analiza. La Tabla 1 muestra los sectores considerados en la implementación de este modelo16. Además, en la tabla se muestra el índice de Ventajas Comparativas Reveladas (VCR) para cada uno de los sectores. El índice de VCR se define como el cociente entre la participación de un producto en las exportaciones de un país y la participación de ese producto en las exportaciones mundiales. Un valor mayor (menor) a uno indica la presencia (ausencia) de VCR en ese sector. 15 Cada uno de estos sectores productivos produce un único bien. La composición de cada uno de los diez sectores productivos considerados es la siguiente: Cereales y Ganadería (G_L): Arroz con cáscara, Trigo, Otros cereales, Lana y Otros productos de origen animal. Otros Productos Agrícolas (OAF): Otros cultivos, Silvicultura y Pesca. Industrias Alimenticias (FIN): Arroz procesado, Productos de la carne, Productos de la leche, Otros productos alimenticios y Bebidas y tabaco. Textiles e Indumentaria (TEX): Textiles y Indumentaria. Manufacturas de Recursos Naturales (NRM): Productos de cuero, Productos de madera y Productos del papel y de la industria gráfica. Minería, Petróleo y Carbón (MPC): Carbón, Petróleo, Gas, Otros minerales y Productos del petróleo y del carbón. Transporte (TRN): Equipo de transporte. Industria Química y Otras Industrias (C_O): Químicos, plásticos y goma, Minerales no metálicos y Otras manufacturas. Maquinaria y Productos Metálicos (M_M): Metales ferrosos, Metales no ferrosos, Productos metálicos y Maquinaria y equipo. Servicios (SVC): Electricidad, gas y agua, Construcción, Comercio y transporte, Otros servicios (privados), Otros servicios (gobierno) y Vivienda. 16 -14- Tabla 1: Sectores considerados y VCR Descripción VCR 1 G_L Cereales y Ganadería 11.3 2 3 OAF FIN Otros Productos Agrícolas Industrias Alimenticias 6.7 11.7 4 TEX Textiles e Indumentaria 0.4 5 NRM Manufacturas de Recursos Naturales 1.2 6 7 MPC TRN Minería, Petróleo y Carbón Transporte 1.8 0.4 8 C_O Industria Química y Otras Industrias 0.8 9 10 M_M SVC Maquinaria y Productos Metálicos Servicios 0.4 - Fuente: Elaboración propia en base al GTAP y COMTRADE. Según el indicador de VCR, Argentina presenta una marcada ventaja comparativa en la producción de productos primarios y alimentos. En términos generales, estos sectores son relativamente intensivos en mano de obra no calificada. A modo de resumen, el Gráfico 2 describe la estructura del Modelo General utilizado en los ejercicios de simulación. Gráfico 2: Estructura del Modelo General AiD εi Ei Di Qi VALOR AGREGADO N L Ωi H Mi ψi RA 0 INSUMOS INTERMEDIOS ξi A1i K D1i χ1i M1i D2i A2i 0 ... A 10i χ2i M2i D10i χ10i M10i El producto del sector i (Qi) se produce utilizando capital (K), trabajo no calificado (L), trabajo calificado (H) e insumos intermedios representados por agregados Armington (Aji). Utilizando el supuesto de Armington (1969), la demanda intermedia del sector i de bien j se representa con un bien compuesto formado por bienes domésticos (Dji) e importaciones (Mji). Un Agregado Armington también se emplea en el consumo privado (AiD). La producción Qi se destina al consumo doméstico de bienes (Di) y a la exportación (Ei). La elección de los -15- productores entre vender al mercado doméstico o exportar al resto del mundo se modela mediante una función de transformación tipo CET (Elasticidad de Transformación Constante). Se supone la existencia de un agente representativo (RA) que tiene una dotación fija de L, H y K, recauda impuestos y demanda AD. Se supone que existe pleno empleo de los factores primarios de producción. Los factores primarios de producción son perfectamente móviles entre sectores y tienen una oferta inelástica. En el gráfico, las letras griegas representan elasticidades y la interpretación de cada una es la siguiente: εi y χji son las elasticidades de sustitución Armington en el consumo final e intermedio, respectivamente; Ωi es la elasticidad de sustitución entre L y H en la producción de i; ξi es la elasticidad de sustitución entre N y K en la producción de i y ψi es la elasticidad de transformación entre las ventas al mercado doméstico (D) y las exportaciones (E) de i. Finalmente, la elasticidad de sustitución entre el valor agregado y los insumos intermedios es cero; es decir, el valor agregado y los insumos intermedios se demandan en proporciones fijas del producto. ESTRUCTURA DEL MODELO GENERAL En esta sección se presentan, de manera resumida, las funciones que determinan el comportamiento optimizador de los agentes del Modelo General. Para construir un modelo de CGE es necesario especificar las formas funcionales que caracterizan las preferencias y la tecnología de producción. Al igual que cuando se presentó el Modelo Simple, para el lector interesado, en las notas al pie de página se muestra la formulación algebraica de las formas funcionales. Adicionalmente, el Apéndice A presenta todas las ecuaciones de este modelo de manera análoga a como fueron codificadas en GAMS. La estructura del modelo y las formas funcionales utilizadas en este trabajo son estándar en la literatura que emplea modelos de CGE. Lado de la producción Cada sector productivo i produce dos tipos de bien: uno para el mercado doméstico (Di) y otro para exportar al resto del mundo (Ei). Se supone que estos bienes son sustitutos imperfectos. Para modelar esta posibilidad se utiliza una función con Elasticidad de Transformación -16- Constante (CET)17. Para producir, cada sector utiliza trabajo (Ni), capital (Ki) y bienes intermedios (Aji). El trabajo y el capital son perfectamente móviles entre sectores. La función de producción del sector i es, entonces, [ Qi = g (Di , E i ) = f K i , N i ( Li , H i ), A ji ] donde g es la función de transformación y f es la función de producción que convierte factores primarios e insumos intermedios en producto. La función de transformación es g (Di , Ei ) = CET (Di , Ei ) La función de producción combina valor agregado e insumos intermedios mediante una función tipo Leontief (LF) o de coeficientes fijos. El trabajo y el capital se combinan mediante una función con Elasticidad de Sustitución Constante (CES)18 para producir el valor agregado. A su vez, el trabajo total utilizado por cada sector es un agregado tipo CES de trabajo no calificado y trabajo calificado. Por su parte, los insumos intermedios de los distintos sectores son incorporados como un agregado tipo Leontief. La función de producción es, entonces, [ ] f = K i , N i (Li , H i ), A ji = LF [CES (K i , N i ), LF ( A1i , A2i ,..., A10 i )] donde Ni es, a su vez, igual a N i = CES (Li , H i ) El insumo intermedio producido por el sector j utilizado en el sector i (Aji) es un bien compuesto (Armington) por las variedades doméstica (DIji) e importada (MIji) del bien j. Los 17 Algebraicamente, la función de transformación tipo CET puede escribirse como ψi 1+ψ i 1+ψ i 1+ψ ⎞ i ⎛ CET (Di , Ei ) = θ i ⎜ηiD Di ψ i + ηiE Ei ψ i ⎟ ⎠ ⎝ donde θi es un parámetro de escala, ηi es un parámetro de distribución y ψi es la elasticidad de transformación. 18 Algebraicamente, la función tipo CES puede escribirse como ξi ξ i −1 ξ i −1 ξ −1 i CES (K i , N i ) = µ i ⎛⎜ν iK K i ξ i + ν Nj N i ξ i ⎞⎟ ⎝ ⎠ donde µi es un parámetro de escala, νi es un parámetro de distribución y ξi es la elasticidad de sustitución entre el capital y el trabajo en la producción del bien i. -17- productores consideran a estas variedades como sustitutos imperfectos. Esto se modela mediante una función tipo CES: A ji = CES (DI ji , MI ji ) Lado del consumo El agente representativo es dueño de toda la dotación factorial de la economía (K, L y H), recibe -como una transferencia de suma fija- todo lo recaudado por el arancel y demanda bienes. La función de utilidad de este consumidor es de tipo Cobb-Douglas (CD)19 y demanda bienes domésticos e importados combinados en un agregado Armington: U = CD( AiD , A2 D ,..., A10 D ) donde U es la utilidad que el agente representativo deriva de consumir los bienes compuestos tipo Armington AiD. Estos bienes compuestos se modelan, al igual que para el consumo intermedio, utilizando una función tipo CES: AiD = CES (DDiD , MDiD ) donde DDiD (MDiD) es la cantidad de la variedad doméstica (importada) de bien i consumida por el consumidor representativo. Gobierno Al igual que en el Modelo Simple, el gobierno se modela como recaudando lo producido por el arancel y devolviendo el total recaudado al consumidor representativo como una transferencias de suma fija. En consecuencia, el agente representativo consolida las demandas privada, pública y de inversión. 19 Algebraicamente, la función de utilidad tipo Cobb-Douglas puede escribirse como 10 CD ( A1D , A2 D ,..., A10 D ) = ∏ AiD i α i =1 donde αi es un parámetro de distribución. -18- Condiciones de equilibrio El equilibrio de este modelo se caracteriza por las siguientes condiciones: Los precios de los factores y los bienes producidos domésticamente son determinados endógenamente. En todos los mercados la oferta se iguala con la demanda. Los beneficios de los productores son cero. Esto significa que los costos de producción de cada sector se igualan con el valor del producto. El consumidor representativo cumple con su restricción presupuestaria. Es decir, el valor de sus gastos se iguala con su ingreso. Este ingreso está dado por la remuneración a los factores productivos que posee más el monto recaudado por los aranceles más la transferencia (igual al déficit comercial) que recibe desde el resto del mundo. Esta transferencia es igual al déficit comercial que se supone fijo en el valor del equilibrio inicial. Asimismo, el sector externo de la economía también se encuentra equilibrado. V. MATRIZ DE CONTABILIDAD SOCIAL Y CALIBRACION Para la calibración de los modelos se construyó una Matriz de Contabilidad Social (SAM) con información para la Argentina correspondiente al año 1992 y estimaciones econométricas para algunas de las elasticidades del modelo obtenidas de la literatura. La SAM construida representa el equilibrio inicial o caso base. La información utilizada para construir la SAM se obtuvo (principalmente) de la versión 3 de la base de datos del Global Trade Analysis Project (GTAP). Esta información se complementó con información de la Matriz Insumo-Producto (MIP) argentina de 1997, del Sistema de Cuentas Nacionales (SNC), de la EPH y del TRade Analysis and INformation System (TRAINS). Se utilizan dos versiones de la SAM: una para calibrar el Modelo Simple y otra para calibrar el Modelo General. En el Apéndice B se presenta una descripción detallada de las fuentes de información utilizadas para construir la SAM. Además, se presentan los valores de las elasticidades del modelo. Una SAM refleja todas las transacciones que tienen lugar en una economía determinada durante un período de tiempo dado (típicamente, un año). En términos generales, una SAM capta el proceso circular de la demanda, que lleva a la oferta, que lleva al ingreso, que lleva nuevamente a la demanda. Para construir una SAM que permita calibrar un modelo de CGE es necesario suponer que los valores observados de las variables constituyen un “equilibrio general”. Es decir, deben -19- cumplirse las siguientes condiciones: (i) las demandas se igualan a las ofertas en todos los mercados, (ii) ningún sector productivo tiene beneficios positivos, (iii) todos los agentes modelados cumplen con su restricción presupuestaria y (iv) el sector externo de la economía está equilibrado. En la práctica, no todas las estadísticas publicadas cumplen estas condiciones, por lo que resulta necesaria la realización de varios ajustes. En la Tabla 2 se presenta una SAM que resume la construida para calibrar el Modelo General. Todas las cifras que aparecen en la tabla están expresadas en millones de dólares de 1992. Tabla 2: Matriz de Contabilidad Social Firmas Factores Flias Gobierno Cuenta K ROW TOTAL 1 2 3 4 5 6 7 14,292 378,899 Firmas 1 145,880 Factores 2 219,247 Flias 3 Gobierno 4 Cuenta K 5 ROW 6 11,522 TOTAL 7 378,899 218,727 219,247 219,247 2,251 3,043 5,421 227,711 793 3,043 5,421 8,192 219,247 227,711 5,421 19,713 3,043 5,421 19,713 Fuente: Elaboración propia en base a datos del GTAP, MIP 1997, SCN, EPH y TRAINS. Para interpretar una SAM se sigue el principio de que las columnas realizan pagos a las filas. Por ejemplo, la celda (1,2)20 expresa que los sectores productivos pagaron 219,247 millones de dólares como remuneración a los factores primarios de producción. Como muestra la celda (3,2), este monto lo recibieron las familias que son dueñas de toda la dotación factorial de la economía. La celda (1,3) expresa que las familias gastaron 218,727 millones de dólares en bienes que producen los sectores productivos. La celda (4,3) expresa que las familias pagaron 793 millones de dólares al gobierno en concepto de aranceles a la importación. La celda (6,3) expresa que las familias importaron bienes desde el resto del mundo por valor de 8,192 millones de dólares. La celda (7,3) expresa que el gasto total de las familias en 1992 fue 227,711 millones de dólares. Este valor es igual al de la celda (3,7) que expresa cual fue el ingreso total de las familias en ese año. Para determinar el valor de los parámetros de participación y de escala de las funciones del modelo se emplea la información de la SAM combinada con valores (obtenidos de la literatura) de las elasticidades de sustitución y transformación. El procedimiento empleado se 20 La celda (i,j) se refiere al valor correspondiente a la intersección de la fila i con la columna j. -20- denomina calibración y consiste en obtener valores para los parámetros del modelo de manera tal que la solución inicial del modelo replique los valores de la SAM (Mansur y Whalley, 1984). Para esto, en lugar de resolver el modelo para obtener un equilibrio, se utiliza el equilibrio observado (el del caso base) para calcular los valores de los parámetros del modelo consistentes con esa observación. En la calibración se distingue entre “parámetros libres” (elasticidades) que pueden ser obtenidos de fuentes externas y “parámetros calibrados” (parámetros de distribución y escala) que son derivados de los anteriores combinados con la SAM de manera tal de reproducir el caso base. VI. EJERCICIOS DE SIMULACION VI.1. COMERCIO Y DESIGUALDAD SALARIAL Los modelos presentados más arriba pueden emplearse para calcular cuánto del cambio en el diferencial salarial se debe al aumento del comercio internacional. Para esto, la apertura comercial argentina se modela como disminuciones en las tasas arancelarias. Como se mencionara más arriba, los datos para la Argentina indican que entre 1992 y 1998, el arancel promedio cayó alrededor de 30%. Durante el mismo período, el cociente wH/wL pasó de 1,70 en 1992 a 1,88 en 1998, indicando un incremento de 10%. RESULTADOS DEL MODELO SIMPLE En esta sección se presentan los resultados de realizar las simulaciones empleando el Modelo Simple. Este modelo considera que existe diferenciación de productos sólo del lado del consumo. Cuando se utiliza el Modelo Simple, se consideran dos sectores productivos: uno que es exportable neto y otro importable neto. Así, teniendo en cuenta que Argentina exporta principalmente productos intensivos en trabajo no calificado y la disminución de la tasa del arancel, se espera que el comercio tenga un efecto negativo sobre la desigualdad salarial. En la Tabla 3 se presentan los resultados de simular la apertura comercial para distintos valores de la elasticidad de sustitución en el agregado (Armington) del bien D y del bien M, ε. En todos los ejercicios, la elasticidades de sustitución entre trabajo calificado y no calificado en la producción de los bienes E y D, ΩE y ΩD, se mantienen iguales a 1,2521. 21 Este es el valor empleado por Abrego y Whalley (1999 y 2000). -21- Tabla 3: Resultados del Modelo Simple 0.2 1 ε 2 5 10 Porcentaje del cambio en w H /w L debido a la apertura comercial 0.1 -0.9 -2.1 -6.0 -13.3 Porcentaje del cambio en w H /w L debido a otros factores 99.9 100.9 102.1 106.0 113.3 La segunda columna de la Tabla 3 indica que -cuando la elasticidad de sustitución entre D y M es 0,2- del aumento del diferencial salarial de 10%, la baja de aranceles sólo puede explicar el 0,1% y el restante 99,9% es explicado por otros factores. Los resultados que se obtienen indican que el comercio no puede explicar el aumento de la desigualdad salarial ocurrido en la última década. De hecho, en este modelo, para valores plausibles de los parámetros la baja de los aranceles hace disminuir la desigualdad salarial. Se observa, además, que cuando los bienes D y M se hacen más sustituibles (aumenta el valor de ε) la porción del aumento en el diferencial salarial que explica el comercio disminuye. RESULTADOS MODELO GENERAL En esta sección se presentan los resultados obtenidos cuando se emplea el modelo más general para analizar el efecto de la apertura comercial sobre el diferencial salarial entre trabajadores con distinta calificación. Una de las ventajas de utilizar este modelo es que permite captar los cambios de las tasas arancelarias que enfrentan las importaciones de cada bien. Durante el período analizado, la baja de aranceles fue mayor para los productos manufactureros que para los productos agrícolas. Los ejercicios de simulación se realizan utilizando distintos valores para los parámetros del modelo. El ejercicio 1 es la especificación central y los ejercicios 2, 3 y 4 constituyen un análisis de sensibilidad de ésta con respecto al valor de los parámetros más relevantes para el problema bajo estudio. En el ejercicio 1 las elasticidades de sustitución (Armington) entre bienes domésticos e importados son el doble de las reportadas en la Tabla B1 del Apéndice B y obtenidas del GTAP, la elasticidad de la frontera de transformación entre ventas al mercado doméstico y exportaciones (CET) es 8 para todos los sectores (Harrison et al., 1997)22, la 22 Varios autores utilizan infinito como valor para esta elasticidad de transformación (Hertel, 1997). -22- elasticidad de sustitución entre trabajo calificado y no calificado es 0,30 (Fallon y Layard, 1975). En todos los ejercicios las elasticidades de sustitución entre trabajo y capital son las mismas y se obtienen del GTAP. Para una descripción de las elasticidades obtenidas del GTAP véase el Apéndice B. La Tabla 4 muestra los resultados y la Tabla 5 los valores asignados a las elasticidades en cada ejercicio de simulación. Tabla 4: Resultados del Modelo General EJ 1 EJ 2 EJ 3 EJ 4 EJ 5 Porcentaje del cambio en w H /w L debido a la apertura comercial 2.9 -0.1 5.9 3.1 0.9 Porcentaje del cambio en w H /w L debido a otros factores 97.1 100.1 94.1 96.9 99.1 La Tabla 4 muestra que cuando se utiliza la especificación central (ejercicio 1), el aumento del comercio producto de la apertura comercial sólo explica el 2,9% del aumento total (igual a 10%) del diferencial salarial wH/wL. El 97,1% restante es explicado por otros factores. Tabla 5: Valores asignados a las elasticidades ELASTICIDAD EJ 1 EJ 2 EJ 3 EJ 4 EJ 5 L vs. H (Ωi) D vs. E (ψi) DD vs. MD (εi) DI vs. MI (χij) 0.3 8.0 GTAP2 GTAP2 0.3 8.0 GTAP GTAP 0.3 0.3 1.1 8.0 3.0 8.0 GTAP3 GTAP2 GTAP2 GTAP3 GTAP2 GTAP2 Los resultados presentados muestran que la apertura comercial explica entre el 0 y el 6%, aproximadamente, del aumento en el diferencial salarial ocurrido en Argentina entre 1992 y 1998. Adicionalmente, de la tabla se deduce que la porción del aumento en el diferencial salarial que explica la disminución de los aranceles es mayor cuando se aumentan las elasticidades de sustitución (Armington) entre bienes domésticos e importados. Lo mismo ocurre cuando se disminuye la elasticidad de transformación (CET) entre ventas al mercado doméstico y exportaciones al resto del mundo. Por el contrario, el efecto de la apertura comercial sobre la desigualdad salarial es menor cuando se incrementa la elasticidad de sustitución entre trabajo calificado y no calificado. El precio doméstico de un bien (el precio de un agregado Armington) determinado es un promedio ponderado de los precios de las variedades doméstica e importada de ese bien. En -23- consecuencia, mayores elasticidades Armington hacen que el sistema de precios doméstico sea más sensible a cambios en las tasas arancelarias. Por lo tanto, mayores elasticidades Armington hacen que la baja de aranceles explique más del aumento en el diferencial salarial. La elasticidad de transformación (CET) determina la facilidad con la que cada sector productivo puede cambiar entre vender al mercado doméstico o exportar al resto del mundo. A mayor valor de esta elasticidad, la variedad que se vende al mercado doméstico y la variedad que se exporta al resto del mundo son más similares por lo que el precio de la variedad doméstica es más sensible a las variaciones en el precio (exógeno) de la variedad que se exporta. En consecuencia, un aumento en el valor de la elasticidad de transformación hace que el precio de la variedad doméstica responda con menor intensidad a cambios en el precio de la variedad importada (a través del agregado Armington) ya que si parte de la producción de la variedad doméstica no se vende, puede más fácilmente transformarse en exportaciones al resto del mundo. Por lo tanto, una menor elasticidad de transformación hace que la baja de aranceles explique una porción mayor del aumento en el diferencial salarial. Adicionalmente, en el Apéndice C se presentan los resultados de simular la apertura comercial argentina pero diferenciando por tipo de importación: bienes de capital, insumos intermedios y bienes para consumo final. VI.2. CALCULO DEL CAMBIO TECNOLOGICO El objetivo de realizar este cálculo es únicamente metodológico ya que los cambios ocurridos en la economía argentina durante los noventa hacen imposible considerar que el único factor, además de la apertura comercial, que explica el aumento de la desigualdad salarial es el cambio tecnológico sesgado contra los trabajadores no calificados. Esta metodología se basa en la propuesta por Abrego y Whalley (1999 y 2000) y puede ser empleada para realizar distintos ejercicios de descomposición con un modelo de CGE. Debido a que el cambio tecnológico es el factor que aparece en la literatura como causa principal del aumento de la desigualdad salarial, luego de calculado el efecto de la apertura comercial sobre el aumento del diferencial salarial, se realiza un ejercicio en el que se determina -de manera residual- cuál es el cambio tecnológico necesario para que el modelo de como solución -cuando se introducen simultáneamente ambos cambios de manera exógenaun cambio en el cociente wH/wL igual al ocurrido en Argentina en el período 1992 a 1998. -24- El tipo de cambio tecnológico que se considera es específico del factor trabajo. En particular, es sesgado contra el trabajo no calificado. El cambio tecnológico se modela como variaciones en los parámetros de participación de las funciones de producción de bienes. Debido a que los parámetros de participación en cada función de producción suman uno, un shock adverso al trabajo no calificado disminuye el parámetro de participación de L en relación al de H para cada sector productivo. A los fines de descomponer el aumento observado en la desigualdad salarial en un componente atribuible al comercio internacional y otro al cambio tecnológico, el modelo se resuelve cinco veces. En la Tabla D1 del Apéndice D se indica cuáles son las variables exógenas y endógenas en cada corrida del Modelo Simple. Para los ejercicios de descomposición del cambio en wH/wL, en primer lugar se determina el cambio en el diferencial salarial producto del cambio en los aranceles, en segundo lugar, se obtiene (resolviendo un modelo donde el cambio tecnológico es una variable endógena y el diferencial salarial está dado exógenamente) el cambio tecnológico necesario para que el diferencial salarial sea igual al observado (en el caso argentino, 10% en el período 1992 a 1998). El resultado de cada uno de los ejercicios de descomposición que se realizan depende de los valores que tomen los parámetros libres del modelo (elasticidades en la producción y en el consumo). VII. CONCLUSIONES Y EXTENSIONES • Del análisis realizado en este trabajo se desprende que la apertura comercial no es una causa importante del aumento de la desigualdad salarial ocurrido durante la década del noventa. Utilizando un modelo de CGE con una estructura estándar se obtuvo que, para valores usuales de los parámetros del modelo, el comercio explica alrededor del 3% del aumento en el diferencial salarial ocurrido en Argentina durante la década del noventa. Este resultado es bajo cuando se los compara con las estimaciones presentes en la literatura. • La comparación de los resultados obtenidos con el Modelo Simple y con el Modelo General permiten apreciar la importancia que tienen la estructura y la dimensión del modelo para obtener un resultado donde el comercio explica parte del aumento del diferencial salarial. -25- • Los modelos de CGE son ampliamente utilizados para el análisis cuantitativo de políticas. Sin embargo, la metodología del CGE es frecuentemente criticada. Los aspectos más criticados son la utilización de valores aparentemente arbitrarios para los parámetros de comportamiento (elasticidades) y la falta de validación empírica de la estructura de los modelos. Estas deficiencias son particularmente problemáticas si se tiene en cuenta que los resultados del modelo son usualmente muy sensibles a estos supuestos. En el caso de las elasticidades de sustitución Armington utilizadas en este trabajo, en un trabajo reciente, Liu et al. (2001) demuestran, en el contexto del modelo estándar del GTAP23, que los valores de las elasticidades Armington de la base de datos del GTAP utilizadas en este trabajo no resultan marcadamente diferentes de las que estiman como correctas en un ejercicio de predicción hacia atrás24. • Se presentó una metodología que puede ser empleada para realizar ejercicios de descomposición con un modelo de CGE. • El modelo utilizado en este trabajo posee una estructura muy simple. Además se trata de un modelo que sólo capta los efectos estáticos de la baja arancelaria. Por lo tanto, una posible extensión del presente trabajo es la inclusión de aspectos no estándar en el modelo de CGE como por ejemplo, la acumulación de capital (Francois y Reinert, 1997), externalidades debidas al comercio (De Melo y Robinson, 1992), costos de transacción (Bussolo, 2001), economías de escala y competencia imperfecta (Francois y Reinert, 1997), etc. Adicionalmente, también podría incorporarse al modelo el sector financiero de la economía. En el caso argentino, esto es particularmente importante ya que, durante los noventa, además de la apertura comercial se produjo una apertura financiera. • Otra posible extensión es la utilización de un modelo de CGE multiregional que permita diferenciar el efecto sobre la desigualdad salarial de la apertura comercial unilateral y del Mercosur. En este sentido, una posibilidad es realizar simulaciones de “predicción hacia atrás” utilizando la versión 5 de la base de datos del GTAP (que contiene información referida al año 1997) y un modelo multiregional similar al presentado por Rutherford y Paltsev (2000). 23 El modelo estándar del GTAP es un modelo de CGE multisectorial y multiregional ampliamente utilizado para el análisis de políticas comerciales desde una perspectiva global. 24 Sin embargo, rechazan la hipótesis de igualdad entre las elasticidades estándar del GTAP y las estimadas. -26- • Teniendo en cuenta que en este trabajo se simuló la apertura comercial únicamente como una baja de aranceles, es probable que los resultados obtenidos subestimen el efecto del comercio sobre el diferencial salarial ya que la apertura comercial argentina se llevó a cabo también a través de la eliminación de restricciones cuantitativas25. 25 Para ver la importancia de las medidas de protección no arancelarias puede consultarse Bouët et al. 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PARAMETROS ELASTICIDADES ξi Elasticidad de sustitución entre el agregado de trabajo y el capital Ωi Elasticidad de sustitución entre trabajo calificado y no calificado σi Elasticidad de sustitución en el consumo final (nivel superior) ψi Elasticidad de transformación (CET) entre ventas al mercado doméstico y exportaciones εi Elasticidad de sustitución (Armington) entre bienes domésticos e importados en el consumo final χ ji Elasticidad de sustitución (Armington) entre bienes domésticos e importados en el consumo intermedio PARAMETROS DE DISTRIBUCION Y ESCALA ι Dji Participación de bienes domésticos en el consumo intermedio (Armington) ι Mji Participación de bienes importados en el consumo intermedio (Armington) ζ ji Parámetro de escala en el consumo intermedio (Armington) δi Participación del trabajo no calificado en el agregado de trabajo φi Parámetro de escala en el agregado de trabajo ν iN Participación del agregado de trabajo en la producción de valor agregado ν iK Participación del capital en la producción de valor agregado -32- µi Parámetro de escala en la producción de valor agregado ηiD Participación de ventas al mercado doméstico en ventas totales ηiE Participación de exportaciones en ventas totales θi Parámetro de escala en la función de transformación (CET) αi Participación de i en el consumo final β iD Participación de bienes domésticos en el consumo final (Armington) β iM Participación de bienes importados en el consumo final (Armington) γi Parámetro de escala en el consumo final (Armington) OTROS K Dotación de capital L Dotación de trabajo no calificado H Dotación de trabajo calificado IO ji Requerimiento de insumo j por unidad de producto i B Déficit comercial en el equilibrio inicial Pi M Precio mundial de las importaciones de i Pi E Precio mundial de las exportaciones de i τ Iji Tasa del arancel al consumo intermedio de j en la producción de i τ iD Tasa del arancel al consumo final de i PREMIO Diferencial salarial A.2. VARIABLES AI ji Consumo intermedio de j en la producción de i DI ji Consumo intermedio de j doméstico en la producción de i MI ji Consumo intermedio de j importado en la producción de i -33- PjiAI Precio del agregado (Armington) j de bienes doméstico j e importado j para consumo intermedio en la producción de i ADi Consumo final del bien i MDi Consumo final del bien importado i DDi Consumo final del bien doméstico i Pi AD Precio del agregado (Armington) i de bienes doméstico i e importado i para consumo final Pi D Precio del bien doméstico QS i Oferta total ES i Oferta para exportaciones DS i Oferta para ventas al mercado doméstico Li Demanda de trabajo no calificado Hi Demanda de trabajo calificado Ni Demanda del agregado de trabajo Ki Demanda de capital WL Remuneración al trabajo no calificado WH Remuneración al trabajo calificado Wi N Remuneración al agregado de trabajo en el sector i WK Remuneración al capital I Ingreso del consumidor R Transferencia del gobierno π Cambio tecnológico sesgado contra el trabajo no calificado A.3. ECUACIONES Para facilitar la lectura, las ecuaciones del modelo fueron agrupadas en seis bloques. En las secciones siguientes se presentan las ecuaciones pertenecientes a cada uno de ellos. -34- BLOQUE DE OFERTA DE BIENES Las ecuaciones de este bloque surgen de la maximización de beneficios del productor. Cada productor maximiza sus beneficios sujeto a una función de transformación tipo CET. Esta función de transformación determina la cantidad que vende en el mercado doméstico y la cantidad que exporta al resto del mundo. La ecuación (1) es la función de oferta de bienes al mercado doméstico y la ecuación (2) es la función de oferta de exportaciones al resto del mundo. La ecuación (3) es la condición de cero beneficios del productor. (η ) (P ) QS DS = ( ) θ [(P ) (η ) + (P )( ) (η ) ] ψi (η ) (P ) QS ES = ( ) (η ) + (P )( ) (η ) ] θ [(P ) ψi D −ψ i i D ψi i i i 1+ψ i D i i D −ψi i E E ψi i i i i 1+ψ i i (1) E −ψi 1+ψ i i i E −ψ i i D 1+ψ i D −ψi i E 1+ψ i (2) E −ψi 1+ψ i i i Pi E ES i + Pi D DS i = Wi N N i + W K K i + ∑ PjiAI AI ji (3) j BLOQUE DE DEMANDA DE FACTORES PRIMARIOS Las ecuaciones de este bloque surgen de la minimización de costos de los productores. En primer lugar, cada productor combina, en proporciones fijas, valor agregado e insumos intermedios. Por el lado del valor agregado, la ecuación (4) es la función de demanda del agregado de trabajo (N) y la ecuación (5) es la función de demanda de capital (K). En la siguiente etapa, el productor determina la composición de trabajo no calificado (L), ecuación (6), y trabajo calificado (H), ecuación (7), del trabajo total que demanda. Por último, la ecuación (8) asegura que el gasto total en el factor trabajo se distribuya entre pagos a las dos variedades de trabajo. (ν ) QS N = ( ) ( ) ] µ (W ) [(ν ) (W ) + ν (W ) (4) (ν ) QS K = ( ) ( ) ] µ (W ) [(ν ) (W ) + ν (W ) (5) N ξi i i N ξi i K ξi i K 1−ξ i K ξi i i K ξi i K ξi i K i 1−ξ i N ξi i N 1−ξ i i ξi ξ i −1 i N ξi i -35- N i 1−ξ i ξi ξ i −1 Li = (δ iπ )Ω N i φi (W ) Hi = L Ωi [(δ π ) (W ) L (1− Ω i ) Ωi i + (1 − δ iπ ) W Ωi (6) ] Ωi H (1− Ω i ) Ω i −1 [(1 − δ iπ )]Ω N i φi (W ) [(δ π ) (W ) H Ωi Ωi L (1− Ω i ) i + (1 − δ iπ ) W Ωi (7) ] Ωi H (1− Ω i ) Ω i −1 Wi N N = W L Li + W H H (8) BLOQUE DE DEMANDA DE INSUMOS INTERMEDIOS Las ecuaciones (9), (10) y (11) definen el agregado Armington de insumos intermedios. La ecuación (9) es la función de demanda intermedia de bienes domésticos y la ecuación (10) es la función de demanda intermedia de bienes importados. La ecuación (11) es la condición de cero beneficios en la producción de este agregado Armington. Por último, la ecuación (12) determina la cantidad demandada de insumo intermedio j por unidad de bien i. El lado derecho de esta ecuación es la función de producción de valor agregado que utiliza como insumos el agregado de trabajo (N) y capital (K). DI ji = (ι ) AI D χ ji ji ζ ji (Pi D ) χ ji MI ji = {(ι ) [P (1 + τ )] M χ ji ji M (1− χ ji ) I ji i ji (ι ) AI M χ ji ji ( ) {(ι ) [P (1 + τ )] ζ ji Pi M χ ji ( M χ ji ji M i ) PjiAI AI ji = Pi M 1 + τ Iji MI ji + Pi D DI ji } i ( ) P +ι D χ ji ji i ( D 1− χ ji } χ ji ) χ ji −1 (9) (10) (11) ξi ξ i −1 ξ i −1 ξ −1 ⎡ ⎤ i = µ i ⎢ν iN N i ξ i + ν iK K i ξ i ⎥ IO ji ⎣ ⎦ AI ji χ ji ji (1− χ ji ) I ji ( ) P + ι Dji χ ji D (1− χ ji ) χ ji −1 (12) BLOQUE DE DEMANDA FINAL Las ecuaciones de este bloque surgen de la maximización de la utilidad del consumidor representativo. Esta maximización de utilidad se realiza en dos etapas. En la primera, el consumidor determina cuánto consume de cada bien, ecuación (13). En la segunda, determina cuánto de la variedad doméstica y cuánto de la variedad importada de cada bien consume, -36- ecuaciones (14) y (15), respectivamente. Por último, la ecuación (16) asegura que el gasto total del consumidor se distribuya completamente entre bienes domésticos e importados. ADi = αi I (13) (P ) ∑α (P )( ) AD σ i 1−σ AD j j j (β ) AD DD = ( ) ( ) } γ (P ) {(β ) P + (β ) [P (1 + τ )] D εi i i i D εi i i D εi i D 1−ε i M εi i i M i D i 1−ε i εi ε i −1 (β ) AD MD = ( ) ( ) } γ (P ) {(β ) P + (β ) [P (1 + τ )] M εi i i M εi i i D εi i ( D 1−ε i i i M εi i M i ) Pi AD ADi = Pi M 1 + τ iD MDi + Pi D DDi D i 1−ε i εi ε i −1 (14) (15) (16) BLOQUE DE CONDICIONES DE EQUILIBRIO Este bloque contiene las condiciones de equilibrio en los distintos mercados del modelo. La ecuación (17) es la condición de equilibrio en el mercado del bien doméstico i. Las ecuaciones (18), (19) y (20) son las condiciones de equilibrio en los mercados de L, H y K, respectivamente. La ecuación (21) es la restricción presupuestaria del agente representativo que expresa que el ingreso total del consumidor es igual a la remuneración a los factores productivos más la transferencia que recibe del gobierno más la transferencia que recibe desde el resto del mundo (exógena e igual al saldo de la balanza comercial). La ecuación (22) es la restricción presupuestaria del gobierno que indica que la transferencia que realiza el gobierno es igual al monto recaudado por el arancel. Finalmente, la ecuación (23) es la condición de equilibrio del sector externo de la economía. DS i = DDi + ∑ IDij (17) j L = ∑ Li (18) i H = ∑ Hi (19) i K = ∑ Ki (20) i I =W H H +W LL +W K K + R + B (21) -37- ⎛ ⎞ ∑ ⎜⎜ P τ MD + ∑ P τ MI ⎟⎟ = R i M i ⎝ D i M i i I ij ij j ⎛ (22) ⎠ ⎞ ∑ P ES + B = ∑ ⎜⎜ P MD + ∑ P MI ⎟⎟ E i i i i ⎝ M M i i ij j ⎠ (23) BLOQUE PARA ESTIMAR EL CAMBIO TECNOLOGICO La ecuación (24) forma parte de la estructura del modelo cuando se lo resuelve para obtener el cambio necesario en la variable que representa el cambio tecnológico (π) para que el modelo replique el cambio en el diferencial salarial observado en el período 1992 a 1998. W H = (PREMIO )W L (24) El cambio tecnológico se modela como una variación en el valor del parámetro δi de las funciones de producción de bienes. Cuando el cambio tecnológico es sesgado a favor de la mano de obra calificada, el valor del parámetro δi disminuirá en una fracción común en todos los sectores productivos calculada como residuo luego de introducido el cambio en las tasas arancelarias. En las ecuaciones referidas al lado de la producción del modelo, el parámetro δi aparece multiplicado por una variable π. Para replicar la SAM, π se fija igual a uno. Luego, cuando se introducen los cambios en las tasas arancelarias se agrega una ecuación que fija el diferencial salarial en 10% ( wH = 1.10wL ) y el valor de la variable π se determina endógenamente cuando se soluciona el modelo. A.4. CALCULO DEL EQUILIBRIO Para computar cada solución del modelo, se debe resolver el sistema de ecuaciones no lineales presentado más arriba. Empleando GAMS/MINOS, esto se puede realizar maximizando una función constante sujeta a restricciones que representan el modelo. -38- APENDICE B: MATRIZ DE CONTABILIDAD SOCIAL Y ELASTICIDADES B.1. MATRIZ DE CONTABILIDAD SOCIAL En este apéndice se describen las fuentes de información utilizadas para construir la SAM empleada para calibrar el modelo de CGE. Además, se describen los ajustes realizados a la información original. La fuente de información principal para la confección de la SAM es la versión 3 de la base de datos del Global Trade Analysis Project (GTAP) que contiene información referida al año 1992. Esta base de datos se encuentra extensamente documentada en Hertel (1997) y McDougall et al. (1998). El GTAP es un consorcio integrado por organizaciones gubernamentales y de investigación que funciona en la Universidad Purdue de Estados Unidos26. Uno de los objetivos básicos del GTAP es facilitar la realización de análisis de CGE sobre cuestiones relacionadas (principalmente) con el comercio internacional. La base de datos del GTAP es ampliamente utilizada por académicos y agencias gubernamentales de varios países del mundo para la realización de trabajos sobre comercio internacional que utilizan modelos de CGE. Esta base de datos está formada por Matrices de Contabilidad Social para los principales países del mundo y para varias regiones agregadas y por los flujos de comercio entre estos países o regiones. Toda la información en esta base de datos está referida a un año particular que se toma como base y todos los datos se encuentran expresados en base a una nomenclatura común. Del GTAP se extrajo información sobre estructura productiva y flujos de comercio para la Argentina. La información obtenida del GTAP se complementó con información de la EPH, la MIP argentina del año 1997 e información sobre tasas arancelarias obtenida de TRAINS27. Una de las características más relevantes para este trabajo de la SAM utilizada es el procedimiento que se empleó para determinar el uso de trabajo calificado y trabajo no calificado en cada sector productivo. Al respecto, se siguió el trabajo “Disaggregating Labor Payments by Skill Level in GTAP” de Jing Liu, Nico van Leeuwen, Tri Thanh Vo, Rod 26 27 El GTAP es dirigido por Thomas Hertel. La información de TRAINS fue originalmente recopilada para utilizar en Berretoni y Cicowiez (2001). -39- Tyers, and Thomas W. Hertel (1998)28. El procedimiento que siguen estos autores para estimar la participación de cada tipo de trabajo en la producción de cada sector consiste en estimar econometricamente la siguiente ecuación: MHP = F (GDPC, TER ) donde MHP es la participación del trabajo calificado en el pago total al factor trabajo, GDPC es el PBI per cápita y TER es el promedio de años de educación terciaria de toda la fuerza de trabajo de la economía. Para la estimación utilizan información para 20 de las 30 regiones consideradas en la versión 3 de la base de datos del GTAP. Con el resultado de la estimación, se predice el valor de MHP para las regiones no consideradas en la muestra. En particular, la información para Argentina es estimada por lo que se la corrigió utilizando información obtenida de la EPH29. Finalmente, para el cálculo del cambio en las tasas arancelarias sectoriales se utilizó información sobre comercio exterior argentino obtenida de TRAINS. B.2. ELASTICIDADES Las elasticidades de sustitución εi, χij y ξi (DD versus MD, DI versus MI y N versus K, respectivamente) se obtienen del GTAP. A su vez, estas elasticidades se originan en el Proyecto SALTER. Este proyecto realizó una extensa revisión de la literatura y algunas estimaciones propias para dar valor a las elasticidades sustitución de un modelo de CGE. Aunque se realiza un análisis de sensibilidad, esta es la fuente principal de las elasticidades empleadas en este trabajo. En la Tabla B1 se muestra el valor que asumen las elasticidades mencionadas. 28 En ese trabajo, el objetivo final de los autores es utilizar el procedimiento para construir la versión 4 de la base de datos del GTAP. 29 Debido a que la EPH es una encuesta urbana, el procedimiento descripto es particularmente importante para determinar el empleo de los distintos tipos de trabajo de los sectores primarios. -40- Tabla B1: Elasticidades de sustitución VA Sector Armington Cereales y Ganadería Otros Productos Agrícolas ξi 0.50 0.50 εi 2.44 2.60 χij 2.44 2.60 Industrias Alimenticias Textiles e Indumentaria 1.10 1.25 2.38 3.30 2.38 3.30 Manufacturas de Recursos Naturales 1.25 3.00 3.00 Minería, Petróleo y Carbón Transporte 0.50 1.25 2.62 5.20 2.62 5.20 Industria Química y Otras Industrias 1.25 2.50 2.50 Maquinaria y Productos Metálicos Servicios 1.25 1.35 2.80 2.05 2.80 2.05 Fuente: GTAP. La primera columna de la Tabla B1 reporta las elasticidades de sustitución en el nivel superior del valor agregado, ξi, para cada uno de los sectores productivos considerados. La elasticidad de sustitución entre factores primarios determina la facilidad de la economía para alterar su mix productivo como respuesta a cambios en el precio relativo de los bienes. Se observa que los sectores primarios presentan una elasticidad de sustitución menor. Esto está relacionado con la utilización de la tierra como factor primario en estos sectores lo que determina una respuesta limitada de la oferta de estos sectores30. El mayor valor para esta elasticidad de sustitución se observa en el sector Servicios. La Tabla B1 también reporta las elasticidades de sustitución entre bienes domésticos e importados en el consumo final e intermedio, εi y χij, respectivamente. Estas elasticidades son utilizadas en la estructura de demanda tipo Armington del Modelo General. Estas elasticidades determinan la facilidad con que los bienes domésticos pueden ser sustituidos por importaciones. Siguiendo a Harrison et al. (1997) y Hillberry et al. (2001), en la especificación central del Modelo General, se emplea el doble de las elasticidades de sustitución Armington del GTAP. Estos autores, entre otros, concluyen que las elasticidades de sustitución Armington del GTAP son bajas, en especial para simulaciones que analizan extensos períodos de tiempo 30 En este trabajo, el factor tierra está incluido en el factor capital. -41- APENDICE C: SIMULACIONES ADICIONALES En este apéndice se utiliza el Modelo General para analizar la apertura comercial argentina pero diferenciando por tipo de importación. Utilizando los parámetros de la especificación central, se realizan tres simulaciones adicionales: En la primera, sólo se simula la baja de los aranceles a la importación de bienes de capital31. En la segunda, sólo se simula la baja de los aranceles a la importación de insumos intermedios32. Por último, en la tercera, sólo se simula la baja de aranceles a la importación de bienes destinados al consumo final. En la Tabla C1 aparecen los resultados. La primera columna reproduce el ejercicio 1 de la Sección VI y se incluye para facilitar la comparación de los resultados. Tabla C1: Resultados EJ 1 Bienes de Consumo Capital Intermedio Consumo Final Porcentaje del cambio en w H /w L debido a la apertura comercial 2.9 0.6 1.3 1.0 Porcentaje del cambio en w H /w L debido a otros factores 97.1 99.4 98.7 99.0 El resultado que se obtiene es que la mayor parte del aumento en el diferencial salarial se debe a la baja de aranceles a la importación de insumos intermedios. La baja de aranceles al consumo intermedio logra explicar 1,3% del aumento total en el diferencial salarial. Se observa también que la baja de aranceles a la importación de bienes de capital explica 0,6% del aumento total del diferencial salarial. Finalmente, la apertura comercial por el lado del consumo final logra explicar 1% el aumento total del diferencial salarial. 31 En este ejercicio, se simula una baja de aranceles al consumo intermedio de importaciones de Maquinaria y Productos Metálicos. 32 Se simula como una disminución de los aranceles al consumo intermedio de importaciones de todos los bienes excepto Maquinaria y Productos Metálicos. -42- APENDICE D: CORRIDAS DEL MODELO Para determinar el cambio tecnológico de manera residual para que el modelo replique el incremento del diferencial salarial ocurrido entre 1992 y 1998, los modelos presentados en la Sección IV son resueltos cinco veces. La Tabla D1 muestra cuáles son las variables endógenas y cuáles las exógenas en cada una de las cinco corridas del modelo. Aunque la tabla se refiere al Modelo Simple, con muy pocas modificaciones puede ser adaptada al Modelo General. Tabla D1: Descripción de cada corrida del modelo Corrida Descripción Objetivo V. Endógenas V. Exógenas 1 Réplica del caso base Verificar la correcta codificación del modelo wH, wL, PD, LD, HD, LE, PE, PM, L, H, τ, π, s s d HE, D , E , E , d D,M 2 Solución sólo con cambios en τ Calcular el efecto sobre los salarios del cambio en el arancel (τ') Idem 1 PE, PM, L, H, τ', π 3 Solución con cambios en τ y se determina el cambio tecnológico Calcular el valor del cambio tecnológico (π∗) consistente con el cambio observado en wH/wL Idem 1 + π y se obtiene π* τ', wH/wL 4 Solución sólo con cambio tecnológico Calcular el efecto sobre los salarios del cambio tecnológico Idem 1 τ, π* 5 Solución con cambios en τ y cambio tecnológico Comprobar que los resultados son correctos Idem 1 τ', π* Referencia: τ es el arancel en el equilibrio inicial y τ' es el arancel en el contrafactual. -43-
https://openalex.org/W3203176244	https://www.frontiersin.org/articles/10.3389/fpain.2021.722820/pdf	English	null	Biased Opioid Ligands: Revolution or Evolution?	Frontiers in pain research	2,021	cc-by	4,556	Biased Opioid Ligands: Revolution or Evolution? Université de Paris, CNRS ERL 3649 Pharmacologie et thérapies des addictions, Inserm UMR-S 1124 T3S Environmental Toxicity, Therapeutic Targets Cellular Signaling and Biomarkers, Paris, France Opioid are the most powerful analgesics ever but their use is still limited by deleterious side effects such as tolerance, dependence, and respiratory depression that could eventually lead to a fatal overdose. The opioid crisis, mainly occurring in north America, stimulates research on ﬁnding new opioid ligands with reduced side effects. Among them, biased ligands are likely the most promising compounds. We will review some of the latest discovered biased opioid ligands and see if they were able to fulﬁll these expectations. INTRODUCTION Opium consumption and its medicinal application dates back to the Neolithic, Bronze, and Iron Age. Ancient Egyptian papyrus records reported the use of opium for pain relief, demonstrating that opioids are used from thousands of years for the treatment of pain. Opium is a substance extracted from Papaver somniferum, and opium poppies produce alkaloids such as morphine (1). In 1973, the notion of “morphine receptors” emerged (2), and since that time numerous studies have been performed on these receptors. In the 1990s, opioid receptors were cloned. They are four of them, MOR, DOR, KOR, and NOR, and are largely found within the central nervous system as well as throughout the peripheral tissues. They are naturally stimulated by endogenous peptides (endorphins, enkephalins, dynorphins, and nociceptin/orphanin FQ). All pharmacological studies conﬁrmed that opioid system plays a key role in the modulation of pain perception, but is also involved in many physiological responses, such as respiration, gastrointestinal motility, endocrine, and immune functions, which may lead in clinical practice to adverse eﬀects when pain must be managed with opioids. Edited by: Mei-Chuan Ko, Wake Forest School of Medicine, United States Reviewed by: Girolamo Calo, University of Padua, Italy Eamonn Kelly, University of Bristol, United Kingdom Reviewed by: Girolamo Calo, University of Padua, Italy Eamonn Kelly, University of Bristol, United Kingdom Correspondence: Nicolas Marie nicolas.marie@parisdescartes.fr Correspondence: Nicolas Marie nicolas.marie@parisdescartes.fr The study of responses of opioid receptors-deﬁcient mice to opioid agonists clariﬁes the biological activity of each receptor. The genetic approach unambiguously demonstrated that MOR are essential for all the biological activities elicited by morphine, including for instance analgesia, reward, respiratory depression, constipation, immunosuppression (3). Thus, a challenge over the past decades has been to try to ﬁnd MOR opioid ligands able to induce a potent analgesic response, with fewer adverse eﬀects. Specialty section: This article was submitted to Pharmacological Treatment of Pain, a section of the journal Frontiers in Pain Research MOR belong to the G protein-coupled receptors (GPCR), and it is now well-established that once the receptor is activated, several intracellular signaling pathways may be activated. Among these pathways, the focus has been made on two of them, G protein (and its subsequent eﬀectors such as adenylate cyclase and ion channels) and beta-arrestin, and it has been suggested that analgesic eﬀects of MOR ligands are mediated by the downstream G-protein-dependent pathway, while beta-arrestin-dependent pathway mediates most of the side eﬀects. Thus, it was speculated that development of biased MOR ligands for G-protein-dependent pathway may have high analgesic potency, but with fewer undesirable eﬀects. The main goal of this review is to brieﬂy describe this strategy and to discuss its advantages and limits. Received: 09 June 2021 Accepted: 27 August 2021 Published: 24 September 2021 Keywords: opioids, biased ligands, analgesia, side effects, TRV130, PZM21 MINI REVIEW MINI REVIEW published: 24 September 2021 doi: 10.3389/fpain.2021.722820 BIASED LIGANDS ARE PROMISING COMPOUNDS This is well- illustrated by the opioid crisis in US where postoperative opioid prescription is a signiﬁcant contributor to opioid epidemic, and more than 350,000 people died from opioid overdose between 1999 and 2016 (https://www.cdc.gov/drugoverdose/epidemic/ index.html). All these side eﬀects are a real impairment in the opioid use, particularly in chronic pain. Among the diﬀerent strategies to ﬁnd potent analgesics with limited side eﬀects, the one based on the particular properties of the opioid receptors and their ligands, the biased signaling, has received great attention since few years even though the concept (as known as functional selectivity) is older (10). Indeed, the notion of functional selectivity was probably ﬁrst suggested in a review on serotoninergic receptors where the ability of a single GPCR to active one or more distinct signaling cascades was described (11). And the term of biased agonist was ﬁrst introduced by Jarpe and co-workers when they observed that a peptide promoted a biased activity on chemokine receptors (12). For opioid receptors, the studies of Bohn and co-workers along with this notion opened the way to ﬁnd new opioid ligands. As rapidly mentioned earlier, following ligand binding, MOR activation could result in the activation of multiple downstream pathways through either G protein dependent processes (e.g., adenylate cyclase inhibition or regulation of ion channels) or G protein independent processes (e.g., beta-arrestin signaling). Beta- arrestins are proteins, existing in two isoforms (beta-arrestin 1 and 2), that bind the activated and phosphorylated receptor and are responsible for its desensitization and internalization (13). In beta-arrestin 2 knockout mice, Bohn and collaborators found that morphine analgesia was enhanced, with abolition of antinociceptive tolerance (14, 15) and reduction of respiratory depression and acute constipation (16). These data suggested that whereas G protein-dependent pathway mediated analgesia, beta-arrestin-dependent signaling pathway promoted side eﬀects. Using diﬀerent strategies [high-throughput screening, Thanks to the progress in GPCR crystal structure, Manglik and co-workers used the MOR crystal structure to virtually dock millions of compounds and they discovered PZM21 (27). PZM21 was found to be biased toward G protein pathway and active in both spinal and supra-spinal analgesia in rodents (27, 28). Interestingly, this compound seemed to induce less respiratory depression and constipation than morphine (27). Finally, the latest notable MOR biased compounds are a series of a piperidine core structure-containing molecules (SR series) generated from a synthesis–driven approach. BIASED LIGANDS ARE PROMISING COMPOUNDS The most notable compound, TRV-130 (Oliceridine) was discovered after a high-throughput screening by Trevena Inc. (18). It has a moderate biased toward cAMP pathway (about 3- fold) and was unable to promote MOR endocytosis according to its very weak ability to recruit beta-arrestin 2. Preclinical studies showed that TRV130 had a higher potency than morphine to induce antinociceptive responses with a reduced respiratory depression and gastrointestinal eﬀects, suggesting a better therapeutic index as compared to morphine (19). Moreover, in the tail immersion test, no tolerance was measured after a 3 days treatment (20). Clinical studies validated these results. Indeed, in a phase 2 study comparing TRV130 with morphine in patients after abdominoplasty, the biased agonist provided not only a quicker analgesia, but was also associated with fewer side eﬀects including hypoventilation, nausea, and vomiting (21). In a phase 3 study, TRV130 was shown to be eﬃcient in reducing pain intensity in two surgical procedures: bunionectomy (APOLLO-1 trial) (22) and abdominoplasty (APOLLO-2 trial) (23). Analysis of side eﬀects in the pooled cohorts of APOLLO trials showed that whereas TRV130 induced a dose dependent respiratory depression, this eﬀect is lower than the one induced by morphine (24), and that TRV130 had a higher probability of producing analgesia rather than respiratory depression (25). These analyses also demonstrated a lower risk of experiencing nausea and vomiting in patients treated with TRV130 compared to morphine-treated patients (26). At present, TRV130 is approved by the Food and Drug Administration (FDA) since august 2020 to treat moderate to severe acute pain. Finally, TRV130 turned to be a good analgesic medication but its advantages, especially regarding side eﬀects, toward classical opioids such as morphine is still matter of debate. Following a protracted exposure, but also after an acute exposure at high dose, a tolerance to opioid-induced pharmacological eﬀects appears. It noteworthy that the level of tolerance depends on the pharmacological eﬀect studied. For instance, there is evidence to suggest that the tolerance to respiratory depression is weak as compared to the antinociceptive tolerance (8) which might explained the fatal overdose that could occur in opioid users (9). Finally, a repeated use of opioids, even in the frame of a pain treatment, could lead to development of addictive behaviors. Citation: Noble F and Marie N (2021) Biased Opioid Ligands: Revolution or Evolution? Front. Pain Res. 2:722820. doi: 10.3389/fpain.2021.722820 September 2021 \| Volume 2 \| Article 722820 Frontiers in Pain Research \| www.frontiersin.org 1 Biased Opioid Ligands Noble and Marie SIDE EFFECTS OF OPIOIDS AND INTEREST IN BIASED AGONISM structure-based virtual screening, or synthesis-driven approach (17)], new biased MOR ligands were discovered. They are mostly biased toward G proteins. With great therapeutic potential, usually comes great side eﬀects. The opiates do not seem to deviate from this rule. Opioid receptors and especially MOR are highly expressed in the brain structures involved in control of breathing including the pre- Bötzinger complex or the Kolliker-Fuse neurons (4). Activation of MOR in the pre-Bötzinger complex with fentanyl promotes a respiratory depression (5) and the knockout of MOR in this complex reduced the eﬀect of morphine on breathing (6). MOR are also expressed in the enteric nervous system and their activation will cause reduction of gastrointestinal motility and thus constipation (7). Frontiers in Pain Research \| www.frontiersin.org CONCLUDING REMARKS It took more than 30 years from the ﬁrst observations of a biased signaling and the release of Oliceridine, the ﬁrst biased opioid receptor compound on the market. In the last few years, some studies have challenged this concept by suggesting that the observed bias is rather the expression of the agonist low intrinsic eﬃcacies (42) or binding kinetics (46). Therefore, the promises of having opioid ligands with virtually a lack of side eﬀects will still take time. Rather than a revolution, the biased compounds are a hopeful evolution in the pharmacological research of analgesics. The pharmacology of GPCRs is very complex, and the simplistic view of one receptor, one signaling pathway, one pharmacological response is a far too simplistic concept, which does not reﬂect the complexity of life sciences. So, should we abandon opiate ligands to treat pain? Certainly not, for now, as they remain the most eﬀective antinociceptive agents. Nevertheless, alternative research strategies should be explored further. The development of non-selective opioid ligands (agonist or mixed agonist/antagonist) could be an interesting approach. For instance, cebranopadol, a mixed MOR/DOR/KOR/NOR agonist was found to be eﬃcient in both acute and chronic pain with a delayed development of tolerance as compared to morphine (47). This compound is actually in phase 3 trials. More recently, Ding and co-workers described the AT-121, a MOR/NOR mixed agonist with analgesic eﬀects in non-human primates and a lack of the most frequent opioid-associated side eﬀects such as physical dependence, abuse potential, respiratory depression, and opioid-induced hyperalgesia (48). Allosteric ligands at the MOR could be also interesting as they could be used to enhance the eﬀects of either the exogenous orthosteric agonists when administrated together or the endogenous opioid peptides released in stress or pain condition (49). Another strategy could be the development of dual-target ligands directed to both MOR and another receptor, including for instance dopamine D3 receptor (50). Whether biased ligands, ligands with low selectivity toward diﬀerent opioid receptors, or bivalent ligands, the development of an analgesic compound free of side eﬀects and with low abuse potential seems to be a way still paved with some diﬃculties. g These data highlight the importance of the model at two level: the species and the experimental model used. Furthermore, in the same species, the choice of the strain of animals used can also have a very important impact on the results. BIASED LIGANDS ARE PROMISING COMPOUNDS Some of these compounds were found to be biased, at diﬀerent levels, toward G protein. They induced analgesia (measured in the hot plate and tail ﬂick tests), showed minor respiratory depression and are brain penetrant (29). Interestingly, SR-17018 was demonstrated to be more potent and eﬃcacious than morphine or oxycodone in a chemotherapeutic-induced neuropathy pain model and displayed a weak tolerance after a repeated administration (30). Finally, when substituted to morphine after a chronic treatment, it prevents the onset of morphine withdrawal (31). September 2021 \| Volume 2 \| Article 722820 2 Biased Opioid Ligands Noble and Marie CONCLUDING REMARKS Thus, the ﬁrst results obtained by Bohn and co-workers on beta- arrestin 2 knockout animals were obtained on a mixed genetic background (beta-arrestin 2 knockout 129/SvJ backcrossed with C57BL6). Previous studies reported that the 129/SvJ mice have a higher sensitivity to morphine-induced antinociception (36), did not develop tolerance to morphine-induced analgesia (37) and were less sensitive to respiratory depression (38). When reassessing the pharmacological responses induced by opioids in beta-arrestin 2 knockout using an homogenous C57BL6 background, Koblich and colleagues did not ﬁnd any diﬀerence in tolerance to analgesic eﬀects of few opioids including morphine (39). More recently, Kliewer and co-workers reported that the knockout of beta-arrestin 2 in C57BL6 mice did not alter the eﬀect of morphine on both respiratory depression and constipation compared to wild-type animals (40). Taken together, these data suggested that the use of 129/SvJ strain might have misled us on the role of beta-arrestin 2 in morphine eﬀects and as a consequence it questions the G protein biased agonists as valuable strategy for safer analgesic medication. More importantly, an increase number of studies challenged the existence of the biased signaling itself (41). Evaluating the bias usually consist of measuring the ability of an agonist to stimulate two signaling pathways. At end, one will compare an ampliﬁed assay of G activation vs. an unampliﬁed assay of beta- arrestin recruitment. Ampliﬁed assay are relatively insensitive BUT THEY REMAIN MOR LIGANDS (WITH THEIR SIDE EFFECTS) to agonist eﬃcacy diﬀerences. Whereas, an agonist with a low intrinsic eﬃcacy will show a maximal eﬀect in G protein assay, it will have a lower maximal eﬀect in beta-arrestin assay. Using newly unampliﬁed probes to measure signaling, several studies demonstrated that the newly biased ligands are in fact agonists with a low intrinsic eﬃcacy relative to morphine (42–44). These recent data show that while biased ligands may have beneﬁts over unbiased ligands, particularly in terms of the reduction of side eﬀects, these may not be due to the biased nature of the compounds (45). Because they bind and activate MOR, all these biased ligands could still promote main eﬀects attributed to MOR stimulation including reward system activation. Indeed, in rats, TRV130 displayed same eﬀects as morphine in intracranial self-stimulation (20). In a drug-discrimination experiment conducted in rats, two biased ligands TRV130 and SR-14968 generalized to fentanyl (32), suggesting that these ligands may produce prototypic MOR agonist abuse-related eﬀects. Using a model closer to the human behavior, intravenous self-administration procedure in rats, Austin Zamarripa and colleagues demonstrated that TRV130 was equi-potent to oxycodone (33). More recently, whereas PZM21 demonstrated a lack of eﬀect in the conditioned place preference paradigm in two independent studies in mice (27, 28), reinforcing eﬀects, here again comparable to those induced by oxycodone, were demonstrated using a self-administration procedure in monkeys (34), but not in rats (28). All these data from animal studies could suggest that the MOR biased compounds could have reinforcing eﬀects in humans. Indeed, in a clinical study assessing the analgesic eﬀects of TRV130 in healthy volunteers, Soergel and collaborators evaluated the abuse-related subjective eﬀects with a drug eﬀects questionnaire. They found that the dose of 3 mg, equianalgesic to 10 mg morphine, induced the same subjective opioid eﬀects such as “high” or “liking” (35). Frontiers in Pain Research \| www.frontiersin.org REFERENCES Mohammed W, Alhaddad H, Marie N, Tardy F, Lamballais F, Risede P, et al. Comparison of tolerance to morphine-induced respiratory and analgesic eﬀects in mice. Toxicol Lett. (2013) 217:251–9. doi: 10.1016/j.toxlet.2012.12.021 26. Beard TL, Michalsky C, Candiotti KA, Rider P, Wase L, Habib AS, et al. Oliceridine is associated with reduced risk of vomiting and need for rescue antiemetics compared to morphine: exploratory analysis from two phase 3 randomized placebo and active controlled trials. Pain Ther. (2021) 10:401– 13. doi: 10.1007/s40122-020-00216-x 9. White JM, Irvine RJ. 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Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform. Biochem Pharmacol. (2020) 177:113910. doi: 10.1016/j.bcp.2020.113910 Copyright © 2021 Noble and Marie. 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. j 45. Azevedo Neto J, Costanzini A, De Giorgio R, Lambert DG, Ruzza C, Calò G. Biased versus partial agonism in the search for safer opioid analgesics. Molecules. (2020) 25:3870. doi: 10.3390/molecules25173870 46. 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https://openalex.org/W2080199104	https://escholarship.org/content/qt9226w0d7/qt9226w0d7.pdf?t=pm5brh	English	null	Pulsed photothermal profiling of hypervascular lesions: some recent advances	Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE	2,000	cc-by	7,502	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 Pulsed photothermal profiling of hypervascular lesions: some recent advances Permalink https://escholarship.org/uc/item/9226w0d7 Authors Majaron, Boris Verkruysse, Wim Tanenbaum, BS et al. Publication Date 2000-05-17 DOI 10.1117/12.386236 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 Pulsed photothermal profiling of hypervascular lesions: some recent advances Permalink https://escholarship.org/uc/item/9226w0d7 Authors Majaron, Boris Verkruysse, Wim Tanenbaum, BS et al. Publication Date 2000-05-17 DOI 10.1117/12.386236 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 Pulsed photothermal profiling of hypervascular lesions: some recent advances Permalink https://escholarship.org/uc/item/9226w0d7 Authors Majaron, Boris Verkruysse, Wim Tanenbaum, BS et al. Publication Date 2000-05-17 DOI 10.1117/12.386236 Copyright Information This work is made available under the terms of a Creative Commons Attribution availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed ABSTRACT Pulsed photothermal radiometry (PPTR) can be used for non-invasive depth profiling of port wine stain (PWS) birthmarks, aimed towards optimizing laser therapy on an individual patient basis. Reconstruction of laser-induced temperature profile from the experimentally obtained radiometric signal involves the skin absorption coefficient in the infrared detection band. In the commonly used 3—5 tm detection band (InSb), the absorption coefficient varies by two orders of magnitude, while assumed to be constant in the reconstruction algorithms used thus far. We discuss the problem of choosing the effective absorption coefficient value to be used under such conditions. Next, we show how to account explicitly for the strong spectral variation of the infrared absorption coefficient in the image reconstruction algorithm. Performance of such improved algorithm is compared to that of the unaugmented version in a numerical simulation of photothermal profiling. Finally, we analyze implementation of a bandpass filter which limits the detection band to 4.5—5 im. This reduces the absorption coefficient variation to a level that permits the use of unaugmented algorithm. An experimental test of the latter approach for in vivo characterization of the depth of PWS lesion and epidermal thickness will be presented, including a novel technique that uses two laser excitation wavelengths in order to separate the epidermal and vascular components of the radiometric signal. Keywords: Pulsed photothermal radiometry (PPTR), heat diffusion, imaging, image reconstruction, infrared absorption, port wine stain. Powered by the California Digital Library University of California eScholarship.org In Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems X, R. Rox Anderson, et al., Editors, Proceedings of SPIE Vol. 3907 (2000) • 1605-7422/00/$1 5.00 Coespondence: 1002 Health Sciences Rd. East, Irvine; e-mail: majaron@bli.uci.edu, boris.majaron@ijs.si; fax: + 1-949-824-6969 Pulsed photothermal profiling of hypervascular lesions: Some recent advances Boris Majaron a,b Wim Verkruysse", B. Samuel Tanenbaumc, Thomas E. Milner' 1. INTRODUCTION Note that in lesions with PWS in close proximity to the epidermal-dermal junction, epidermal heating due to broad melanin absorption may prevent determination of PWS depth, owing to limited spatial resolution of PPTR.6'9 In order to overcome this problem, we use an approximation technique, which utilizes two laser excitation wavelengths to separate the epidermal and vascular components of the radiometric signal.1° In this way, the epidermal thickness and PWS depth can be assessed with adequate accuracy and reliability. Alternatively, a bandpass filter can be implemented, which narrows the JR detection band to a level that permits the use of the unaugmented algorithm. An experimental test of the latte:r approach using a 4.5—5 tm acquisition band for in vivo characterization of PWS will be presented. Note that in lesions with PWS in close proximity to the epidermal-dermal junction, epidermal heating due to broad melanin absorption may prevent determination of PWS depth, owing to limited spatial resolution of PPTR.6'9 In order to overcome this problem, we use an approximation technique, which utilizes two laser excitation wavelengths to separate the epidermal and vascular components of the radiometric signal.1° In this way, the epidermal thickness and PWS depth can be assessed with adequate accuracy and reliability. 1. INTRODUCTION 1000 100 10 0.1 I 2 3 4 5 6 7 Wavelength (tim) 8 Figure 1 : Infrared absorption coefficient of water, displaying a variation of two orders of magnitude within the detection band of the lnSb detector (? = 3—5 im).8 Alternatively, a bandpass filter can be implemented, which narrows the JR detection band to a level that permits the use of the unaugmented algorithm. An experimental test of the latte:r approach using a 4.5—5 tm acquisition band for in vivo characterization of PWS will be presented. Note that in lesions with PWS in close proximity to the epidermal-dermal junction, epidermal heating due to broad melanin absorption may prevent determination of PWS depth, owing to limited spatial resolution of PPTR.6'9 In order to overcome this problem, we use an approximation technique, which utilizes two laser excitation wavelengths to separate the epidermal and vascular components of the radiometric signal.1° In this way, the epidermal thickness and PWS depth can be assessed with adequate accuracy and reliability. 1000 100 10 0.1 I 2 3 4 5 6 7 Wavelength (tim) 8 Figure 1 : Infrared absorption coefficient of water, displaying a variation of two orders of magnitude within the detection band of the lnSb detector (? = 3—5 im).8 1000 100 10 0.1 I 2 3 4 5 6 7 Wavelength (tim) 8 Figure 1 : Infrared absorption coefficient of water, displaying a variation of two orders of magnitude within the detection band of the lnSb detector (? = 3—5 im).8 Figure 1 : Infrared absorption coefficient of water, displaying a variation of two orders of magnitude within the detection band of the lnSb detector (? = 3—5 im).8 Figure 1 : Infrared absorption coefficient of water, displaying a variation of two orders of magnitude within the detection band of the lnSb detector (? = 3—5 im).8 Alternatively, a bandpass filter can be implemented, which narrows the JR detection band to a level that permits the use of the unaugmented algorithm. An experimental test of the latte:r approach using a 4.5—5 tm acquisition band for in vivo characterization of PWS will be presented. 1. INTRODUCTION Port-wine stain birthmarks (PWS) are hypervascular lesions in human skin, which consist of an excess of ectatic blood vessels. These are usually fully contained within the most superficial millimeter of the skin. The exact depth varies from patient to patient, but on average, the highest fractional blood content is found 200—400 tm below the epidermal-dermal un' PWS are currently treated by selective photocoagulation ofthe ectatic vasculature using a pulsed green (KTP/YAG, 532 nm) or yellow/orange laser (flashlamp-pumped dye, 577nm, 585-600 nm). In order to optimize therapy on an individual patient basis, determination ofboth PWS depth and epidermal thickness is required, especially when cryogen spray cooling is applied.2'3'4 Pulsed photothennal radiometry (PPTR), which is based on time-resolved acquisition of infrared (IR) radiant emission following pulsed laser exposure, was recently introduced for assessment of laser-induced temperature profiles in PWS.5'6 The ability ofPPTR to determine the depth of sub-surface chromophores has been demonstrated by profiling layered tissue phantoms, and by comparison with histological assessment ofPWS depth.7 Reconstruction of laser-induced temperature profiles from experimental radiometric signals involves the value of the tissue absorption coefficient at the infrared detection wavelength. While assumed to be constant in the reconstruction algorithms developed thus far, the absorption coefficient of water, which is the primary absorber in the commonly used 3—5.tm detection *Coespondence: 1002 Health Sciences Rd. East, Irvine; e-mail: majaron@bli.uci.edu, boris.majaron@ijs.si; fax: + 1-949-824-6969 114 band (InSb), varies by two orders of magnitude (Fig. 1).8 We discuss below, how to obtain the effective value of the JR absorption coefficient under such conditions. Next, we show how to account explicitly for such strong spectral variation of the absorption coefficient in the image reconstruction algorithm. Performance of such an improved algorithm is compared to that ofthe unaugmented version in a numerical simulation ofphotothermal profiling of PWS. band (InSb), varies by two orders of magnitude (Fig. 1).8 We discuss below, how to obtain the effective value of the JR absorption coefficient under such conditions. Next, we show how to account explicitly for such strong spectral variation of the absorption coefficient in the image reconstruction algorithm. Performance of such an improved algorithm is compared to that ofthe unaugmented version in a numerical simulation ofphotothermal profiling of PWS. 2.1. Theory The basic theory of PPTR profiling will be reviewed first.5'6 The temperature field evolution following a pulsed laser exposure of skin can be treated by one-dimensional heat-diffusion equation (using thermal diffusivity value D = 1.l• 10 7 m2/s). Such an approach is reasonable, since the laser irradiated spot is typically much larger (>5mm) than the dermal depths considered in this study (<1 mm). Using the Green's function approach, the time evolution of temperature increase AT(z,t) can be calculated from the initial laser-induced temperature profile AT(z ',O) as: T(z,t) JAT(z',O) KT(z',z,t) dz', (1) where KT(z',z,t) represents the thermal point spread function of the problem. where KT(z',z,t) represents the thermal point spread function of the problem. where KT(z',z,t) represents the thermal point spread function of the problem Note that, owing to the finite penetration depth of the emitted infrared radiation, JR detectors do not measure the surface temperature. The radiometric signal obtained from a non-uniformly heated body is in general a sum of contributions from different depths z (measured from the sample surface inward): 115 S(t) = C IRJB(T(Zt)) e_1RZ dz . (2) Here, constant C accounts for skin emissivity, geometry and transmission of the collection optics, sensitivity of the detector, and similar factors, .tIR is the tissue absorption coefficient at the detection wavelength, and B(T) is given by Planck's law of radiation. For relatively small temperature increases encountered in our experiments, Planck's law can be linearized around the initial skin temperature I'0, resulting in S(t) = CB(T0) + C Bx(To) lRJAT(Zt) e_1RZ dz . (3) S(t) = CB(T0) + C Bx(To) lRJAT(Zt) e_1RZ dz (3) For the discussed case of pulsed laser heating, the dynamic component of the radiometric signal AS(t) can now be written by inserting the temperature increase z\T(z,t) (1) into (4), and reordering: S(t) = C Bx(To) IRfAT(Z'O) :LKT(ZZt) e1RZ dz dz' . (4) (4) After the point spread function of the problem K1(z',z,t) is established, integration over z can be performed, and it becomes clear that the PPTR signal (minus the background) AS(t) is linear in the initial temperature profile AT(z ',O): AS(t) = JK(z',t) AT(z' ,O) dz' . AS(t) = JK(z',t) AT(z' ,O) dz' . (5) (5) Defmition of the kernel function K(z',t) is evident from (4). 2.1. Theory In the examples below, we use a mixed (Robin) boundary condition to represent convective and radiative heat transfer at the skin surface (reduced heat transfer coefficient h = 0.02 m'). The kernel function then has a form:6 K(z' ,t) = ! c Bx (T0 ) IR e 4Th {erfcx(u ) + erfcx(u ) — 2h erfcx(u1 )}} , (6) 2 (hta) where erfcx(u) exp(u2)(l - erf(u)), u IR[i z'/(2,.j5) , and u1 h /i5 z'I(2,jñ). (6) S = KT; K K(z'1,tj). 2.2. Experimental setup and methods A PWS lesion on a volunteer patient is irradiated with a 1.5 ms long pulse from a flashlamp-pumped dye laser (ScleroPLUS, Candela, Wayland, MA), which allows a choice of four wavelengths (585 to 600 nm in increments of 5 nm). Energy density at the center of a 6 mm diameter laser spot is 5—6 J/cm2. The transient increase in infrared radiant emission from the central 1 .9x 1 .9 rmn2 area is recorded with an JR camera sensitive to a wavelength band of 3—5 tm (Gallileo, Raytheon, Dallas, TX), at a rate of 500 frames per second. PPTR signals AS(t) are obtained by calibrating the response of the camera's 64x64 array detector elements with a computer-controlled blackbody (BB7O1, Omega Engineering, Stamford, CT), averaging over the JR camera array, and subtracting the radiant emission level prior to the pulsed laser exposure (background). Reconstruction of the initial temperature profile T(z ',O) from the experimental PPTR signals AS(t) is a severely ill-posed inverse problem. We solve it using a dedicated iterative algorithm based on a nonnegative-constrained conjugate-gradient method.6 Since the PPTR signals, as well as the numerical solution in terms of the estimated initial temperature profile, are inevitably discretized, the linear mapping (5) of the initial temperature profile vector T (T =iT(z,O)) onto the signal vector S (S, = AS(t)) can be represented by a kernel matrix j: (7) S = KT; K K(z'1,tj). 116 The computational load is somewhat reduced by the fact that the kernel matrix elements K have to be calculated only once for each image reconstruction example. Nevertheless, a characteristic inversion run involving a 400-element signal vector and a 64-element temperature profile vector requires a few minutes CPU time on a Sun Sparc II workstation. The computation is regularized by early termination.6 Rather than dwelling on the issue of determining the optimal number of iteration steps, we show in the discussed example several iterative solutions, which cover the whole range from smooth, blurred temperature profiles in under-iterated solutions, near optimal and often also over-iterated solutions. As discussed previously by Mimer et al.,6 the latter display characteristic oscillations, as the algorithm attempts to fit experimental noise and other mismatches between the experimental and theoretically predicted signals. 3.1. Response function R(z) In order to analyze the influence of the strong spectral variation of the JR absorption coefficient IR(X), radiometric signals must be calculated by adding radiative contributions (2) within the detection band (?i to ?.h): Xh z= AS(t) = C' J D(?) J Bx(To) IRO) zT(z,t) e1R( dz d? , (8) xl z=O Xh z= AS(t) = C' J D(?) J Bx(To) IRO) zT(z,t) e1R( dz d? , xl z=O (8) where we have also taken into account the spectral response ofthe detector D(?.). Equation (8) can be rewritten as: where we have also taken into account the spectral response ofthe detector D(?.). Equation (8) can be rewritten as: Z=3 Xh z=c13 AS(t) = C $ z\T(z,t) JD(X)B(To) IRO) erR( dz = $iT(Z,t) R(z) dz . (9) z=O ?L/ z=O (9) The response function R(z), which is obtained by integrating over the detection wavelengths, represents the radiometric signal resulting from a unity temperature increase in a delta-layer at a depth z. In general, its functional dependence deviates from the previously assumed exponential decrease with depth (3). Figure 2 presents an example of such a response function 30 , b 20 10 . C.) c '—I c 110 1 o.ooo 0.050 OAOO 0.150 z (mm) z (mm) Figure 2: (a) Response function R(z) calculated by taking into account the spectral variation of the IR absorption coefficient of skin (70% water content) and spectral response of the InSb detector (. = 3—5 tm; solid curve). Response functions for various values of the spectrally constant absorption coefficient are presented for comparison (dashed and dotted curves; see the legend). (b) Same four response functions presented on a logarithmic scale, demonstrating straight lines for examples with constant lIR LIR =IR t,=25mm' tt=22mm' = 19mm' 1tIR •.••• t25mm1 p=22mm' l9mm' 30 20 . C.)c 110 z (mm) LIR =IR t,=25mm' tt=22mm' = 19mm' , b 10 c '—I 1 o.ooo 0.050 OAOO 0.150 z (mm) 1tIR •.••• t25mm1 p=22mm' l9mm' Figure 2: (a) Response function R(z) calculated by taking into account the spectral variation of the IR absorption coefficient of skin (70% water content) and spectral response of the InSb detector (. = 3—5 tm; solid curve). Response functions for various values of the spectrally constant absorption coefficient are presented for comparison (dashed and dotted curves; see the legend). 3.1. Response function R(z) (b) Same four response functions presented on a logarithmic scale, demonstrating straight lines for examples with constant lIR 117 R(z),calculated for tu9) variation of skin(70%water content), detector (InSb) spectral response proportional to wavelength, 3—5 tm detection band, and initial temperature of T0 =36 °C (solid line). Three response functions obtained with various constant absorption coefficient values are also presented for comparison, displaying the exponential decay with depth (dashed and dotted lines; see the legend for values). 3.2. Effective absorption coefficient In the next step, we attempt to determine which effective value of the JR absorption coefficient in should be used in PPTR image reconstruction algorithms that ignore its spectral dependence within the detection band. By introducing a spectrally constant effective value integration over 2. in (8) yields a constant, which can be taken out ofthe integral over depth z: Xh z=cc S(t) = C' teff JD(?) Bx(To) d . JLT(z,t) dz . (10) x1 z=O (10) By equating the response functions R(z) in (9) and (10), the effective absorption coefficient value tfl is given by the implicit equation: SD()Bx(To) IRO) eM1R( effe'° Xh . (11) SD(2) Bx(To) Figure 3: The effective absorption coefficient value l1eff(Z), as calculated from (13) for the detection band of 3—5 tm (solid line). The solution varies strongly with depth z and is double-valued for all depths. The dashed line presents the same function calculated for the detection band reduced to 4.5—5.0 tm. 0.10 The numerical solution of (1 1) for the 3—5 .tm detection band and spectral response of the InSb detector is presented in Figure 3 (solid curves). Note that the effective absorption coefficient value varies strongly with depth (z), and is double- valued. While the latter effect is intuitively quite surprising, it does not present a computational problem, as any of the two branches can evidently be used to calculate the signal from (10) (or (4)). The strong depth dependence, however, indicates that there is no constant value of that would yield a correct time dependence of the PPTR signal under the discussed experimental conditions. Consequently, the reconstruction algorithm has to be augmented by taking into account the spectral dependence according to (9) or, alternatively, the detection bandwidth should be decreased to a level that permits the use of the unaugmented algorithm. Both approaches are discussed in sections 4 and 5, respectively. (mm) SD()Bx(To) IRO) eM1R( effe'° Xh . SD(2) Bx(To) 0.10 (mm) (11) Figure 3: The effective absorption coefficient value l1eff(Z), as calculated from (13) for the detection band of 3—5 tm (solid line). The solution varies strongly with depth z and is double-valued for all depths. The dashed line presents the same function calculated for the detection band reduced to 4.5—5.0 tm. Figure 3: The effective absorption coefficient value l1eff(Z), as calculated from (13) for the detection band of 3—5 tm (solid line). 4. IMPROVED KERNEL MATRIX In the following, we compare the performance of an image reconstruction algorithm that explicitly accounts for the spectral variation ofthe JR absorption coefficient jt1R(A) with the unaugmentedversion (using a constant value J-teff). This comparison is performed in a numerical simulation of photothermal profiling. First, an initial temperature distribution AT(z ',O) ("object") is chosen, and the corresponding PPTR signal AS(t) calculated using the kernel function K(z',t), which involves the "correct" response function R(z) (9). Then, this signal, with some noise superimposed onto it, is used as an input to the iterative image reconstruction algorithm. The reconstruction is performed first with the kernel matrix elements K (7) calculated using the simplified response function (10) with teff 22 1111Ti, Ifld then using the correct response function R(z) (9) (see Fig. 2). Results from one such comparison are presented in Fig. 4. As the "object", we use a hyperGaussian temperature profile AT(z ',O) = M'0 exp(—2(x---xo)4/w4) with AT0 = 1 °C, xo 300 pm, w = 100 tm (dashed curves in Fig. 4), which vaguely resembles a laser-heated PWS layer. The corresponding signal AS(t), which includes also a small amount of normal noise (SNR = 1000) is calculated at 250 equidistant time points (At =2 ms), and the solution (depth) space which represents the most superficial millimeter of the skin is divided into 64 intervals. As a matter of convenience, the simulation was performed on a PC (Pentium 11-350), using a slightly adapted reconstruction method (details to be presented elsewhere). 1.5 1.0 0.5 II 'n=;O 1.0 ,,1'T\ n100 :: , \. I I 1.0 i\ n200 :; 0.6 • Depth (mm) Figure 4: Convergence of the calculated temperature profile AT(z ',O), reconstructed using the algorithm based on a constant absorption coefficient kR 22 mm1 (a), and the correct spectral dependence pIR(X) (b). Solid curves represent the solution approximations obtained after 20, 50, 1 00 and 200 iteration steps of the image reconstruction algorithm. The hyperGaussian initial temperature profile used to calculate the input radiometric signal is plotted for comparison (dashed curves). a n =20 U I,. I 4 j— I- U 0.8 1.0 0.4 0.6 Depth (mm) 1.5 1.0 0.5 II 'n=;O 1.0 ,,1'T\ n100 :: , \. I I 1.0 i\ n200 :; 0.6 • Depth (mm) a n =20 U I,. 3.2. Effective absorption coefficient The solution varies strongly with depth z and is double-valued for all depths. The dashed line presents the same function calculated for the detection band reduced to 4.5—5.0 tm. Figure 3: The effective absorption coefficient value l1eff(Z), as calculated from (13) for the detection band of 3—5 tm (solid line). The solution varies strongly with depth z and is double-valued for all depths. The dashed line presents the same function calculated for the detection band reduced to 4.5—5.0 tm. The numerical solution of (1 1) for the 3—5 .tm detection band and spectral response of the InSb detector is presented in Figure 3 (solid curves). Note that the effective absorption coefficient value varies strongly with depth (z), and is double- valued. While the latter effect is intuitively quite surprising, it does not present a computational problem, as any of the two branches can evidently be used to calculate the signal from (10) (or (4)). The strong depth dependence, however, indicates that there is no constant value of that would yield a correct time dependence of the PPTR signal under the discussed experimental conditions. Consequently, the reconstruction algorithm has to be augmented by taking into account the spectral dependence according to (9) or, alternatively, the detection bandwidth should be decreased to a level that permits the use of the unaugmented algorithm. Both approaches are discussed in sections 4 and 5, respectively. 118 4. IMPROVED KERNEL MATRIX I 4 j— I- 0.8 1.0 U 0.4 0.6 Depth (mm) Figure 4: Convergence of the calculated temperature profile AT(z ',O), reconstructed using the algorithm based on a constant absorption coefficient kR 22 mm1 (a), and the correct spectral dependence pIR(X) (b). Solid curves represent the solution approximations obtained after 20, 50, 1 00 and 200 iteration steps of the image reconstruction algorithm. The hyperGaussian initial temperature profile used to calculate the input radiometric signal is plotted for comparison (dashed curves). The under-iterated solution obtained after 20 iteration steps (n = 20) is a blurred and deformed image of the actual temperature profile, and is very similar for both approaches. When using the simplified kernel function (Fig. 4a), increasing the number of iteration steps (n) seems to temporarily improve the result, but then very rapidly produces an oscillatory The under-iterated solution obtained after 20 iteration steps (n = 20) is a blurred and deformed image of the actual temperature profile, and is very similar for both approaches. When using the simplified kernel function (Fig. 4a), increasing the number of iteration steps (n) seems to temporarily improve the result, but then very rapidly produces an oscillatory The under-iterated solution obtained after 20 iteration steps (n = 20) is a blurred and deformed image of the actual temperature profile, and is very similar for both approaches. When using the simplified kernel function (Fig. 4a), increasing the number of iteration steps (n) seems to temporarily improve the result, but then very rapidly produces an oscillatory 119 solution, which indicates the presence of an additional PWS layer starting at 120 im — clearly a computational artifact. In contrast, the solution obtained using the corrected kernel matrix converges rapidly toward a significantly more representative image of the actual temperature profile (Fig. 4b). The position of the shallower edge of the profile, which is the most important parameter in the targeted application, is reproduced with remarkable accuracy. The peak temperature value is overestimated by only 8 %, compared to 43 % when using the simplified kernel function. The convergence of the reconstruction is also improved remarkably; when using the corrected matrix, the residual norm (which is minimized in the iteration) reaches a value of 9.6 i09 after 50 iteration steps, and changes only minimally (< 1%) by iteration step 200. 4. IMPROVED KERNEL MATRIX 4a) well above the noise level. 4. IMPROVED KERNEL MATRIX In contrast, with the simplified kernel function, the residue norm after 200 iteration steps is still 2.7 106 (!) a 3 % decrease fromstep 100. The difference between these two results is illustrated further in Fig. 5, where the radiometric signal calculated by each method from its last solution (n =200) is compared to the signal used as the input to the reconstruction algorithm. Using the simplified kernel function (Fig. 5a), the mismatch between the input ("object") and predicted signal ("image") is particularly large in the first 40 ms, where the error exceeds 30 % of the signal value at that point, and 3 % of the maximalsignal value overall. In contrast, with the corrected kernel function, the two signals are undistinguishable (Fig. 5b), and even the 100-times expanded residue can be hardly observed (see the inset). 0.0 0.10.2 0.3 0.4 0.5 0.3 0.4 0.5 Figure 5: Radiometric signal S(t), calculated from the image reconstruction solutions iT(z ',O) obtained using IR 22 mm' (a), and the correct spectral dependence tIRQ) (b). Dashed curves represent the signal calculated from the actual initial temperature profile ("object"), which was used as the input to the reconstruction algorithm for both examples. The insets shows the residue (expanded 100 x). Residue ( x 100) 0.0 0.1 0.2 0.3 0.4 Time (s) Time (s) 0.0 0.10.2 0.3 0.4 0.5 0.3 0.4 0.5 Residue ( x 100) Time (s) 0.0 0.1 0.2 0.3 0.4 Time (s) Figure 5: Radiometric signal S(t), calculated from the image reconstruction solutions iT(z ',O) obtained using IR 22 mm' (a), and the correct spectral dependence tIRQ) (b). Dashed curves represent the signal calculated from the actual initial temperature profile ("object"), which was used as the input to the reconstruction algorithm for both examples. The insets shows the residue (expanded 100 x). Our general experience from other similar simulations is that higher levels of noise in the PPTR signal prevent successfu convergence ofboth methods. The benefit ofthe corrected kernel function may thus not show up as clearly as in the presented example. This is particularly true for more complex initial temperature profiles. Nevertheless, we are confident that the performance of PPTR profiling should benefit from the suggested correction of the kernel matrix in many realistic experimental situations. In our PPTR profiling experiments, we have regularly observed a structure of the residue (similar to the one presented in the inset ofFig. 5. BANDPASS FILTERING When using the reduced detection band, the camera integration time was increased from 0.5to 0.7 s, in order to compensate for the anticipated reduction of the radiometric signal. These two signals were used as input to the image reconstruction algorithm based on the simplified approach with a constant jtIR (4). In accordance with results presented in Figs. 2a, 3, the value of 1eff 22 mm was used to analyze the signal "a", and = 26 mm' for signal "b". 8 6 4 2 0 Time (s) 8 6 4 2 0 Time (s) Figure 6: Comparison of PPTR signals obtained from a pws lesion in vivo using: a — full detection bandwidth of the detector, and b —a detection bandwidth reduced to 4.5 — 5.0 pm. (Excitation wavelength 585 nm) Figure 6 presents two PPTR signals obtained from the same PWS lesion on the forearm of a volunteer patient using the excitation wavelength of 585 nm, and a detection bandwidth of 3.0 —5.0 im (curve a), and 4.5 — 5.0 im (b). When using the reduced detection band, the camera integration time was increased from 0.5to 0.7 s, in order to compensate for the anticipated reduction of the radiometric signal. These two signals were used as input to the image reconstruction algorithm based on the simplified approach with a constant jtIR (4). In accordance with results presented in Figs. 2a, 3, the value of 1eff 22 mm was used to analyze the signal "a", and = 26 mm' for signal "b". Figure 6 presents two PPTR signals obtained from the same PWS lesion on the forearm of a volunteer patient using the excitation wavelength of 585 nm, and a detection bandwidth of 3.0 —5.0 im (curve a), and 4.5 — 5.0 im (b). When using the reduced detection band, the camera integration time was increased from 0.5to 0.7 s, in order to compensate for the anticipated reduction of the radiometric signal. These two signals were used as input to the image reconstruction algorithm based on the simplified approach with a constant jtIR (4). In accordance with results presented in Figs. 2a, 3, the value of 1eff 22 mm was used to analyze the signal "a", and = 26 mm' for signal "b". Figure 7: Longitudinal temperature profiles immediately after pulsed laser exposure of a PWS in vivo, as calculated iteratively from PPTR signals in Fig. 6. 5. BANDPASS FILTERING As discussed above, an alternative approach to using the more accurate (and complex) formula in calculation of the kernel matrix elements could be a reduction of the detection bandwidth to a level that permits the use of the unaugmented algorithm. Using the equation (1 1), we have calculated t(z) for watery tissue for several detection bands. Based on the results of such an analysis, and wavelength dependence of Planck's factor B(T), we decided to reduce the detection bandwidth of our system to 4.5 — 5.0 pin. A custom long-pass IR filter (cut-on at 4.50 jim, Barr Associates, Westford MA) was fitted to the collection 120 optics of the InSb-array camera. As indicated by Fig. 3 (dashed curve), radiometric signals obtained with such a modified detector can be accurately described by the simplified formula (10) and effective absorption coefficient equal to teff 26.5 mm' . We have estimated that by using the additional filter, the amplitudes of the acquired signals AS(t) would decrease roughly by a factor of two. Provided that performance of the image reconstruction was limited primarily by error due to spectral variation of t(A), rather than the level of experimental noise, such modification should improve the overall performance ofPPTR profiling. optics of the InSb-array camera. As indicated by Fig. 3 (dashed curve), radiometric signals obtained with such a modified detector can be accurately described by the simplified formula (10) and effective absorption coefficient equal to teff 26.5 mm' . We have estimated that by using the additional filter, the amplitudes of the acquired signals AS(t) would decrease roughly by a factor of two. Provided that performance of the image reconstruction was limited primarily by error due to spectral variation of t(A), rather than the level of experimental noise, such modification should improve the overall performance ofPPTR profiling. Figure 6: Comparison of PPTR signals obtained from a pws lesion in vivo using: a — full detection bandwidth of the detector, and b —a detection bandwidth reduced to 4.5 — 5.0 pm. (Excitation wavelength 585 nm) Figure 6 presents two PPTR signals obtained from the same PWS lesion on the forearm of a volunteer patient using the excitation wavelength of 585 nm, and a detection bandwidth of 3.0 —5.0 im (curve a), and 4.5 — 5.0 im (b). 5. BANDPASS FILTERING The excitation wavelength was 585 nm, and the detection bandwidths were 3.0—5.0 .tm(a), and 4.5—5.0 tm (b). 25 a 25 c-) < 10 Iteration steps: n=10 n=20 00.0 0.2 0.4 0.6 0.8 1.0 Depth (mm) Depth (mm) 25 a c-) < 10 Iteration steps: n=10 n=20 00.0 0.2 0.4 0.6 0.8 1.0 Depth (mm) 25 Depth (mm) Figure 7: Longitudinal temperature profiles immediately after pulsed laser exposure of a PWS in vivo, as calculated iteratively from PPTR signals in Fig. 6. The excitation wavelength was 585 nm, and the detection bandwidths were 3.0—5.0 .tm(a), and 4.5—5.0 tm (b). 121 The two families of iterative solutions (Figs. 7a, b) run from smooth, blurred temperature profiles in under-iterated solutions (see n = 2 in Fig. 7b), to over-iterated solutions. The latter display characteristic oscillatory artifacts, resulting from the ill- posedness of the inverse problem, combined with the experimental noise and other mismatches between the experimental and theoretically predicted signals. This is particularly pronounced in example "b", which corresponds well to the approximately two times lower SNR in the PPTR signal (Fig. 6). In both examples, the most reliable solution seems to be found after approximately five iteration steps (solid curves). It is evident, however, that with either approach, the temperature profile in this specific PWS lesion cannot be resolved from the epidermal temperature rise, resulting from melanin absorption. This may happen when the two layers lie in close proximity to each other, and determination of PWS depth and epidermal thickness, which are required to optimize laser treatment, is thus prevented due to the limited spatial resolution of PPTR profiling. To overcome this problem, we have developed an approximation technique, which achieves improved selectivity by utilizing two excitation 5.1. Two-wavelength excitation technique This approximation technique allows us to differentiate between the PWS and epidermal contribution to the PPTR signal by exploring different spectral properties of the two chromophores (hemoglobin, melanin) at two excitation wavelengths. This enables detennination of PWS depth and epidermal thickness even when the two layers are in close proximity to each other. Moreover, it breaks down the relatively complex initial temperature profile into two simpler "objects", which are easier to reconstruct. The method is based on the linearity between the PPTR signal AS(t) and the initial temperature distribution AT(z,O) (5). An experimental signal obtained, for example, following pulsed irradiation at 585 nm, can thus be expressed as a sum of two components, originating from the heated PWS layer [x(t)], and epidermis [v(t)]: AS585 (t) = x(t) + y(t) . (12) (12) AS585 (t) = x(t) + y(t) . At 600 nm, the absorption and scattering properties of the epidermis and dermis are essentially equivalent to those at 585 nm' As a result, the temperature profile in the epidermis and, consequently, the corresponding signal component, is practically the same with both excitation wavelengths. In contrast, light absorption by blood at 600 nm is significantly weaker than at 585 12 This results in a lower PWS contribution to the PPTR signal obtained after 600 nm irradiation, which can therefore be approximated to the first order by AS600(t) =ax(t) +3y(t) . (13) (13) AS600(t) =ax(t) +3y(t) . AS600(t) =ax(t) +3y(t) . Figure 8: Non-normalized temperature profile in the PWS, as reconstructed from signals f3 AS585(t) -iS600(t) obtained using both experimental approaches (see Fig. 7 for details). Depth (mm) Depth (mm) Depth (mm) Depth (mm) Figure 8: Non-normalized temperature profile in the PWS, as reconstructed from signals f3 AS585(t) -iS600(t) obtained using both experimental approaches (see Fig. 7 for details). 122 Based on the above discussed spectral properties, we can expect a < 1 and 3 1. Based on the above discussed spectral properties, we can expect a < 1 and 3 1. From (12) and (13), the PWS component ofASs8s(t) can be derived as x(t) = J3 AS585 (t)— AS600(t) • (3—a) (14) By using x(t) as input to the image reconstruction algorithm, the temperature profile resulting exclusively from blood absorption could be obtained, but values of a and f3 need to be determined first. We rely on the fact that the radiometnc signal immediately following pulsed irradiation results predominantly from temperature increase in the superficial layer of tissue -a few penetration depths of the detected JR radiation (40-50 trn). Since the blood vessels are located deeper in the skin, we can estimate f3 by requiring that the PWS signal component starts from x(O) =0. A signal 3 s585(t) -s600(t), proportional to x(t) (14), can then be calculated. The initial temperature profile calculated from such signal is proportional to the temperature profile in PWS, and shows its top boundary very clearly (Fig. 8). Comparison of the two examples in Fig. 8 shows that with the reduced detection band (and thus more constant the iteration can be run longer before the non-physical oscillations appear in the solution (n = 20 in Fig. 8b vs. n = 10 in Fig. 8a). Furthermore, the residual norm reaches a lower value (12.3 at steps 10 and 20, vs. 13 .8 in example "a"), despite the significantly higher noise level and absolute signal level in the corresponding input PPTR signal ("b" in Fig. 6). This indicates a closer match between the theoretically predicted signal shapes and the experimentally obtained PPTR signals when using the appropriately reduced detection band. Note that, whereas the PWS depth (most superficial boundary) is the same in both examples (within the estimated accuracy of the solution), the depths of the temperature peak are significantly different (220 im in Fig. 8b vs. 190 tm in Fig. 8a). Similarly as above, the epidermal contribution to 1S585(t) can he derived as Similarly as above, the epidermal contribution to 1S585(t) can he derived as y(t) = AS600(t) — aAS585 (t) (15) (f3 —a) t) — aAS585 (t) (15) (f3 —a) y(t) = AS600(t) — aAS585 (t) (f3 —a) (15) To determine the value of a, we use the knowledge that the pure epidermal (melanin) temperature profile must be zero at depths deeper than 150-200 tm into the skin. Based on the above discussed spectral properties, we can expect a < 1 and 3 1. We therefore calculate signals y(t) (15) with increasing values of a, starting To determine the value of a, we use the knowledge that the pure epidermal (melanin) temperature profile must be zero at depths deeper than 150-200 tm into the skin. We therefore calculate signals y(t) (15) with increasing values of a, starting Figure 9: Initial temperature profiles in the PWS (solid line), and in the epidermis (dashed), as determined using the two- wavelength excitation technique in vivo and detection bandwidths of 3.0—5.0 tm (a), and 4.5—5.0 im (b). All solutions are representative only at depths up to -M.25 mm. 20 15 10 5 C) H—Pws - - - - epidermis 0.4 0.6 C-) Depth (mm) I .0 Depth (mm) 1.0 20 15 10 5 C) H—Pws - - - - epidermis 0.4 0.6 Depth (mm) I .0 C-) Depth (mm) 1.0 Figure 9: Initial temperature profiles in the PWS (solid line), and in the epidermis (dashed), as determined using the two- wavelength excitation technique in vivo and detection bandwidths of 3.0—5.0 tm (a), and 4.5—5.0 im (b). All solutions are representative only at depths up to -M.25 mm. 123 from 0, and observe the resulting temperature profiles. These usually differ little in the superficial layer, while temperatures deeper in the skin decrease monotonically with increasing a, and fmally disappear. At this value of a, the solution in general oscillates less, and the residue norm reaches a lower value, than at lower (and sometimes also higher) values. Thus obtained estimate of a can then be used to renormalize the previously calculated PWS profiles. from 0, and observe the resulting temperature profiles. These usually differ little in the superficial layer, while temperatures deeper in the skin decrease monotonically with increasing a, and fmally disappear. At this value of a, the solution in general oscillates less, and the residue norm reaches a lower value, than at lower (and sometimes also higher) values. Thus obtained estimate of a can then be used to renormalize the previously calculated PWS profiles. The final result, displaying both PWS and epidermal temperature profiles, is plotted in Figure 9. The depth of the PWS most superficial boundary, which is clearly distinguished from the epidermis, is determined with reasonable accuracy. 7. ACKNOWLEDGEMENT This work was supported by a research grant from the Institute of Arthritis and Musculoskeletal and Skin Diseases (AR43419) at the National Institutes of Health, and by the Slovenian Ministry of Science and Technology (BM). Institutional support from the Office of Naval Research, Department of Energy, National Institutes of Health, and the Beckman Laser Institute and Medical Clinic Endowment is also acknowledged. 6. CONCLUSIONS PPTR can be used for non-invasive depth profiling of PWS, with a potential for on-line guidance of laser therapy on an individual patient basis. In reconstruction of the laser-induced temperature profiles from the experimentally obtained radiometric signals, variation of the tissue absorption coefficient within the commonly used 3—S tm IR detection band (InSb) has to be taken into account when calculating the kernel function of the problem. Alternatively, a bandpass filter can be implemented to reduce the absorption coefficient variation in the detection band and allow the use of the simplified kernel function. The epidermal and PWS contributions to the PPTR signals can be separated using two laser excitation wavelengths, which enables determination of the epidermal thickness and PWS depth in vivo, even when the two layers are in close physical proximity to each other. Based on the above discussed spectral properties, we can expect a < 1 and 3 1. The overlap of the two profiles at lOO tm most likely results from the uneven boundary between the epidermis and dermis, but could in part result also from broadening of one or both profiles by the reconstruction algorithm. The epidermis profile deeper than 0.25 mm is an artifact of the method and should be disregarded. We attribute it to differences between the temperature profiles induced in PWS with 585vs. 600 nm irradiation. Since the latter penetrates deeper into the PWS lesion, the validity of proportionality between the two PWS temperature profiles, assumed in (13), deteriorates with depth. However, such a first order approximation definitely holds in the thin top layer of the PWS, where both temperature profiles (using 585 and 600 mu excitation) can be linearized. The solutions presented in Fig. 9 are thus representative up to a depth of '.O.25 mm. At greater depths, the PWS profile solution may differ from the actual temperature profile induced by the 585 nm excitation, as some contribution from deeper blood vessels is contained in signal y(t). More experimental examples and a thorough discussion of the strengths and limitations of this approach will be presented elsewhere. 7. REFERENCES 1 . S. H. Barsky, S. Rosen, D. E. Geer, and J. M. Noe, "The nature and evolution of port wine stains: a computer-assisted study,"J. Invest. Dermatol. 74, pp. 154-157, 1980. 2. B. Anvari, T. E. Mimer, B. S. Tanenbaum, S. Kimel, L. 0. Svaasand, and J. S. Nelson, "Selective cooling of biological tissues for thermally mediated therapeutic procedures," Phvs. Med. Biol. 40, pp. 241-252, 1995. 3. B. Anvari, B. S. Tanenbaum, T. E. Milner, S. Kimel, L. 0. Svaasand, and J. S. Nelson, "A theoretical study of the thermal response of skin to cryogen spray cooling and pulsed laser irradiation: implications for the treatment of port wine stain birthmarks," Phys. Med. Biol. 40,pp. 1451-1465, 1995. 4. W. Verkruysse, B. Majaron, B. S. Tanenbaum, J. S. Nelson, "Optimal cryogen spray cooling parameters for pulsed laser treatment of port wine stains," submitted to Lasers Surg. Med. 124 5. S. L. Jacques, J. S. Nelson, W. H. Wright, and T. E. Mimer, "Pulsed photothermal radiometry of port-wine-stain lesions," Appi. Opt. 32, pp. 2439-2446, 1993. 6. T. E. Mimer, D. M. Goodman, B. S. Tanenbaum, and J. S. Nelson, "Depth profiling of laser-heated chromophores in biological tissues by pulsed photothermal radiometry," I Opt. Soc. Am. A 12, pp. 1479-1488, 1995. 7. T. E. Milner, D. J. Smithies, D. M. Goodman, A. Lau, and J. S. Nelson, "Depth determination of chromophores in human skin by pulsed photothermal radiometry," App!. Opt. 35, pp. 3379-3385, 1996. 8. E. D.Palik ed., Handbook ofOptical Constants in Solids II, Academic Press, 1991. 8. E. D.Palik ed., Handbook ofOptical Constants in Solids II, Academic Press, 1991. 9. D. J. Smithies, T. E. Milner, B. S. Tanenbaum, D. M. Goodman, and J. S. Nelson, "Accuracy of subsurface distributions computed from pulsed photothermal radiometry," Phys. Med. Biol. 43, pp. 2453-2463, 1998. 10. B. Majaron, W. Verkruysse, B. S. Tanenbaum, T. E. Mimer, s. A. Telenkov, D. M. Goodman, and J. S. Nelson, "Combining two excitation wavelengths for pulsed photothermal profiling of hypervascular lesions in human skin," submitted to Phys. Med. Rio!. 11. S. Wan, R. R. Anderson, and J. A. Parrish, "Analytical modeling for the optical properties of the skin with in vitro and in vivo applications," Photochem. Photobiol. 34, pp. 493-499, 1981. 12. E. J. van Kampen and W. G. Zijlstra, "Determination of hemoglobin and its derivatives", in Advances in Clinical Chemistiy, H. Sobotka and C. P. 7. REFERENCES Stewart, eds., (Academic Press, New York, 1965), p. 158. 125
https://openalex.org/W1974920983	https://europepmc.org/articles/pmc3673477?pdf=render	English	null	The impact of insecticide resistance on <i>Culex pipiens</i> immunity	Evolutionary Applications	2,012	cc-by	11,492	Correspondence J. Vezilier, Centre de Recherche IRD, MIVEGEC (CNRS UMR 5290), 911 Avenue Agropolis, 34394 Montpellier, France. Tel.: +33 467 41 63 73; fax: +33 467 41 62 99; e-mail: julien.vezilier@ird.fr Received: 2 June 2012 In Revised form: 30 October 2012; Accepted: 5 November 2012 doi:10.1111/eva.12037 Received: 2 June 2012 In Revised form: 30 October 2012; Accepted: 5 November 2012 doi:10.1111/eva.12037 doi:10.1111/eva.12037 a very speciﬁc environment in which the parasites differen- tiate, proliferate, and migrate to the correct tissues to ensure transmission to the next host. A modiﬁcation in any of the factors that make up this complex physiological environment can drastically alter the vectorial competence of mosquitoes (Dong et al. 2006; Garver et al. 2009). Argu- ably, the mosquito immune system is one of the most important of these factors. © 2012 The Authors. Published by Blackwell Publishing 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. Evolutionary Applications ISSN 1752-4571 Evolutionary Applications Evolutionary Applications ISSN 1752-4571 Evolutionary Applications Keywords y acetylcholinesterase, anopheles, antimicrobial peptide, carboxylesterases, nitric oxide, transferrin. y acetylcholinesterase, anopheles, antimicrobial peptide, carboxylesterases, nitric oxide, transferrin. Because of their role as vectors of diseases, the evolution of insecticide resistance in mosquitoes has been intensively investigated. Insecticide resistance is associ- ated to a wide range of pleiotropic effects on several key life-history traits of mos- quitoes such as longevity and behavior. However, despite its potential implications in pathogen transmission, the effects of insecticide resistance on mosquito immunity have received little, if any, attention. Here, we investigate the impact of insecticide resistance in Culex pipiens, an epidemiologically important vector of a wide array of pathogens. Using both isogenic laboratory strains and ﬁeld-caught mosquitoes, we investigate the impact of two main insecticide resis- tance mechanisms (metabolic detoxiﬁcation and target site modiﬁcation) on the relative transcription of several genes involved in the immune response to patho- gens, at both their constitutive and inducible levels. Our results show a discrep- ancy between the isogenic laboratory lines and ﬁeld-collected mosquitoes: While in the isogenic strains, insecticide-resistant mosquitoes show a drastic increase in immune gene expression, no such effect appears in the ﬁeld. We speculate on the different mechanisms that may underlie this discrepancy and discuss the risks of making inferences on the pleiotropic effects of insecticide-resistant genes by using laboratory-selected insecticide-resistant lines. Evolutionary Applications Evolutionary Applications Evolutionary Applications ISSN 1752-4571 Introduction Many of the most dangerous infectious diseases such as malaria, ﬁlariasis, or dengue fever, are transmitted to humans by mosquitoes. Since their introduction in the sec- ond half of World War II, insecticides have played a central role in reducing disease transmission. Their efﬁciency is, however, threatened by the evolution and spread of insecti- cide resistance. Today, insecticide resistance has been reported in all main mosquito vector species and geograph- ical regions with high parasite-related mortality and mor- bidity (Roberts and Andre 1994; Ranson et al. 2011). One obvious way in which insecticide resistance impacts on the transmission of diseases is by increasing the number of mosquitoes in the population. However, it has been recently suggested that insecticide resistance may also have an impact on the quality of these mosquitoes (McCarroll et al. 2000; Rivero et al. 2010). Mosquitoes indeed provide Mosquitoes rely on a suite of immune responses to com- bat infection. These responses can be classiﬁed into two types: constitutive (which are always present and ready to act) and induced (which are expressed only after the host has been exposed to an infection, Hamilton et al. 2008). Endogenous innate immune molecules of mosquitoes have been shown to hinder the development of malarial (Luck- hart et al. 1998), ﬁlarial (Shiao et al. 2001), and viral para- sites (Sanchez-Vargas et al. 2009). In a recent microarray study comparing insecticide resistant and susceptible 497 Insecticide resistance and mosquito immunity Vezilier et al. Anopheles mosquitoes, Vontas et al. found a differential expression of some of these immune effectors genes (Von- tas et al. 2005, 2007) suggesting a potential link between insecticide resistance and the insect immune system. 2007; Michalski et al. 2010), and protozoa (Votypka et al. 2002; Kimura et al. 2010). It has also a well-deserved repu- tation for being one of the mosquito species where the molecular and genetic bases of insecticide resistance to organophosphate insecticides are best understood (see Fig. 1, Labbe et al. 2007; Raymond et al. 2001). Two main mechanisms of insecticide resistance have evolved in mosquitoes: (i) the overproduction of detoxify- ing enzymes that sequester and/or degrade the insecticide before it reaches the nervous system (metabolic resistance) and (ii) mutations in the insecticide neural targets that ren- der them less sensitive to the insecticide’s active ingredient (target site resistance, Fig. 1). Introduction These insecticide resistance mechanisms could interfere with mosquito immunity in at least two ways (Rivero et al. 2010). First, insecticide resis- tance genes or genes linked to them as a result of hitchhik- ing could have a pleiotropic effect on one of the genes involved in the complex immune cascades, from the recog- nition of the parasite as foreign to the transduction of the signal and the deployment of the killing mechanism. Sec- ond, insecticide resistance may interfere with immunity through resource-based trade-offs (Rivero et al. 2010). Indeed, both insecticide resistance (Rivero et al. 2011) and immunity (Moret and Schmid-Hempel 2000) have been shown to be energetically costly. The predictions arising from each of these two processes are not necessarily the same: While resource-based trade-offs will, by deﬁnition, curtail mosquito immunity, the direct pleiotropic effects of insecticide resistance genes could have either a positive or a negative effect on immunity depending on, among other things, the nature of the immune genes concerned. Comparative studies of insecticide-resistant and suscep- tible mosquitoes are faced with several experimental chal- lenges. The ﬁrst one is that in order to obtain meaningful conclusions, the insecticide-resistant and susceptible mos- quitoes must be sympatric. Allopatric comparisons (Vontas et al. 2004 Vontas et al. 2005, 2007; Okoye et al. 2007) can- not disentangle the effects of insecticide resistance genes from other differences that inevitably arise during divergent evolutionary history. However, in areas with a long and complex history of insecticide use, fully susceptible mos- quitoes are very hard to ﬁnd, so comparative studies of sympatric insecticide-resistant and susceptible mosquitoes are few and far behind (but see McCarroll et al. 2000). Many studies thus have resorted to using laboratory- selected lines (McCarroll and Hemingway 2002), but this raises a second experimental difﬁculty: The conclusions from laboratory-selected insecticide-resistant strains may not be directly applicable to the conditions in the ﬁeld (due to unnaturally high insecticide selection pressures, or inad- vertent selection for other mosquito traits, McCarroll and Hemingway 2002; Curtis 2001). Here, to disentangle the effect of insecticide resistance on mosquito immunity, we use both approaches. In the Montpellier region, repeated treatments of Cx. pipiens larval sites with organophosphate insecticides (initiated 40 years ago) have resulted in the evolution of two types of insecticide resistance (carboxyles- terase overproduction and acetylcholinesterase modiﬁca- tion). Introduction In this region, there is an insecticide-treated area (a 20-km band close to the sea), a nontreated area (further Here, we investigate the effect of insecticide resistance on immunity in the mosquito Cx. pipiens. Cx. pipiens is a geo- graphically widespread and abundant species that is an epi- demiologically important vector of a wide array of pathogens including several arboviruses (Hamer et al. 2008; Kilpatrick et al. 2010), ﬁlarial worms (Morchon et al. (A) (B) Figure 1 Insecticide resistance in Cx. pipiens. (A) Metabolic resistance. It consists in the overproduction of a large amount of detoxifying carboxyles- terases (Raymond et al. 2001), which is achieved by the tandem ampliﬁcation of two paralogous esterase loci esterase-3 (encoding for the esterase A) and esterase-2 (esterase B). These two genes are in strong linkage disequilibrium and are commonly referred to as an Ester superlocus (Berticat et al. 2001). (B) Target site resistance. The modiﬁcation of the acetylcholinesterase enzyme in Cx. pipiens is controlled by the locus ace-1. The most prevalent alleles for this locus are the wild-type susceptible ace-1S and the insecticide resistant ace-1R, which contains a single point mutation that renders the acetylcholinesterase enzyme insensitive to the insecticide. This point mutation is identical to the one found in acetylcholinesterase resis- tant Anopheles gambiae and A. albimanus mosquitoes (Weill et al. 2003; Weill et al. 2004). (A) (B) (B) (B) (A) Figure 1 Insecticide resistance in Cx. pipiens. (A) Metabolic resistance. It consists in the overproduction of a large amount of detoxifying carboxyles- terases (Raymond et al. 2001), which is achieved by the tandem ampliﬁcation of two paralogous esterase loci esterase-3 (encoding for the esterase A) and esterase-2 (esterase B). These two genes are in strong linkage disequilibrium and are commonly referred to as an Ester superlocus (Berticat et al. 2001). (B) Target site resistance. The modiﬁcation of the acetylcholinesterase enzyme in Cx. pipiens is controlled by the locus ace-1. The most prevalent alleles for this locus are the wild-type susceptible ace-1S and the insecticide resistant ace-1R, which contains a single point mutation that renders the acetylcholinesterase enzyme insensitive to the insecticide. This point mutation is identical to the one found in acetylcholinesterase resis- tant Anopheles gambiae and A. albimanus mosquitoes (Weill et al. 2003; Weill et al. 2004). Figure 1 Insecticide resistance in Cx. pipiens. (A) Metabolic resistance. © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Introduction It consists in the overproduction of a large amount of detoxifying carboxyles- terases (Raymond et al. 2001), which is achieved by the tandem ampliﬁcation of two paralogous esterase loci esterase-3 (encoding for the esterase A) and esterase-2 (esterase B). These two genes are in strong linkage disequilibrium and are commonly referred to as an Ester superlocus (Berticat et al. 2001). (B) Target site resistance. The modiﬁcation of the acetylcholinesterase enzyme in Cx. pipiens is controlled by the locus ace-1. The most prevalent alleles for this locus are the wild-type susceptible ace-1S and the insecticide resistant ace-1R, which contains a single point mutation that renders the acetylcholinesterase enzyme insensitive to the insecticide. This point mutation is identical to the one found in acetylcholinesterase resis- tant Anopheles gambiae and A. albimanus mosquitoes (Weill et al. 2003; Weill et al. 2004). Figure 1 Insecticide resistance in Cx. pipiens. (A) Metabolic resistance. It consists in the overproduction of a large amount of detoxifying carboxyles- terases (Raymond et al. 2001), which is achieved by the tandem ampliﬁcation of two paralogous esterase loci esterase-3 (encoding for the esterase A) and esterase-2 (esterase B). These two genes are in strong linkage disequilibrium and are commonly referred to as an Ester superlocus (Berticat et al. 2001). (B) Target site resistance. The modiﬁcation of the acetylcholinesterase enzyme in Cx. pipiens is controlled by the locus ace-1. The most prevalent alleles for this locus are the wild-type susceptible ace-1S and the insecticide resistant ace-1R, which contains a single point mutation that renders the acetylcholinesterase enzyme insensitive to the insecticide. This point mutation is identical to the one found in acetylcholinesterase resis- tant Anopheles gambiae and A. albimanus mosquitoes (Weill et al. 2003; Weill et al. 2004). 498 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Vezilier et al. Insecticide resistance and mosquito immunity north), and an intermediate area where metabolic and tar- get-site-resistant mosquitoes coexist with susceptible ones (Lenormand et al. 1999). In addition, through a series of back-crossings carried out at the Institute des Sciences de l’Evolution de Montpellier, the different insecticide resis- tance alleles found in the region have been separately intro- gressed into a common (insecticide-susceptible) genetic background to produce different isogenic insecticide-resis- tant mosquito lines (Berticat et al. 2002). Mosquito rearing and collections Isogenic mosquito lines Isogenic mosquito lines Three different isogenic strains of Cx. pipiens mosquitoes sharing the same SLAB genetic background but differing by the alleles at the insecticide resistance loci were used in the experiments. Details of these strains are given in Table 1. Eggs of each of the different mosquito strains were obtained from the Institute des Sciences de l’Evolution de Montpellier and set up to hatch under our standard insec- tary conditions (25 1°C, 70 5% RH and 12L:12D photoperiod). On the hatching day, larvae were haphaz- ardly seeded into plastic trays (four trays per genotype, dimensions: 25 cm 9 35 cm 9 7 cm) containing 1 L of mineral water (Eau de Source Carrefour, France) at a con- stant density of 300 individuals per tray. Larvae were pro- vided with a half-tablet of concentrated yeast on the day of the hatching, 200 mg of TetraMin® ﬁsh ﬂakes (Tetra GmbH, Melle, Germany) the following day, and from then on 400 mg TetraMin every 2 days until pupation. Tray We investigate immunity by measuring the constitutive and inducible expression of several immune-related genes using a quantitative PCR approach. This technique is increasingly used in the ﬁeld of invertebrate ecological immunity (Wigby et al. 2008; Fellous and Lazzaro 2010) and relies on the fundamental assumption that the levels of immune gene transcripts measured are directly propor- tional to the amount of immune proteins that are trans- lated (Greenbaum et al. 2003; Guo et al. 2008; but see Bartholomay et al. 2004). y We chose six candidate genes, all of which have been shown to be important components of the mosquito’s immune system: four antimicrobial peptides (defensin, cecr- opin A, cecropin B, and gambicin), the nitric oxide synthase (NOS), and transferrin. Antimicrobial peptides (AMPs) are an essential component of the defense machinery of mos- quitoes against bacteria (Bartholomay et al. 2003), fungi (Dimopoulos et al. 2001), malarial (Lowenberger 2001), and ﬁlarial parasites (Lowenberger et al. 1996; but see Bar- tholomay et al. 2003). Nitric oxide (synthesized by the NOS) is an ubiquitous and powerful pathogen-killing mechanism (Rivero 2006) which, in mosquitoes, has been shown to be effective against Plasmodium (Lim et al. 2005), bacteria (Hillyer and Estevez- Lao 2010), and viruses (Ra- mos-Castaneda et al. 2008). Transferrin is a key regulator of the iron metabolism that seems to play a key role in innate immunity (Yoshiga et al. Introduction Combined, these two approaches provide a powerful test of the role of insec- ticide resistance on immunity within the mosquito as well as of the validity of using laboratory-selected strains to make inferences about mosquito immunity (Rivero et al. 2010). immune-related genes? (ii) Does this effect depend on the insecticide resistance mechanism involved (esterase over- production versus acetylcholinesterase modiﬁcation)? (iii) Is this effect consistent at both their constitutive and induc- ible expression levels? and (iv) Do laboratory-reared and ﬁeld-collected mosquitoes give similar answers to these questions? We discuss the potential implications of our results for disease transmission. © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Mosquito rearing and collections 1997; Yun et al. 2009). Transferrin upregulation following infection is believed to result in the sequestration of iron away from pathogens, thus limiting their growth (Law 2002). Transferrin has also been shown to have a direct antimicrobial activity against a variety of pathogens (Yun et al. 2009). In addition, to com- pare the level of insecticide resistance in isogenic and ﬁeld- caught mosquitoes, we also quantiﬁed the relative expres- sion of the esterase-2 gene (which encodes for one of the ampliﬁed carboxylesterase enzyme conferring metabolic resistance to Cx. pipiens, see Fig. 1). Table 1. Insecticide-resistant and susceptible strains used in the iso- genic strain experiment. The overproduction of esterases is controlled by the Ester superlocus. Alleles for this locus are the wild-type suscepti- ble Ester0 and the insecticide-resistant Ester4 allele (most common allele in the Montpellier region which overproduces the esterase A4 and B4 isozymes). The modiﬁcation of the acetylcholinesterase is controlled by the locus ace-1. Alleles for this locus are the wild-type susceptible ace- 1S and the insecticide resistant ace-1R. For more details on these strains, see Berticat et al. (2002). Since their creation, the SLAB, SA4B4, and SR mosquito strains have been kept in culture in the laboratory. To avoid genetic drift and due to the occasional contamination of the lines, they have been regularly backcrossed over the years (to obtain newly crossed SA4B4 and SR lines). The lines used in this study had been last crossed <1 year before the beginning of the experiment. Table 1. Insecticide-resistant and susceptible strains used in the iso- genic strain experiment. The overproduction of esterases is controlled by the Ester superlocus. Alleles for this locus are the wild-type suscepti- ble Ester0 and the insecticide-resistant Ester4 allele (most common allele in the Montpellier region which overproduces the esterase A4 and B4 isozymes). The modiﬁcation of the acetylcholinesterase is controlled by the locus ace-1. Alleles for this locus are the wild-type susceptible ace- 1S and the insecticide resistant ace-1R. For more details on these strains, see Berticat et al. (2002). Since their creation, the SLAB, SA4B4, and SR mosquito strains have been kept in culture in the laboratory. To avoid genetic drift and due to the occasional contamination of the lines, they have been regularly backcrossed over the years (to obtain newly crossed SA4B4 and SR lines). Field-caught mosquitoes g q More than 50 Cx. pipiens egg rafts were collected in Octo- ber 2010 from a population where insecticide susceptible (Ester0, ace-1s), esterase-resistant (Ester4, ace-1s), and ace- tylcholinesterase-resistant mosquitoes (Ester0, ace-1R) coex- ist in sympatry (Vezilier et al. 2010 for details). Eggs were brought to our insectary for hatching and the resulting lar- vae reared following the same protocol as for the isogenic strain experiment. Eggs were collected instead of larvae because larval condition has been shown to have a key effect on mosquito immunity and vectorial capacity (Ok- ech et al. 2007; Fellous and Lazzaro 2010). One week after emergence, 360 adult females were haphazardly assigned to one of the three injection treatments (120 females per treat- ment). Quantitative PCR analysis We set out to investigate the relative expression of six immune-related genes (cecropin A, cecropin B, gambicin, defensin, transferrin, and NOS) and the esterase-2 gene by quantitative PCR (qPCR). Brieﬂy, total RNA was extracted from whole mosquitoes (n = 270 isogenic and 153 ﬁeld- caught mosquitoes) using Trizol Reagent following the manufacturer’s protocol (Invitrogen). RNA integrity was electrophoretically veriﬁed by ethidium bromide staining before quantiﬁcation using a NanoDrop spectrophotometer (NanoDrop Thermo Fisher Scientiﬁc). Oligo-dT primed cDNAs were produced from 1 lg of total RNA using M-MLV reverse transcriptase according to manufacturer’s protocols and reagents (Invitrogen). The qPCR assays were performed with LightCycler480 (Roche) in 384-well qPCR plates. The qPCR reaction consisted in a 1 9 Light-Cycler 480 master mix, 0.5 lM of each primer, and 1 lL of cDNA (1/8 dilution) to obtain a ﬁnal volume of 5 lL. The primer sequences used for the qPCR reactions are given in Table 2. Primers were designed on available Cx. pipiens sequences (partial or complete cDNAs, see GenBank references in Table 2) in conserved gene regions after alignment with several other sequences from closely related species. The qPCR program used was the following: 10 min at 95°C, fol- lowed by 40 cycles of 10 s at 95°C, 20 s at 57°C, and 25 s at 72°C. A ﬁnal melting curve was systematically produced to control for ampliﬁcation speciﬁcity. Relative expression of each immune-related gene was calculated using 2DDCT method (Pfafﬂ2001) using the G6pdh (glucose 6-phosphate dehydrogenase) gene as a reference. This method relies on the assumption that the ampliﬁcation efﬁciencies of the tar- Mosquito rearing and collections The lines used in this study had been last crossed <1 year before the beginning of the experiment. Strain IR mechanism Alleles Genetic background SLAB None Ester0, ace-1S SLAB SA4B4 Overproduction of esterases A4 and B4 Ester4, ace-1S SLAB SR Insensitive acetylcholinesterase Ester0, ace-1R SLAB We address the following four questions: (i) Does insec- ticide resistance alter the level of expression of these 499 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Vezilier et al. Insecticide resistance and mosquito immunity water was changed on feeding days to avoid bacterial growth on the water surface. On pupation, trays were placed inside an emergence cage (27 9 40 9 35 cm) and provided with an ad libitum source of 10% sugar solution for the emerged adults. One week after emergence, 90 females from each insecticide-resistant strain were haphaz- ardly chosen from the different emergence cages and ran- domly assigned to one of three experimental treatments (uninjected, Ringer, and LPS injected, 30 females per treat- ment). separately frozen to identify their insecticide resistance sta- tus (see Molecular methods, below). Injection of LPS was preferred to the injection of live bacteria as an immune challenge because it allows controlling for the eventual dif- ferences that could exist in bacterial growth between the strains. Molecular methods Insecticide resistance status of ﬁeld-caught mosquitoes Insecticide resistance status of ﬁeld-caught mosquitoes Genotyping at the Ester and ace-1 loci was performed on mosquito head homogenates using an RFLP analysis as described in Vezilier et al. (2010). As the number of target- site-resistant females (Ester0, ace-1R) present in our initial pool of 360 wild mosquitoes was too low to achieve a satis- fying number of replicates for the three injection treat- ments, only fully susceptible (Ester0, ace-1S, n = 21 uninjected, 25 Ringer injected, and 21 LPS injected) and metabolic resistant (Ester4, ace-1S, n = 29 uninjected, 27 Ringer injected, and 30 LPS injected) females were retained in for the qPCR analysis. © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Constitutive versus induced gene expression Co st tut ve ve sus duced ge e e p ess o In the isogenic mosquito lines, the relative expression of all but one of the genes was found to be signiﬁcantly induced in response to the injection treatment (main treatment effect, cecB: v2 2 = 24.32, P < 0.001; gamb: v2 2 = 25.41, P < 0.001; def: v2 2 = 13.13, P = 0.001; transf: v2 2 = 113.76, P < 0.001; see Fig. 2A–D). For cecropin B, gambicin, and transferrin, a posteriori contrasts conﬁrmed that the enhanced gene expression resulted from the exposure to the LPS rather than from the physical stress induced by (or the opportunistic infections that come with) mosquito injection (signiﬁcant Ringer-LPS contrast, cecB: v1 2 = 19.97, P < 0.001; gamb: v1 2 = 18.50, P < 0.001; transf: v3 2 = 63.62, P < 0.001). Defensin expression, how- ever, was stimulated by the injection itself and not by the LPS immune elicitor (nonsigniﬁcant Ringer-LPS contrast, v1 2 = 0.82, P = 0.366). The results for the NOS also showed a signiﬁcant treatment effect on gene expression (v2 2 = 17.91, P < 0.001), although this seemed to stem from a reduction in NOS expression in Ringer-injected females (nonsigniﬁcant uninjected-LPS contrast, v1 2 = 0.41, P = 0.524, see Fig. 2E). Mosquito experimental injections from the model. A posteriori contrasts were carried out by aggregating factor levels together and by testing the ﬁt of the simpliﬁed model using an LRT (Crawley 2007). The validity of the G6pdh gene as an endogenous control was analyzed by ﬁtting the mean G6pdh CT values obtained for each individual on the different plates as a response vari- able (glm model), using mosquito treatment, mosquito genotype, and their interaction as ﬁxed explanatory vari- ables (see Fig. S2). get genes and the reference genes are approximately equal (Livak and Schmittgen 2001). To assess the validity of this assumption, we compared the DCT values (CT-targetCT-g6pdh) under different dilutions of the template (1/1 to 1/32). For most target genes, DCT values were not signiﬁcantly affected by dilutions, which indicate that the ampliﬁcation efﬁciencies are indeed similar. After testing four different couples of primers, the cecropin A gene failed to meet these efﬁciency criteria and was thus discarded from the study (see Fig. S1 for details). To ensure that mean gene expres- sion, mosquito treatment, and mosquito insecticide resis- tance status would not be confounded with the microplate effect, we designed the qPCR plates according to two crite- ria: (i) the same individuals were simultaneously assayed for the expression of several genes on the same plate and (ii) qPCR plates included all combinations of insecticide- resistant categories and treatments for each gene. Mosquito experimental injections The injection protocol was identical for the isogenic lines and ﬁeld-caught mosquitoes. Adult females were brieﬂy anesthetized using a CO2 pad. Mosquitoes were either: (i) uninjected, to measure constitutive gene expression levels in the absence of any immune stimulation, (ii) injected with the LPS immune elicitor (Sigma Aldrich E. coli 055:B5 LPS, lot L5418 phenol-extracted and gel ﬁltration puriﬁed, 0.5 mg/mL Ringer), to measure inducible gene expression levels, or (iii) injected with physiological saline (Drosophila Ringer) as a trauma control. Injections of 69 nL of liquid per mosquito were performed intrathoracically by using a Nanoinjector (Drummond) equipped with a sterile, ﬁnely drawn glass capillary needle. Mosquitoes were then individ- ualized into numbered dry 30-ml drosophila plastic tubes covered with a mesh and kept under our standard insectary conditions. Food was provided in the form of a cotton pad soaked in a 10% glucose solution placed on top of each tube. To match the induction peak of most of the immune genes investigated (Bartholomay et al. 2003), females Cx. pipiens were killed 24 h after injection using a CO2 pad. Mosquitoes were placed into an eppendorf containing 1 mL of Trizol reagent (Invitrogen Corp.) and immediately frozen at 80°C. Wild caught females were ﬁrst decapi- tated before freezing in Trizol and mosquito heads were 500 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Insecticide resistance and mosquito immunity Vezilier et al. Table 2. Quantitative PCR primers. Table 2. Quantitative PCR primers. Gene Primer Sequence (5′–3′) Amplicon size (bp) Reference Cecropin B Forward TGGCAGCCCTGTTGCTGCTG 133 Genbank AY189810 (Bartholomay et al. 2003) Reverse GCCTGCACTCCTGCTGCAAC Defensin Forward AGTGGATTCGGCGTCAACGA 102 Genbank AY191319 (Bartholomay et al. 2003) Reverse GTTTCGGCACACGCAAACCT Gambicin Forward CTGTGACGACTGCAGGAGAC 100 Genbank XM_001866164 Reverse AATCCTCGCTGAGCTCTCGT Transferrin Forward AAGTACTCTCCGAACGACGA 109 Genbank XM_001865823 Reverse CCGAGTACTTGTCCGGGTAG NO Synthase Forward CGAGAAGGCCCACATCTACG 126 Genbank XM_001841984 Reverse CGACAGCATGTACTTCTCCA Esterase-2 Forward CCGACGAGCTGTCCTATCTG 216 Weill et al. (2000) Reverse CGTCGTTGGCAATGTTCAG G6pdh Forward CGCGCACGAGGAAAAGTACG 131 Genbank CPU09034 Reverse GGTTTGCGGTCTTCCCAACC from the model. A posteriori contrasts were carried out by aggregating factor levels together and by testing the ﬁt of the simpliﬁed model using an LRT (Crawley 2007). The validity of the G6pdh gene as an endogenous control was analyzed by ﬁtting the mean G6pdh CT values obtained for each individual on the different plates as a response vari- able (glm model), using mosquito treatment, mosquito genotype, and their interaction as ﬁxed explanatory vari- ables (see Fig. S2). © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Statistical methods Analyses were conducted using the R statistical package (v. 2.12.0, http://cran.r-project.org). Target gene expression (expressed as 2DDCT) was analyzed using mixed effect lin- ear models (lme, nlme package) ﬁtting plate identity as a random explanatory variable, and mosquito strain (iso- genic mosquitoes) or genotype (ﬁeld-collected mosqui- toes), experimental treatment, and their interaction as ﬁxed explanatory variables. Maximal models were simpliﬁed by sequentially eliminating nonsigniﬁcant terms and interac- tions to establish a minimal model (Crawley 2007). The signiﬁcance of explanatory variables in mixed effect models was established using a likelihood ratio test (LRT), which is approximately distributed as a chi-square distribution (Bolker 2008). The signiﬁcant v2 values given in the text are for the minimal model, while nonsigniﬁcant values cor- respond to those obtained before deletion of the variable The injection treatment also had a signiﬁcant effect on the relative expression of the defensin and transferrin genes 501 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Insecticide resistance and mosquito immunity Vezilier et al. Isogenic Cecropin B Gambicin Defensin NO Synthase Field-caught Transferrin NI RINGER LPS 0 1 2 3 NI RINGER LPS 0 5 10 15 20 NI RINGER LPS 0 5 10 15 20 NI RINGER LPS 0 4 8 12 16 NI RINGER LPS 0 4 8 12 16 NI RINGER LPS 0 1 2 3 NI RINGER LPS 0 1 2 3 NI RINGER LPS 0 4 8 12 16 NI RINGER LPS 0 4 8 12 16 NI RINGER LPS 0 1 2 3 Fold change in expression Fold change in expression Fold change in expression Fold change in expression Fold change in expression (A) (F) (B) (G) (C) (H) (D) (I) (E) (J) e effect of insecticide resistance on immune-related gene expression. The cecropin B, gambicin, defensin, transferrin sion were measured at their constitutive level (noninjected: NI), or after injection with Ringer or LPS in both isogenic quitoes (F–J). Symbols represent the mean SE change in gene expression compared with the reference, that is, the usceptible mosquitoes from the noninjected treatment group. White circles and full lines: insecticide susceptible mosqu ine: metabolic-resistant mosquitoes; dark triangles and dotted lines: target-site-resistant mosquitoes. Statistical methods Isogenic B Field-caught NI RINGER LPS 0 1 2 3 NI RINGER LPS 0 1 2 3 Fold change in expression (A) (F) Field-caught Isogenic Cecropin B NI RINGER LPS 0 4 8 12 16 NI RINGER LPS 0 4 8 12 16 Fold change in expression (B) (G) Gambicin NI RINGER LPS 0 5 10 15 20 NI RINGER LPS 0 5 10 15 20 Fold change in expression (C) (H) Defensin NI RINGER LPS 0 4 8 12 16 NI RINGER LPS 0 4 8 12 16 Fold change in expression (D) (I) NI RINGER LPS 0 4 8 12 16 Fold change in expression (D) NI RINGER LPS 0 4 8 12 16 (I) Fold change in expression Transferrin NO Synthase NI RINGER LPS 0 1 2 3 Fold change in expression (E) NI RINGER LPS 0 1 2 3 (J) NI RINGER LPS 0 1 2 3 Fold change in expression (E) Figure 2 The effect of insecticide resistance on immune-related gene expression. The cecropin B, gambicin, defensin, transferrin, and NO synthase gene expression were measured at their constitutive level (noninjected: NI), or after injection with Ringer or LPS in both isogenic line (A–E) and wild caught mosquitoes (F–J). Symbols represent the mean SE change in gene expression compared with the reference, that is, the expression level of insecticide susceptible mosquitoes from the noninjected treatment group. White circles and full lines: insecticide susceptible mosquitoes; gray squares and dashed line: metabolic-resistant mosquitoes; dark triangles and dotted lines: target-site-resistant mosquitoes. © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 502 Vezilier et al. Insecticide resistance and mosquito immunity in ﬁeld-caught mosquitoes (def: v2 2 = 49.89, P < 0.001; transf: v2 2 = 35.57, P < 0.001, Fig. 2H, I). The gambicin gene also responded to the treatment but only in Ester4 metabolic-resistant females (genotype 9 treatment, v2 2 = 8.41, P = 0.015, Fig. 2G). While for defensin, this effect was independent of the LPS immune challenge (Ringer-LPS contrast, def: v1 2 = 3.23, P = 0.072), transfer- rin and gambicin transcriptional activation appeared to be speciﬁc to the injection of LPS (Ringer-LPS contrast, transf: v1 2 = 4.56, P = 0.033; gamb: v2 2 = 7.86, P = 0.020). Mos- quito injection had, however, no discernible effect on the cecropin B (v2 2 = 1.61, P = 0.445, see Fig. Statistical methods 2F) or NOS (v2 2 = 0.18, P = 0.912; see Fig. 2J) expression. In sharp contrast to what happens in the isogenic labora- tory lines, in ﬁeld-caught mosquitoes insecticide resistance had no effect on the relative expression of most of the immune-related genes investigated: cecropin B (v1 2 = 0.19, P = 0.664), defensin (v1 2 = 0.86, P = 0.35), transferrin (v1 2 = 0.09, P = 0.768), and NOS (v1 2 = 2.08, P = 0.150). The only exception was the gambicin, where Ester4 meta- bolic-resistant females had signiﬁcantly higher expression levels after the LPS induction than insecticide susceptible mosquitoes (signiﬁcant genotype 9 treatment interaction, see above). As expected, in both the laboratory and the ﬁeld-caught mosquitoes, the relative expression of the esterase-2 gene was higher in mosquitoes carrying the metabolic insecti- cide-resistant (Ester4) allele than in mosquitoes carrying the wild-type susceptible (Ester0) one (laboratory: v2 2 = 265.99, P < 0.001; ﬁeld: v1 2 = 132.01, P < 0.001), independently of the experimental treatment applied (strain 9 treatment interaction, laboratory: v4 2 = 6.17, P = 0.187; genotype 9 treatment interaction, ﬁeld: v2 2 = 1.91, P = 0.384, see Fig. 3). However, while in the ﬁeld the level of esterase expression in insecticide-resistant mosquitoes is ﬁvefold that of susceptible ones (Fig. 3B), in the isogenic laboratory strains, the difference between resis- tant and susceptible strains is as high as tenfold (isogenic – ﬁeld-caught resistant contrast, F1,130 = 44.79, P < 0.001, Fig. 3). Insecticide resistance effect on immune-related gene expression In the laboratory isogenic mosquito lines, insecticide resis- tance was found to have a very signiﬁcant effect on the rela- tive expression of gambicin (v2 2 = 45.05, P < 0.001), defensin (v2 2 = 23.39, P < 0.001), transferrin (v2 2 = 43.70, P < 0.001), and NOS (v2 2 = 11.15, P = 0.004) but not of cecropin B (v2 2 = 3.43, P = 0.180, Fig. 2A–E). Indeed, unexpectedly, for four of the ﬁve genes investigated, meta- bolic-resistant (SA4B4) females had expression levels which were up to four times higher than those of susceptible (SLAB) mosquitoes (SLAB-SA4B4 contrasts, gamb: v1 2 = 44.09, P < 0.001; def: v1 2 = 23.35, P < 0.001; transf: v1 2 = 42.12, P < 0.001; NOS: v1 2 = 10.56, P = 0.001, Fig. 2B–E). There was also a higher relative transferrin expression in SR females compared with SLAB ones (SLAB-SR contrast, transf: v1 2 = 15.61, P < 0.001, see Fig. 2D). These strain effects were constant across treat- ments for all genes (strain 9 treatment interaction, cecB: v4 2 = 3.94, P = 0.413; gamb: v4 2 = 6.42, P = 0.170; def: v4 2 = 2.54, P = 0.637; NOS: v4 2 = 3.33, P = 0.504), except for transferrin (v4 2 = 12.27, P = 0.016). Discussion Insecticide resistance in Cx. pipiens has been previously shown to be associated to a plethora of pleiotropic effects on the ﬁtness of both ﬁeld-caught and laboratory-reared mosquitoes. These pleiotropic effects have invariably taken the shape of life history costs and include decreases in pre- imaginal survival (Berticat et al. 2008), adult longevity (Agnew et al. 2004), fecundity (Duron et al. 2006), and Isogenic NI RINGER LPS 0 2 4 6 8 10 12 14 Fold change in expression (A) Isogenic Field-caught NI RINGER LPS 0 2 4 6 8 10 12 14 NI RINGER LPS 0 2 4 6 8 10 12 14 Fold change in expression (A) (B) Figure 3 The effect of insecticide resistance on the esterase-2 gene expression. Gene expression was measured both at its constitutive level (Nonin- jected: NI) or after injection of Ringer or LPS in both isogenic line (A) and ﬁeld-caught mosquitoes (B). Symbols represent the mean SE change in gene expression compared with the reference (as above). White circles and full lines: insecticide susceptible mosquitoes; gray squares and dashed line: metabolic-resistant mosquitoes; dark triangles and dotted lines: target-site-resistant mosquitoes. Field-caught NI RINGER LPS 0 2 4 6 8 10 12 14 (B) Isogenic Figure 3 The effect of insecticide resistance on the esterase-2 gene expression. Gene expression was measured both at its constitutive level (Nonin- jected: NI) or after injection of Ringer or LPS in both isogenic line (A) and ﬁeld-caught mosquitoes (B). Symbols represent the mean SE change in gene expression compared with the reference (as above). White circles and full lines: insecticide susceptible mosquitoes; gray squares and dashed line: metabolic-resistant mosquitoes; dark triangles and dotted lines: target-site-resistant mosquitoes. © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 503 Vezilier et al. Insecticide resistance and mosquito immunity predator escape (Berticat et al. 2004). However, despite the potential key implications for disease transmission, the effects of insecticide resistance on mosquito immunity has received little attention (but see Vontas et al. 2005, 2007). We quantiﬁed immune-related gene expression in both iso- genic laboratory strains and ﬁeld-collected Cx. pipiens female mosquitoes. The results from our isogenic strain mosquitoes were unexpected in that they showed that mos- quitoes resistant to insecticides through the overproduction of esterases had signiﬁcantly higher constitutive and induc- ible transcription levels of virtually all the immune-related genes investigated compared to their insecticide susceptible counterparts. Discussion Their constitutive immunity was overall quite low so it is uncertain how costly it is to maintain, or whether it can explain why metabolic resistance brings about lower energetic resources (Rivero et al. 2011) and reduced adult longevity in the absence of infection (Vezilier et al. 2012). Field-collected insecticide-resistant and sus- ceptible mosquitoes, however, showed no signiﬁcant differ- ences in immune expression. melanisation cascade (phenoloxidase and prophenoloxi- dase) are signiﬁcantly higher in esterase-resistant (SA4B4) females than in susceptible (SLAB) ones. In addition, using microarray analyses, Vontas et al. found the defensin and cecropin genes to be constitutively expressed at a higher level in laboratory-maintained insecticide-resistant strains of Anopheles gambiae compared with their insecticide sus- ceptible counterparts (Vontas et al. 2005), and the NOS gene to be constitutively overexpressed in insecticide- resistant Anopheles stephensi (Vontas et al. 2007). These Anopheles laboratory strains seem to be resistant to insecti- cides through a complex combination of insecticide- resistance mechanisms, which have been only partially elucidated. In contrast, in Cx. pipiens, the molecular and genetic basis for resistance in both the isogenic lines and in ﬁeld-collected mosquitoes are well established (Raymond et al. 1998; Labbe et al. 2007; see also Fig. 1), which renders the task of explaining the discrepancy in the results obtained more tractable. We suggest three different scenarios that could explain these results. The results from the isogenic lines are in agreement with two other studies comparing the immunity of insecticide- resistant and susceptible laboratory mosquito populations. S. Cornet et al. (unpublished manuscript) have shown that the activities of two key enzymes involved in the Cx. pipiens The ﬁrst scenario involves the existence of an immuno- regulatory factor at the amplicon level (see Fig. 4). Indeed, the high level of the esterase-2 transcripts in the isogenic lines (Fig. 3) strongly suggests that, under the strong insec- ticide selective pressures exerted in the laboratory and the Figure 4 Esterase overproduction in Cx pipiens mosquitoes and the SA4B4 isogenic strain. The overproduction of detoxifying carboxylesterases in Cx pipiens is achieved through the tandem ampliﬁcation of two paralogous esterase loci esterase-3 (encoding for the esterase A) and esterase-2 (ester- ase B). These two genes are in strong linkage disequilibrium and are commonly referred to as an Ester superlocus (Berticat et al. 2001). Discussion The amplicon on which this superlocus occurs is however much larger (30–60 kb) than the esterase containing region ( 10 kb, Hemingway et al. 2002; Guillemaud et al. 1997). The ensemble of the esterase-containing amplicons that are repeated plus their ﬂanking region in the mosquito constitutes an Ester-resistant allele. To construct the SA4B4 strain, a homozygous strain for the Ester4 allele (Poirie et al. 1992) was introgressed into a suscepti- ble reference line (SLAB) by a repeated backcross procedure (Berticat et al. 2002). Several scenarios may explain the higher immune phenotype observed in the SA4B4 strain. A ﬁrst scenario (option 1) involves the existence of an immunoregulatory gene within the amplicon, which would result in it being ampliﬁed to a higher extent in SA4B4 mosquitoes than in ﬁeld-caught mosquitoes. Other genes have already been shown to be hitchhiked and co-ampliﬁed by this tandem repetition (Guillemaud et al. 1997; Hemingway et al. 2002). A second scenario (option 2) involves the existence of a strong immunoregulatory allelic variant in linkage disequilibrium with the Ester4 allele. Such strong immunoregulatory variant may have been present in the original (VIM) strain. A third scenario (option 3) is that the immune phenotype is the result of epistatic interactions between one of these immu- noregulatory factors (option 1 or option 2) and the SLAB genetic background. Dashed lines represent the SLAB genetic background in which the Ester4 allele is expressed. Figure 4 Esterase overproduction in Cx pipiens mosquitoes and the SA4B4 isogenic strain. The overproduction of detoxifying carboxylesterases in Cx pipiens is achieved through the tandem ampliﬁcation of two paralogous esterase loci esterase-3 (encoding for the esterase A) and esterase-2 (ester- ase B). These two genes are in strong linkage disequilibrium and are commonly referred to as an Ester superlocus (Berticat et al. 2001). The amplicon on which this superlocus occurs is however much larger (30–60 kb) than the esterase containing region ( 10 kb, Hemingway et al. 2002; Guillemaud et al. 1997). The ensemble of the esterase-containing amplicons that are repeated plus their ﬂanking region in the mosquito constitutes an Ester-resistant allele. To construct the SA4B4 strain, a homozygous strain for the Ester4 allele (Poirie et al. 1992) was introgressed into a suscepti- ble reference line (SLAB) by a repeated backcross procedure (Berticat et al. 2002). Several scenarios may explain the higher immune phenotype observed in the SA4B4 strain. © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Discussion This is consistent with the predominant role of defensin against gram-positive bacteria (Dimopoulos et al. 2001). The gam- bicin gene was found to be speciﬁcally activated on the LPS challenge in the isogenic line experiment, supporting previ- ous reports that its encoded peptide is involved in the humoral response against gram-negative bacteria (Fig. 2B) (Vizioli et al. 2001; Bartholomay et al. 2003). This ﬁnding was, however, not fully supported by the ﬁeld-caught mos- quito experiment where Ringer injection had a similar effect on gambicin expression (Fig. 2G), although this weak response might have stemmed from an overall lower immunogenic capacity of the LPS in ﬁeld-caught versus isogenic line mosquitoes, as also suggested by the transfer- rin gene expression proﬁles (Fig. 2D, I). Transferrin tran- scription was signiﬁcantly induced by the LPS challenge in both experiments, supporting previous reports showing the direct involvement of this gene product in the mosquito innate immune response (Yoshiga et al. 1997; Fig. 2D, I). In contrast, although NOS expression has already been shown to be induced following LPS injection (Choi et al. 1995), no such effect was apparent in both our experiments where uninjected and LPS-injected mosquitoes had similar NOS expression levels (Fig. 2E, J). Note, however, that as the immune response was quantiﬁed at a single time point (24 h after immune challenge), some of the differences pointed out here may reﬂect differences in the expression kinetics between the genes (Lemaitre et al. 1997; Magalhaes et al. 2008). Although gene expression studies are one of the most A second, and perhaps more parsimonious scenario, is that the immunoregulation takes place at the allelic, rather than the amplicon, level (option 2 in Fig. 4). The Ester4 allele was indeed originally kept in the laboratory at the homozygous state within the VIM strain (Poirie et al. 1992) and later introgressed into a susceptible reference line (SLAB) by a repeated backcross procedure to create the SA4B4 strain (Berticat et al. 2002). One cannot exclude the possibility that this original Ester4-resistant allele was in linkage disequilibrium with a strong immunoregulatory allelic variant, and that the backcross procedure used to introgress this strain within the SLAB genetic background was not sufﬁcient to break this linkage. Discussion A ﬁrst scenario (option 1) involves the existence of an immunoregulatory gene within the amplicon, which would result in it being ampliﬁed to a higher extent in SA4B4 mosquitoes than in ﬁeld-caught mosquitoes. Other genes have already been shown to be hitchhiked and co-ampliﬁed by this tandem repetition (Guillemaud et al. 1997; Hemingway et al. 2002). A second scenario (option 2) involves the existence of a strong immunoregulatory allelic variant in linkage disequilibrium with the Ester4 allele. Such strong immunoregulatory variant may have been present in the original (VIM) strain. A third scenario (option 3) is that the immune phenotype is the result of epistatic interactions between one of these immu- noregulatory factors (option 1 or option 2) and the SLAB genetic background. Dashed lines represent the SLAB genetic background in which the Ester4 allele is expressed. 504 Insecticide resistance and mosquito immunity Vezilier et al. low associated costs, these lines have maintained a higher number of Ester4 amplicons than their wild counterparts (amplicons number within a given metabolic-resistant allele is known to vary in the ﬁeld allowing mosquitoes to rapidly adjust their insecticide resistance levels, Callaghan et al. 1998; Guillemaud 1997). The amplicon-level immu- noregulation could happen through the existence of a gene within the amplicon encoding a regulator common to the different immune-related genes investigated (for instance, a transcription factor from the NFjB family, Antonova et al. 2009; Yun et al. 2009) (option 1 in Fig. 4). This amplicon- level scenario is, however, unlikely as in this case ﬁeld-col- lected mosquitoes should have also overexpressed the immune-related genes, albeit to a lesser extent. immune effectors genes 24 h after an immune insult. As expected, most genes were up-regulated in response to an LPS injection. Among the three AMPs investigated, the cecropin B gene was the one showing the lowest induction levels in both isogenic and ﬁeld-caught mosquito experi- ments, conﬁrming previous ﬁndings that this gene responds poorly to an immune insult (Bartholomay et al. 2003; Fig. 2A, F). Both experiments were also congruent in showing that the defensin gene expression levels were simi- lar between the Ringer and LPS treatments, suggesting that cuticle piercing per se, or the opportunistic infections that come with it, are sufﬁcient to activate this gene’s transcrip- tion, and that the gene does not speciﬁcally respond to the (Escherichia coli – derived) LPS insult (Fig. 2C, H). © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Discussion Finally, the strong immune phenotype observed in the isogenic SA4B4 strain could be the result of epistatic interactions between these immunoregulatory factors (an immune regulator at the amplicon or allelic level) and the SLAB genetic background (option 3 in Fig. 4). The ﬁnd- ing, however, that selection for high resistance levels in laboratory strains from two other mosquito species also results in an upregulation of the immune system (Vontas et al. 2005, 2007) suggests that our results are not speciﬁc to a particular genetic background and that the effect may be a common artifact of laboratory strains. Indeed, our results also showed a higher transferrin expression in tar- get-site-resistant (SR) mosquitoes. We do not have a clear mechanistic explanation for how a single point mutation in the acetylcholinesterase gene could bring about this change. Target site resistance mutates key components of the vector’s neural network and is thus mostly expected to have an effect on mosquito behavior (Rivero et al. 2010). While there is some evidence that these behavioral modiﬁ- cations indeed take place (Berticat et al. 2002, 2004), other pleiotropic effects of this mutation such as reduc- tions in fecundity (Duron et al. 2006) and longevity (Ag- new et al. 2004) have proven more difﬁcult to explain mechanistically. Although gene expression studies are one of the most common tools available for estimating immunocompe- tence, it is not always clear how well they reﬂect the actual ability of individuals to defend themselves against parasites (Fedorka et al. 2007). This is indeed a key question for its potential consequences for the vectorial capacity of mos- quitoes. In a recent paper, we have shown that both ﬁeld- collected and isogenic insecticide resistant and susceptible Cx. pipiens mosquitoes are equally susceptible to P. relictum (one of the etiological agents of avian malaria, Vezilier et al. 2010). McCarroll et al. (2000), McCarroll and Hem- Insecticide resistance effects aside, our results provide new insights into the response of different mosquito 505 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 Vezilier et al. Insecticide resistance and mosquito immunity ingway (2002), however, showed that the development of the ﬁlaria Wuchereria bancrofti larvae was arrested in insec- ticide-resistant Cx. quinquefasciatus mosquitoes, although the role of the immune system in this result has not been established. Data archiving statement Data for this study are available at Dryad - doi:10.5061/ dryad.1pp2c In conclusion, this study is, to our knowledge, the ﬁrst one to investigate the impact of insecticide resistance on the mosquito immune system comparing both isogenic strain mosquitoes (the approach most frequently used to investi- gate the pleiotropic effects of insecticide resistance) and sym- patric ﬁeld-caught-mosquitoes from a population where insecticide-resistant and susceptible mosquitoes coexist. Our results lead us to make two distinct conclusions. The ﬁrst one is that, under the speciﬁc conditions used in our experi- ments, insecticide resistance does not have any immune expression costs in ﬁeld-caught mosquitoes. This result con- trasts with previous studies that have shown that insecticide resistance in Culex pipiens trade-offs with virtually all other life-history traits investigated (Berticat et al. 2002, 2004; Ag- new et al. 2004; Bourguet et al. 2004; Duron et al. 2006; Hardstone et al. 2009), and which explain the sharp decline in insecticide resistance allele frequencies in insecticide-free areas. It is possible, however, that immune gene transcrip- tion per se has no costs (but see Libert et al. 2006; Garver et al. 2009) and that the trade-offs take place post-transcrip- tionally. The second conclusion is more practical by nature. The discrepancy between the results obtained using ﬁeld- caught and isogenic mosquitoes (where we measured increased immune expression levels in insecticide-resistant mosquitoes) adds experimental weight to the risks of making inferences on the pleiotropic effects of insecticide resistance from laboratory-selected lines recently highlighted in the lit- erature (McCarroll et al. 2000; Curtis 2001; McCarroll and Hemingway 2002; Rivero et al. 2010). For many mosquito populations, however, the difﬁculty in obtaining sympatric resistant and susceptible mosquitoes from the ﬁeld renders the use of isogenic insecticide-resistant and susceptible strains unavoidable. Thus, whenever possible, efforts should be made to use several laboratory-selected isogenic mosquito strains with different insecticide-resistant alleles expressed in different genetic backgrounds. Admittedly, this approach might be cumbersome to implement, but the logistic difﬁ- Discussion Although immune expression may or may not reﬂect protection to pathogens, immune expression studies are interesting in their own right as they represent an investment in a trait that is likely to trade-off with other life-history traits, some of which may be relevant for trans- mission (such as longevity, see, e.g., Libert et al. 2006). Many pathogens can be transmitted by Culex mosquitoes (such as several arboviruses including the West Nile agent, Hamer et al. 2008; Kilpatrick et al. 2010), strengthening the need for further work to be carried out on the impact insecticide resistance on the quality of mosquitoes as vec- tors of diseases. culties do not mean the problems associated to laboratory lines can be ignored. Acknowledgements We thank the high throughput qPCR platform at the Uni- versity of Montpellier 2 and, in particular, to Philippe Clair for his help with treatment of the samples. 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(C–D) Mosquito insecticide-resistant status had no effect on the g6pdh expression in either the isogenic strain (main strain effect: F2,204 = 0.7048, P = 0.4954; strain 9 treatment: F4,200 = 1.1029, P = 0.3563) or the ﬁeld-caught mosquito experiments (main genotype effect: F1,149 = 0.087, P = 0.7685; genotype 9 treatment: F2,147 = 1.3908, P = 0.2521). Supporting Information Sanchez-Vargas, I., J. C. Scott, B. K. Poole- Smith, A. W. E. Franz, V. Barbosa-Solomieu, J. Wilusz, K. E. Olson et al. 2009. Dengue Virus type 2 infections of Aedes aegypti are modulated by the Mosquito’s RNA interference pathway. PLoS Pathogens 5:2:e1000299. Additional Supporting Information may be found in the online version of this article: Additional Supporting Information may be found in the online version of this article: Figure S1. Ampliﬁcation and melting curves for several cecropin A primers tested that failed to meet either qPCR efﬁciency or speciﬁcity criteria. 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Vezilier, J., A. Nicot, S. Gandon, and A. Rivero. 2012. Plasmodium infec- tion decreases fecundity and increases survival of mosquitoes. Pro- ceedings of the Royal Society of London, Series B: Biological Sciences 279:4033–4041. Vezilier, J., A. Nicot, S. Gandon, and A. Rivero. 2010. Insecticide resis- tance and malaria transmission: infection rate and oocyst burden in Culex pipiens mosquitoes infected with Plasmodium relictum. Malaria Journal 9:1:379. Figure S2. Injection treatment and mosquito insecticide resistance status effects on the control (g6pdh) gene expression. Box and whisker plot of the median CT values (horizontal black bars) at which the control g6pdh gene was found to reach its optimal ﬂuorescence threshold after Vizioli, J., P. Bulet, J. A. Hoffmann, F. C. Kafatos, H. M. Muller, and G. Dimopoulos. 2001. Gambicin: a novel immune responsive antimicro- 508 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 © 2012 The Authors. Published by Blackwell Publishing Ltd 6 (2013) 497–509 no injection (NI), injection of physiological saline (Ringer), or injection of lipopolysacharide (LPS). Boxes below and above the median indicate the ﬁrst and third quartiles, respectively. Dashed lines delimit 1.5 times the interquartile range on both side of the box, above which individual counts are considered outliers and marked as dots. (A–B) Mosquito injection was found to slightly increase g6pdh expression by 0.41 0.15 cycles on average (F2,206 = 3.9521, P = 0.02 069) in the isogenic mos- quito experiment only (F2,149 = 1.8983, P = 0.1534 for the ﬁeld-caught mosquito experiment). This marginal (albeit statistically signiﬁcant) Insecticide resistance and mosquito immunity Insecticide resistance and mosquito immunity Vezilier et al. no injection (NI), injection of physiological saline (Ringer), or injection of lipopolysacharide (LPS). Boxes below and above the median indicate the ﬁrst and third quartiles, respectively. Dashed lines delimit 1.5 times the interquartile range on both side of the box, above which individual counts are considered outliers and marked as dots. (A–B) Mosquito injection was found to slightly increase g6pdh expression by 0.41 0.15 cycles on average (F2,206 = 3.9521, P = 0.02 069) in the isogenic mos- quito experiment only (F2,149 = 1.8983, P = 0.1534 for the ﬁeld-caught mosquito experiment). This marginal (albeit statistically signiﬁcant) 509
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https://openalex.org/W4292536540	https://zenodo.org/records/7013561/files/ICHA_38.pdf	English	null	Assessing the accuracy of Visible Infrared Imaging Radiometer (VIIRS) and MultiUltrahigh Resolution (MUR) Sea-Surface Temperature products for inclusion of a model to forecast blooms of Alexandrium catenella in south-central Alaska	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,614	Assessing the accuracy of Visible Infrared Imaging Radiometer (VIIRS) and MultiUltra- high Resolution (MUR) Sea-Surface Temperature products for inclusion of a model to forecast blooms of Alexandrium catenella in south-central Alaska Timothy T. Wynne1, Steven R. Kibler2, Alexandria Sabo2,3, Kaytee Pokrzywinski 1 National Ocean and Atmosphere Administration, National Centers for Coastal Ocean Sciences, HAB Forecasting Branch, 1305 East-West Highway, Silver Spring, MD 20910, U.S.A; 2 National Ocean and Atmosphere Administration, National Centers for Coastal Ocean Sciences, HAB Forecasting Branch, 101 Pivers Island Rd., Beaufort, NC, U.S.A.; 3 CSS, Inc., 101 Pivers Island Rd., Beaufort, NC, U.S.A. 1 National Ocean and Atmosphere Administration, National Centers for Coastal Ocean Sciences, HAB Forecasting Branch, 1305 East-West Highway, Silver Spring, MD 20910, U.S.A; 2 National Ocean and Atmosphere Administration, National Centers for Coastal Ocean Sciences, HAB Forecasting Branch, 101 Pivers Island Rd., Beaufort, NC, U.S.A.; 3 CSS, Inc., 101 Pivers Island Rd., Beaufort, NC, U.S.A. * corresponding author’s email: timothy.wynne@noaa.gov * corresponding author’s email: timothy.wynne@noaa.gov Keywords: water temperature, sea surface temperature, Alaska, Alexandrium catenella, forecasting Abstract The toxic harmful algal bloom species Alexandrium catenella has been observed to bloom in Alaskan coastal waters when sea surface temperatures (SST) exceed approximately 8oC. Consequently, acquiring, fine scale SST data for Alaskan coastal waters represents a critical first step in developing ecological models capable of predicting the occurrence of toxic Alexandrium blooms in this region. Remotely sensed satellite SST records represent the most comprehensive SST data set, but before those data can be used, the satellite data require validation which was the goal of this study. Specifically, we compare a remotely sensed monthly sea surface climatological data set produced by NASA’s Jet Propulsion Laboratory with seven meteorological buoys provided by the National Ocean and Atmosphere Administration (NOAA) National Data Buoy Center (NDBC). The comparisons were done on a point-to-pixel basis as well as an aerial estimation method. The selected study area, the Kodiak Archipelago in Alaska (U.S.A.), is characterized as possessing a varied coastline, filled with a number of coves and embayments. Three of the NDBC buoys are located in embayments and an additional one is in the ~40 km wide Shelikof Strait, with the remaining three offshore. The remotely sensed and in situ measurements were highly correlated providing the basis for the extraction fine scale SST data for the Alaska region over the past 10+ years which can be subsequently incorporated into bloom prediction models. ater temperature, sea surface temperature, Alaska, Alexandrium catenella, ywords: water temperature, sea surface temperature, Alaska, Alexandrium c Keywords: water temperature, sea surface temperature, Alaska, Alexandrium catenella, forecasting 238 Sea Surface Temperature Data Sea Surface Temperature Data Monthly SST data were acquired from NOAA PolarWatch (2021), from NASA Jet Propulsion Laboratory (JPL) Multi-scale Ultra-high Resolution (MUR) SST product. This is a global product combining an array of data, including microwave data, and mapped to a 0.01o spatial resolution (JPL, 2021). It should be noted that part of the data stream into the MUR modelled SST product was from the NDBC buoys (Chin et al., 2017). Daily Sea Surface Temperature (SST) was acquired from the NOAA Coastwatch program from the Visible Infrared Imaging Radiometer (VIIRS) at a spatial resolution of 750 m (Coastwatch, 2021). Fig. 1. Locations of the NDBC buoys used in this manuscript. The polygon shown is what was used to extract the data from the MUR imagery for the comparison shown in figure 3. Material and Methods Sea-Surface Temperature (SST) is one of the most easily quantifiable environmental parameters in the marine environment, and one of the most used in biophysical modeling exercises. Many biological properties are influenced by SST, including phytoplankton growth rates and nutrient uptake kinetics (Lomas and Glibert, 1999). Work by Vandersea et al. (2017) found temperature was the factor best predicting when toxic Alexandrium catenella blooms occur in Alaskan coastal waters. If accurate models predicting the likelihood of Alexandrium blooms are to be developed, it is imperative to have reliable and detailed estimates of SST for this region. Synoptic remote sensing represents the most robust way to quantify this parameter. Consequently, an intensive validation for satellite sensed Alaskan sea surface temperatures was undertaken as the first key step in developing predictive models for A. catenella bloom occurrence. Water Temperature Data The water temperature (WTMP) data were acquired from the National Data Buoy Center (NDBC, 2021). These data are collected as frequently as every quarter of an hour. Daily and monthly means were calculated with missing data values discarded. Seven stations were considered that are in the vicinity of the Kodiak Archipelago (Fig. 1). Comparison Methods The MUR monthly data were compared to the NDBC water temperatures in two ways. The first was a traditional point-to-pixel matchup. A 3 x 3 box of pixels was extracted from each monthly image and the median of this 3 x 3 box was calculated and compared directly with the monthly median water temperature from the NDBC data. Fig. 1. Locations of the NDBC buoys used in this manuscript. The polygon shown is what was used to extract the data from the MUR imagery for the comparison shown in figure 3. The second method was adopted by Wynne et al. (2021) where a polygon was drawn around the seven buoys in figure 1 and then all of the 239 Fig. 3. Relationship between the monthly NDBC water temperature and the monthly MUR SST from the integrated technique (orange line). Each month is reduced to a single point. included data were extracted and averaged. This polygon is shown in figure 1. In this manner, each month had a single point. This average was compared to the mean of the seven different NDBC stations. This process was termed the integrated method by Wynne et al. (2021). The mean daily water temperature was calculated from the NDBC data. A 3 x 3 box around the point of interest was extracted from the daily VIIRS SST. The median was calculated from the extracted points. A daily matchup was achieved if a single point from the 3 x 3 matchup was available. Fig. 3. Relationship between the monthly NDBC water temperature and the monthly MUR SST from the integrated technique (orange line). Each month is reduced to a single point. Results and Discussion A least squares regression from the point-to- pixel match-ups was used to assess differences in monthly MUR data (Fig. 2). The regression line fit well, indicating relatively low error and bias. Using this method, the monthly mean NDBC water temperature data and the monthly MUR SST data were also compared using a least- squares regression analysis for the integrated technique (Fig. 3). Fig. 2. Regression between the monthly NDBC water temperatures and the combined monthly MUR SST for the stations in figure 1 based on a point-to-pixel matchup technique and the monthly MUR data water temperatures. The daily water temperature and from the NDBC and the daily VIIRS regression is shown in figure 4. The slopes from the monthly point-to-pixel matchup (Fig. 2) and the monthly integrated matchup (Fig. 3) vary by less than 2%. This indicates that the integrated method produces a reliable representation of water temperatures around the Kodiak Archipelago. This could have important ramifications for subsequent HAB forecasting work in that datasets with a course spatial resolution may be sufficient for forecasting across southern Alaska. Fig. 2. Regression between the monthly NDBC water temperatures and the combined monthly MUR SST for the stations in figure 1 based on a point-to-pixel matchup technique and the monthly MUR data water temperatures. Fig. 2. Regression between the monthly NDBC water temperatures and the combined monthly MUR SST for the stations in figure 1 based on a point-to-pixel matchup technique and the monthly MUR data water temperatures. 240 Fig. 4. Correlation between the daily NDBC water temperature and the daily VIIRS SST for the stations in figure 1 based on a point-to-pixel matchup technique. Acknowledgements. Bryan Eder provided support to create figure 1. Jennifer Sveadijian from Coastwatch helped obtain and process the MUR and VIIRS datasets. References Chin, T.M., Vazquez-Cuervo, J., Armstrong, E.M. (2017). Remote Sens. of Environ. 200, 154-169. Coastwatch (2021). //coastwatch.noaa. gov/cw/satellite-data-products/sea-surface- temperature/noaa-coastwatch-cogridded. html. Accessed November 11th, 2021. Fig. 4. Correlation between the daily NDBC water temperature and the daily VIIRS SST for the stations in figure 1 based on a point-to-pixel matchup technique. Fig. 4. Correlation between the daily NDBC water temperature and the daily VIIRS SST for the stations in figure 1 based on a point-to-pixel matchup technique. Lomas, M.W. and Gilbert, P.M. (1999). Limnol. Oceanogr. 44, 556-572. NDBC, National Data Buoy Center (2021). //www.ndbc.noaa.gov. Accessed September 9th, 2021. The slopes between the regression lines of the VIIRS daily imagery and the MUR monthly imagery using the point-to-pixel matchup are also within good agreement varying by ~3%. Vandersea, M.W., Kibler, S.R., Van Sant, S.B., Tester, P.A., et al., (2017). Phycol. 58, 303-320. It appears that the two different SST products both underestimate the water temperatures by approximately 10%. This bias can be corrected relatively easily, by dividing the SST by the slope of the regression line. The results here indicate that the VIIRS daily product, with high spatial resolution (750 m pixel vs 1 km for MUR) shows good agreement with field water temperatures around the Kodiak Archipelago. Similarly, the MUR monthly SST product shows good agreement with NDBC water temperature, and with the high spatial resolution VIIRS data. The results of this analysis indicate ability to provide gap filled coverage would be a good choice for a monthly SST product in the Gulf of Alaska for any potential biophysical model to predict the occurrence of Alexandrium blooms. Wynne, T.T., Mishra, S., Meredith, A., Litaker, R.W., Stumpf, R.P. (2021). Remote Sens. 13, 2305. 241
https://openalex.org/W2052954307	https://ccsenet.org/journal/index.php/elt/article/download/20357/13430	English	null	A Comparison of the Effects of Reflective Learning Portfolios and Dialogue Journal Writing on Iranian EFL Learners’ Accuracy in Writing Performance	English language teaching	2,012	cc-by	9,055	English Language Teaching; Vol. 5, No. 11; 2012 ISSN 1916-4742 E-ISSN 1916-4750 Published by Canadian Center of Science and Education English Language Teaching; Vol. 5, No. 11; 2012 ISSN 1916-4742 E-ISSN 1916-4750 Published by Canadian Center of Science and Education A Comparison of the Effects of Reflective Learning Portfolios and Dialogue Journal Writing on Iranian EFL Learners’ Accuracy in Writing Performance Fatemeh Hemmati1 & Fatemeh Soltanpour1 1 Department of English Language, Tehran Payam-e-Noor University, Tehran, Iran Correspondence: Fatemeh Hemmati, Department of English Language, Tehran Payam-e-Noor University, Tehran, 19395-3697, Iran. Tel: 98-912-378-9248. E-mail: fatemehhemmati2002@yahoo.co.uk Fatemeh Hemmati1 & Fatemeh Soltanpour1 1 Department of English Language, Tehran Payam-e-Noor University, Tehran, Iran Correspondence: Fatemeh Hemmati, Department of English Language, Tehran Payam-e-Noor University, Tehran, 19395-3697, Iran. Tel: 98-912-378-9248. E-mail: fatemehhemmati2002@yahoo.co.uk Received: July 22, 2012 Accepted: August 28, 2012 Online Published: September 6, 2012 doi:10.5539/elt.v5n11p16 URL: http://dx.doi.org/10.5539/elt.v5n11p16 Abstract This study aimed at comparing the effects of reflective learning portfolio (RLP) and dialogue journal writing (DJW) on the Iranian EFL learners’ grammatical accuracy in writing as well as their overall writing performance. 60 Iranian EFL learners between the ages of 17 to 30 who were studying at general English courses were selected based on their performance on the Nelson English Language Test. They were assigned randomly into two experimental groups: DJW and RLP. Each group received 14-sessions of treatment. Two samples of Task 2 of General Module of IELTS were used for the pretests and posttests. Each essay was scored independently by three raters. The final score consisted of the average score of the three raters.The findings revealed that the gains in the RLP group’s grammatical accuracy and overall writing performance were significantly better than that of the DJW group. This could have been due to the influence of reflection with support of a mentor or collaborator as well as the efficacy of intentional learning over incidental one and explicit learning over implicit one. The results have some main implications for syllabus designers, material developers, and language teachers. Keywords: grammatical accuracy, writing performance, dialogue journal, learning portfolio, reflection 1. Introduction Keywords: grammatical accuracy, writing performance, dialogue journal, learning portfolio, reflection 1. Introduction L2 writers usually encounter significant challenges in developing their writing skills (Evans, Hartshorn, McCollum, & Wolfersberger, 2010). As most writing instructors frequently observe and Leki, (1992, as cited in Howrey & Tanner, 2008) stated, the main challenge writing teachers encounter is to see that learners learn from their previous mistakes, and acquire writing fluency as well as accuracy. In spite of the given instructions, learners often show slight or no improvement in their writing. As Howrey & Tanner (2008) argued, learners often do not learn that they need to take serious responsibility for improving their own writing, and often neglect teacher feedback. The accuracy of L2 writing, according to Evans et al. (2010) may be influenced by a number of variables such as the learning environment, learner differences, and instructional methodologies. Among all the mentioned variables, they pointed to the weaknesses in instructional methodologies which may play a significant role in preventing EFL/ESL learners from maximizing their ability to write accurately. As Voit (2009) assured, the dialogue journal could provide a social opportunity for great linguistic achievements. On the other hand, according to Zubizarreta (2009), learning portfolio that requires reflection with support and under the guidance of a collaborator and mentor can be helpful for learner’s acquisition of language. .2 The Base of Dialogue Journal Writing and Reflective Learning Portfolios Halliday and Hasan (1989) emphasized on learning as a social process and believed that there is a strong connection between language and its social context. Halliday’s “social-semiotic perspective” (Lingley, 2005) is relevant to various kinds of studies of interaction including the interaction in the DJW [and probably RLP]. Moreover, the above-mentioned techniques are consistent with Lev Vygotsky’s “sociocultural theory” which assumes that language develops as a result of social interaction. Vygotsky believed in the Zone of Proximal Development (ZPD). He assured that learning takes place through the learner’s participation in completing tasks with a more experienced partner (Kumpulainen & Wray, 2002; Lightbown & Spada, 2006; O’Donoghue & Clarke, 2010). Furthermore, the RLP has roots in “Constructivism” which, based on Razak and Asmawi (2004) and O’Donoghue & Clarke (2010), has great focus on the context and learning process. Constructivists believe that learning always involves both analyzing and transforming new information (O’Donoghue & Clarke, 2010). Students learn in problem-solving environments that challenge their knowledge and encourage them to reflect on what they know and whether or not their knowledge is accurate and profound enough based on the content of the course (Nelson, 2002). Learners are in charge and control of what, when, and how they learn (Driscoll, 2000 and Hannafin, 1992, both cited in Callele, 2008). As a result, they need to be aware of their own thinking and learning processes (Driscoll, 1994, as cited in Razak & Asmawi, 2004). When learners are in charge and control of their learning (Gilbert, 1989, as cited in Callele, 2008), they take responsibility for the quality of it as well (Driscoll, 2000, as cited in Callele, 2008). In addition, the DJW and RLP are in line with Merrill Swain’s comprehensible output hypothesis. Swain (2000, as cited in Lightbown & Spada, 2006) stated that successful second language acquisition depends on learners producing oral or written language. She believed that through collaborative dialogue, which is a cognitive and a social activity, language use mediates language learning. The concept of reflective practice dates back to John Dewey’s notion of reflection (Akbari, 2007; Kocoglu, Akyel, & Ercetin, 2008). Reflection is both an educational outcome and a means to life-long learning. The unique values of reflection need to be realized through educational practices in the learning context (Richards et al., 2008). .2 The Base of Dialogue Journal Writing and Reflective Learning Portfolios Portfolio-based learning, as Elango, Jutti, and Lee (2005) stated, is “an approach firmly rooted in the principles of experiential learning, which is a cyclical process of recording, reviewing/reflecting, and learning from events” (p. 511). 1.3 Dialogue Journal Writing 1.1 The Significance of the Present Study The majority of the Iranian EFL learners are unable to produce a piece of writing that is accurate. This is a challenge for not only the students enrolled in English programs, but for many university students as well. As it was argued by Evans et al. (2010), the weaknesses in instructional methodologies can be an important reason for this problem. As it will be argued below, two techniques of keeping the learning portfolio and DJW are among the techniques that claim to be effective in helping the learners acquire the language skills, and among all the skills and components of the language, they have special focus on improving writing skills. Both techniques try to develop 16 English Language Teaching Vol. 5, No. 11; 2012 www.ccsenet.org/elt the reflective practice through social interactions. The primary concern for the researcher in this study was to see how such an opportunity to write reflectively via writing dialogue journals as well as the opportunity for critical reflection and self-assessment of learning under the guidance and with support of a collaborator and mentor via technique of using learning portfolio is effective in helping the learners improve their writing accuracy as well as their grammatical accuracy in writing. It should be noted that due to the fact that the learning portfolio puts strong emphasis on the reflection and because of the researcher’s interest and attention on this point, the term reflective learning portfolio has been used for the technique of the learning portfolio in this study. 1.2 The Base of Dialogue Journal Writing and Reflective Learning Portfolios 1.2 The Base of Dialogue Journal Writing and Reflective Learning Portfolios 1.4 Reflective Learning Portfolio Moreover, it should be noted that the student assessment portfolios focus on the product, the finished document; however, learning portfolios focus on both process and the product (Zubizarreta, 2008). 1.5 Research Questions and Hypotheses Based on the above-mentioned literature, this study addressed the following research questions: 1. Is there any significant difference between the RLP and DJW in improving Iranian EFL learners’ grammatical accuracy in writing? 2. Is there any significant difference between the RLP and DJW in improving Iranian EFL learners’ overall writing performance? Based on the above questions, the following research hypotheses were formulated: 1. There is no significant difference between the RLP and DJW in improving Iranian EFL learners’ grammatical accuracy in writing. 2. There is no significant difference between the RLP and DJW in improving Iranian EFL learners’ overall writing performance. 2. There is no significant difference between the RLP and DJW in improving Iranian EFL learners’ overall writing performance. 2. Methods writing performance. 2. Methods g p 2. Methods 1.4 Reflective Learning Portfolio The learning portfolio is a flexible tool that involves learners in a process of continuous reflection and collaboration which has focus on selective evidence of learning. It provides an opportunity for both improvement and assessment of students’ learning (Zubizarreta, 2008). Zubizarreta (2009) pointed out that, “deep reflection – not a learning log – is at the very heart of the learning portfolio” (p. xxv). Learners’ reflection on their cognition process is proved as a vital component of education (McCombs 1987; Wolf and Reardon 1996, both cited in Nunes, 2004). Zubizarreta (2009) stated through reflective portfolios, students become involved in self-evaluation and begin to monitor their own progress over time. The learning portfolio can come in different forms such as written text, electronic display, or other creative project (Zubizarreta, 2008). Based on Zubizarreta (2008, 2009), during the process of developing the learning portfolio, there is an interplay among three essential elements of reflection, evidence, and collaboration or mentoring. The mentor attempts to train or develop the learners’ thinking skills and support them in aspects of the ecision making and learning (Malderez, 2009). Documentation and evidence of learning in the model require the students to write reflective narrative. In selecting information to be included in the appendix as the concrete evidence of learning, the students and collaborators establish some criteria in the beginning of the work and apply them during the practice of learning portfolio development (Zubizarreta, 2009). One of the big differences between student assessment portfolios and learning portfolios, based on Zubizarreta (2009), is the learning portfolios’ intentional focus on the students’ learning – what is “left out of the formula in student [assessment] portfolios” (p. 5). Such learning includes not only learning the content of the lesson but also as Zubizarreta (2009) claimed, through developing the learning portfolio, the learners will gain insights into their own learning styles, and also the strategies they can adopt to promote their learning. On the other hand, as Cameron et al. (1998, as cited in Davies & Le Mahieu, 2003) claimed, students’ self-assessment is one essential component in the process of learning portfolio development which is served to promote learning. In learning portfolio, assessment is not the goal, but one means to achieve the real goal which is effective learning. 1.3 Dialogue Journal Writing A dialogue journal, often used as a supplementary activity (Yoshihara, 2008), is a written conversation between a student and teacher who write regularly to each other over a course of study. Students initiate writing. They make decisions about topics, length, style, and format (Peyton, 1993). Thus, the interaction becomes student-generated. The goal is to communicate through writing, not on form (Jones, 1991). Peyton (1993) stated the teacher does not overtly correct errors. Thus, students can write freely, without focusing on form. The teacher’s response in the journal is used as a model of correct English usage. Effective dialogue journal use is a system with three equally important components: “(a) the written communication itself, (b) the dialogic conversation, and (c) the responsive relationship” between a learner and a more competent person in the foreign language (Staton, 1991, p. xvii). Hiemstra (2002) stated that journal writing is a learning method that can help solve problems about the learner’s writing ability. As Jones (1991) believed, improvement of the written forms of language and syntax can be made in at least two ways: First, the willingness to express the thoughts and ideas while taking part in real dialogue may encourage and lead the students to search for the correct use of a grammatical structure, spelling, or meaning of the word. Second, according to Burling (1982) and Krashen (1982) (both cited in Jones, 1991), by 17 Vol. 5, No. 11; 2012 English Language Teaching www.ccsenet.org/elt taking part in a communicative act through writing, students may acquire the written linguistic structures unconsciously. taking part in a communicative act through writing, students may acquire the written linguistic structures unconsciously. Kreeft (1984) argued that writing dialogue journal provides students with a large number of comprehensible texts to read. As a result, it can help students build fluency in writing. Previous studies on the DJW have proved its efficacy in improving the writing skill for reluctant writers (Kreeft, 1984; Reid, 1997), limited English proficiency students (Peyton, Staton, Richardson, & Wolfram, 1990), development of writing fluency (McGrail, 1991) and (Holmes and Moulton, 1997), writing quality, reading comprehension, and writing apprehension (Minjong, 1997), developing language functions (Nassaji and Cumming, 2000 as well as Shuy, 1993, as cited in Yoshihara, 2008), decreasing the grammatical errors (Crumley 1998 as well as Peyton 1986, as cited in Mirhosseini, 2009). 2. Methods 2.1 Participants 2.3 Instrumentation The test 200 B from Book 2 (Intermediate) of “Nelson English Language Tests” was used for homogenizing the students’ general English. No item of the test was excluded; the students answered all 50 items. Based on the authors’ recommendation, the test takers needed to answer at least 30 questions correctly to get the pass mark (Fowler & Coe, 1976). In order to consider the important factors of content validity, face validity, and test comparability that is one of the important factors of internal validity, two samples of the Task 2 of the General Writing Module of IELTS were used for both pretest and posttest of writing. The exam topics required the participants to present and justify their opinions (See Appendix A). It should be noted that the participants in this study had already experienced writing in argumentative format, so they were familiar with the format of the test. Moreover, due to the fact that the criterion-related validity of the test was important, the researcher has tried to use a test that is comparable to a well-established test of writing. To evaluate the learners’ essays, a modified version of Cohen’s (1994) and Jacobs et al.’s (1981) scale was used (as cited in Ghafarian, 2011) (See Appendix B). This scale was used in the study because based on Bacha (2001) and Weigle (2002), analytic scales separate scores which provide the researcher with more useful diagnostic information and a more accurate picture of the individuals’ writing ability, and the researcher can identify writers’ strengths and weaknesses. As a result, as Becker (2010/2011) assured, the reliability of scoring is improved when analytic rubrics are used. 2.1 Participants p The participants were 60 Iranian EFL learners between the ages of 17 to 30 who were selected among a total of The participants were 60 Iranian EFL learners between the ages of 17 to 30 who were selected among a total of The participants were 60 Iranian EFL learners between the ages of 17 to 30 who were selected among a total of 18 Vol. 5, No. 11; 2012 English Language Teaching www.ccsenet.org/elt 121 learners. They were studying at general English courses at Jahad e Daneshgahi of Khaarazmi University in Karaj, Iran. There were 24 women and seven men in the RLP, and 25 women and five men in the DJW group; thus, among 60 participants, there were 48 women and 12 men. They all had already passed eight semesters and were studying at the intermediate level. 2.3 Procedure The participants in both treatment groups named the RLP and DJW had two classes each week. Each class session lasted one hour & 45 minutes. During the study, the students studied the book Top Notch 3 written by Saslow and Ascher (2006). At the beginning of the study, all the participants filled out a questionnaire concerning their personal information (See Appendix C). However, they were assured that their anonymity would be preserved. The participants took the pretest in the second session and the posttest in session 19. Two points should be noted: (1) No student took the tests twice, and (2) The topics of essays were different in pretests and posttests. However, the topics were the same for both experimental groups. The sampling method used in this study was convenience sampling. However, random assignment to groups was used by the researcher. As Mackey and Gass (2005) argued, to assess the feasibility and usefulness of the data collection methods and make the necessary revisions, the whole study was piloted before the beginning of the main study. In order to prevent the possibility of the researcher’s bias and considering the rater reliability, two other raters scored the students’ essays. Each essay was scored independently by the three raters. The final score consisted of the average score of the three raters. 2.2 Sampling Procedures The study was a pretest-posttest as well as a comparison-group one. It was quasi-experimental because the convenience sampling was used based on the participants’ performance on the Nelson English Language Test. However, they were randomly assigned into two experimental groups called the RLP and DJW. Thus, there were two independent variables named the RLP and DJW as well as two dependent variables named grammatical accuracy in writing and overall writing performance. Each of the RLP and DJW groups consisted of 30 participants totaled 60. Both groups were taught by the researcher herself. 3.2 The Nelson Test A Mann-Whitney U test was run to compare the RLP and DJW groups on the Nelson Test in order to prove that both groups enjoyed the same level of general language proficiency prior to the administration of the treatments. Based on the results, it could be concluded that the RLP group (Mdn= 16) did not differ significantly from the DJW group (Mdn= 16.00) on the Nelson Test, (U = 447, z = −.052, ns, r = −.006). Based on these results, it could be concluded that the two groups enjoyed the same level of general language proficiency prior to the main study. 3.1 The Normality Tests In the RLP group, the following tests did not enjoy normal distribution: pretest of grammatical accuracy in writing, posttest of grammatical accuracy in writing, pretest of overall writing performance, and the Nelson Test. Their outcomes were not within the ranges of +/- 1.96. As a result, the data did not enjoy normal distribution (Field, 2009). Pretest of overall writing performance was the only non-normal data in the DJW group. That is why the tests that enjoyed normal distribution were analyzed through the parametric independent t-test, while the other tests which did not meet the assumption of normality were analyzed through the non-parametric test of Mann-Whitney U test. 2.3.1 Reflective Learning Portfolio Every session during the term, the RLP group had a break of about 20 minutes at the end of the session. The participants were required to think about that session: what they studied; what they learned; how they learned; how they felt, what surprised them; what concerned them, etc. and filled out reflection questionnaires related to their learning processes (See Appendix D).The questionnaire also included reflections on mistakes produced in written essays and compositions, or in exercises from books and worksheets. The students cooperated with the teacher or their fellow students and tried to solve their problems. To help the students be familiar with writing 19 English Language Teaching Vol. 5, No. 11; 2012 www.ccsenet.org/elt reflections, thinking aloud method was used in the beginning of the term. The students were asked to save all their work – worksheets done in class, notes kept after reflection on teacher’s feedback to their essays, drafts of their essays, homework and their learning portfolio forms. The treatment in this group also lasted for 14 sessions. What was done (except for the number of treatment sessions) had been suggested by Zubizarreta (2008, 2009) as well as Nunes (2004). 2.3.2 Dialogue Journal Group Every session the DJW group had a break of about 20 minutes at the end of the class. The participants were required to write to their teacher in their notebooks. The learners decided about topic, length, style, and format (Staton, 1991). After the class, the teacher read what the learner had written and wrote back in their journals. According to Mirhosseini’s (2009) suggestion, learners were told not to worry about grammar or spelling, and to focus on expressing their thoughts and feelings freely. On the other hand, Mirhosseini and Peyton (1991) stated that teachers can at times correct written forms in the journals or comment on the communicative aspects of dialogues without inhibiting the dialogue. According to Peyton’s (1991) suggestion, the teacher tried to model the correct usage of the error in her responses. In addition, she occasionally addressed the problem areas common to many students separately in class. The treatment lasted for 14 sessions. 3.3 Pretest of Grammatical Accuracy A Mann-Whitney U test was run to compare the RLP and DJW groups on the pretest of grammatical accuracy in writing in order to prove that both groups enjoyed the same level of grammatical accuracy prior to the administration of the treatments. Based on the results, it could be concluded that the RLP group (Mdn= 11.67) did not differ significantly from the DJW group (Mdn= 11.67) on the pretest of grammatical accuracy, (U = 440.50, z = −.150, ns, r = −.019). Based on these results, it could be concluded that the two groups enjoyed the same level of grammatical accuracy in writing prior to the main study. 3.4 Pretest of Overall Writing Performance 3.4 Pretest of Overall Writing Performance A Mann-Whitney U test was run to compare the RLP and DJW groups on the pretest of overall writing performance in order to prove that both groups enjoyed the same level of overall writing performance prior to the administration of the treatments. Based on the results, it could be concluded that the RLP group (Mdn= 11.53) did not differ significantly from the DJW group (Mdn= 11.61) on the pretest of overall writing performance, (U = 431.50, z = −.276, ns, r = −.035). Based on these results, it could be concluded that the two groups enjoyed the same level of overall writing performance prior to the main study. The Cronbach alpha indices were calculated as inter-rater reliability coefficients. The Cronbach alpha indices were calculated as inter-rater reliability coefficients. 20 Vol. 5, No. 11; 2012 English Language Teaching www.ccsenet.org/elt Table 1. Inter-rater reliability indices Tests Indices Pretest of Grammatical Accuracy in Writing Posttest of Grammatical Accuracy in Writing Pretest of Overall Writing Performance Posttest of Overall Writing Performance .86 .91 .87 .91 As shown in Table 1, the indices ranged from a high of .91 for both the posttest of posttest of grammatical accuracy in writing and overall writing performance to a low of .86 for the pretest of grammatical accuracy. Table 1. Inter-rater reliability indices Tests Indices Pretest of Grammatical Accuracy in Writing Posttest of Grammatical Accuracy in Writing Pretest of Overall Writing Performance Posttest of Overall Writing Performance .86 .91 .87 .91 As shown in Table 1, the indices ranged from a high of .91 for both the posttest of posttest of grammatical accuracy in writing and overall writing performance to a low of .86 for the pretest of grammatical accuracy. 3.6 The first Research Question Because the posttest of grammatical accuracy in writing did not meet the assumption of normality, it was analyzed through the non-parametric test of Mann-Whitney U test. The test was run to compare the RLP and DJW groups on the posttest of grammatical accuracy in writing in order to probe the effect of the two types of writing techniques on the improvement of the grammatical accuracy of the students after the administration of the treatments. Because the posttest of grammatical accuracy in writing did not meet the assumption of normality, it was analyzed through the non-parametric test of Mann-Whitney U test. 3.4 Pretest of Overall Writing Performance The test was run to compare the RLP and DJW groups on the posttest of grammatical accuracy in writing in order to probe the effect of the two types of writing techniques on the improvement of the grammatical accuracy of the students after the administration of the treatments. Table 2. Mann-Whitney U posttest of grammatical accuracy in writing Mann-Whitney U 154.000 Wilcoxon W 619.000 Z -4.695 Asymp. Sig. (2-tailed) .000 Table 2. Mann-Whitney U posttest of grammatical accuracy in writing Based on the results displayed in Table 2, it could be concluded that RLP group (Mdn= 15) outperformed the DJW group (Mdn= 11.67) on the posttest of grammatical accuracy, (U = 154, z = −4.69, s, r = −.60). The p value associated with this z is .000. Based on these results, it could be concluded that the first null-hypothesis as there is not any significant difference between the RLP and DJW in improving the Iranian EFL learners’ grammatical accuracy in writing was rejected. 4. Discussion and Conclusion The research questions in this study were concerned with the significance of differences in the grammatical accuracy as well as overall writing performance between the RLP and DJW groups. The analyses indicated that the RLP group of the EFL Iranian learners outperformed the DJW group in posttests, i.e., the gains in the RLP group’s grammatical accuracy in writing as well as their overall writing performance were significantly better than that of the DJW group. The findings of this study support the scholars’ claim that developing learning portfolios promote reflective thinking. This was supported in previous studies such as (Cardona, 2005; Davies & Willis, 2001; Tillema& Smith, 2000; Wade & Yarbrough, 1996; Winsor & Ellefson, 1995; Zubizaretta, 2004; all cited in Kocoglu et al., 2008) as well as Orland-Barak (2005). On the other hand, the efficacy of the RLP technique in this study are in line with Zubizarreta’s (2009) claim about the importance of using the power of collaboration and mentoring, as well as the findings of some of the previous studies on the effect of collaboration and collaborative learning on writing such as Liang (2002), Ekawat (2010), and Jafari & Nejad Ansari, (2012) as well as the effect of mentoring (Motallebzadeh, 2011). Consequently, the result of this study regarding the RLP supports the positive effects of reflection with support of a mentor or collaborator. That is, reflection with support of a collaborator could have affected the learners’ improvement positively. On the other hand, as it was already explained, the goal of writing dialogue journals is to communicate through writing (Jones, 1991) and as Peyton (1993) stated, the teacher does not overtly correct the student’s writing. Consequently, students can write freely, without focusing on form. That is, the teacher’s response in the journal can be used as a model of correct English usage. According to some researchers such as Burling (1982) and Krashan (1982) (both cited in Jones, 1991), the act of communicating in writing can help learners acquire the written grammatical structures unconsciously. The researchers of the present study tried to provide the learners with the correct model of the errors committed by the subjects in the responses without referring explicitly to the errors. Thus, the technique of DJW included the concepts of incidental learning (Schmidt 1994a, as cited in Hulstijn, 2003) and implicit learning (Deykeyser, 2003) and (Schmidt, 2001). 3.7 The Second Research Question Thus, the null-hypothesis as there is not any significant difference between the RLP and DJW in improving the Iranian EFL learners’ overall writing performance was rejected. p g g p j It should be noted that, the assumption of homogeneity of variances was met (Levene’s F = .46, P = .49 > .05). That is why the first row of Table 4, “Equal variances assumed” is reported. It should be noted that, the assumption of homogeneity of variances was met (Levene’s F = .46, P = .49 > .05). That is why the first row of Table 4, “Equal variances assumed” is reported. Figure 1. Posttest of overall writing performance 4 Di i d C l i Figure 1. Posttest of overall writing performance 3.7 The Second Research Question An independent t-test was run to compare the mean scores of the RLP and DJW groups on the posttest of overall writing performance in order to probe the effect of the two types ofwriting techniques on the improvement of the overall writing performance of the students after the administration of the treatments. Table 3. Descriptive statistics posttest of overall writing performance by groups Group N Mean SD SEM Reflective Learning Portfolio Dialogue Journal Writing 30 30 13.8923 12.9407 .78910 .73591 .14407 .13436 Based on the results displayed in Table 3, it could be concluded that on average, the RLP group (M = 13.89) outperformed the DJW group (M = 12.94). able 3. Descriptive statistics posttest of overall writing performance by groups Table 4. Independent t-test posttest of overall writing performance by groups Table 4. Independent t-test posttest of overall writing performance by groups Levene's Test for equality of variances t-test for equality of means F Sig. t df Sig. (2-tailed) Mean difference Std. error difference 95% Confidence interval Lower Upper Equal variances assumed .46 .49 4.83 58 .000 .95 .19 .55 1.34 Equal variances not assumed 4.83 57.72 .000 .95 .19 .55 1.34 Table 4. Independent t-test posttest of overall writing performance by groups Levene's Test for equality of variances t-test for equality of means F Sig. t df Sig. (2-tailed) Mean difference Std. error difference 95% Confidence interval Lower Upper Equal variances assumed .46 .49 4.83 58 .000 .95 .19 .55 1.34 Equal variances not assumed 4.83 57.72 .000 .95 .19 .55 1.34 21 English Language Teaching Vol. 5, No. 11; 2012 www.ccsenet.org/elt As shown in Table 4, the difference between the mean scores of the RLP and DJW groups was significant (t (58) = 4.83, p< .05); the p-value for this t was .000 (Sig (2-tailed) = .000 < .05); it represented a large-sized effect (r = .53). Thus, the null-hypothesis as there is not any significant difference between the RLP and DJW in improving the Iranian EFL learners’ overall writing performance was rejected. As shown in Table 4, the difference between the mean scores of the RLP and DJW groups was significant (t (58) = 4.83, p< .05); the p-value for this t was .000 (Sig (2-tailed) = .000 < .05); it represented a large-sized effect (r = .53). 4. Discussion and Conclusion On the other hand, literature has supported the importance of attention on learning morphology and syntax (Schmidt, 2001). Van Patten (1994, as cited in Schmidt, 2001) argued that attention is not only necessary but also sufficient for learning L2 structure. Carr and Curran (1994, as cited in Schmidt, 2001, p. 8) assured that “focused attention is required for some types of structural learning”, when the goal of learning 22 English Language Teaching Vol. 5, No. 11; 2012 www.ccsenet.org/elt is learning complicated or ambiguous structures. The technique of RLP in this study had special focus on attention and reflection. The RLP created awareness of the quality of learning among students; as a result, their attention was focused on their errors and problems, and because of the existence of a collaborator or mentor, they had the opportunity to find the answers to their problems. The findings of this study is in line with several previous studies such as Norris and Ortegra (2000) and (Doughty and Williams, 1998; Long and Robinson, 1998; Spada, 1997, all cited in Deykeyser, 2003) which have already proved the efficacy of some kind of attention to form; such attention can be through explicit teaching of grammar and explicit error correction, or through input enhancement which were available for the participants in the RLP group. Moreover, considering the context of Iran, some previous studies such as Dabaghi (2008) and Khatib and Ghannadi (2011) supported the efficacy of intentional learning over incidental one, and the efficacy of explicit learning over implicit one. Thus, the greater efficacy of RLP in this study could have been due to the greater efficacy of intentional learning over incidental one as well as higher efficiency of explicit learning over implicit one. Another important factor regarding the DJW should be noticed here. Although the factor of individual differences was not investigated in this study, based on the literature, it can be stated that individual differences could be a factor which affects the effectiveness of the DJW on improving the students’ language skills. Some of the previous studies such as, Peyton (1990) and Casanave (1994), (both cited in Farrell, 2005) as well as Farrell’s (2005) study revealed that some of the students, but not all improved their sentence accuracy. 4. Discussion and Conclusion It is argued in the literature that individual differences cause the learners to be different, for instance, in their noticing abilities; i.e., some learners may notice some qualities of input more than others (Sawyer & Rants, 2001, as cited in Dörnyei & Skehan, 2003). Considering this point, it can be said that some of the participants in this study might have failed to notice the use of the correct forms in their teachers’ response to their letters. On the other hand, studies such as (MacIntyre & Noels, 1996; Oxford & Nyikos, 1989; Schmidt, Jacues, Kassabgy & Boraie, 1997) (all cited in Schmidt, 2001) proved the existence of strong links between motivation and learning strategies, especially cognitive and metacognitive strategies which focus attention on the aspects of the L2. The aptitude factor is also related to attention. Skehan (1998a, as cited in Schmidt, 2001) stated that “the ability to notice what is in input is one of three factors in foreign language aptitude” (p. 10). Moreover, short-term or working memory capacity is related to attention. The ‘central executive’ component of working memory in the model of Baddley (1986, as cited in Schmidt, 2001) is “explicitly related to attention and responsible for controlling the flow of information into working memory” (p. 10). As a result, the results of this study regarding the using of the DJW might have been due to the individual differences, i.e., only learners with certain type of characteristics may benefit from the DJW and improve their writing fluency and grammatical complexity of their writing. The third factor which should be noted regarding the using of the DJW is the context of Iran which is a context of EFL. The point is that although vast body of literature on the use of journals in ESL settings has concluded that the DJW improves linguistic and writing ability, some of the studies in Japan, such as Casanave’s (1994, as cited in Yoshihara, 2008) and Duppenthaler (2004) revealed that most of the students did not demonstrate considerable improvements in linguistic and writing ability. As the Iranian English learners study in an EFL context, the result of this study concerning the using of the DJW could suggest the existence of differences in the efficacy of the DJW on improving the students’ linguistic and writing ability between the EFL and ESL contexts. 4. Discussion and Conclusion This difference might be due to the lack of sufficient exposure to L2 in the context of the EFL. Therefore, the findings of this study regarding the grammatical accuracy are in line with Yoshihara’s (2008) suggestion which pointed that more research into the DJW needs to be carried out to “deepen our understanding of its effects and whether or not it is equally effective in ESL and EFL contexts” (p. 4). Based on the above-mentioned discussion, it can be concluded that the greater efficacy of intentional learning over incidental one as well as higher efficiency of explicit learning over implicit one could have caused the RLP technique to be more effective than the DJW in improving the participants’ grammatical accuracy in writing as well as their overall writing performance. On the other hand, the factor of individual differences might have caused the DJW to be less effective than the RLP. Finally, learners in the ESL contexts may indicate different results regarding the using of the DJW than the students in the EFL contexts, and such a difference might be due to the lack of exposure to L2 out of the context of classrooms. 5. Implications Syllabus designers and material developers can gain insights from the results of this study. Learning portfolio can be included in syllabus and materials, to enable students to acquire the habit of self-reflection, self-direction, and self-evaluation which are embedded in the portfolio process. Including learning portfolio in the textbooks can help learners enhance their writing skill and probably other skills. University students can benefit from the practice of learning portfolio and develop their writing as well as their ability to think critically through the 23 English Language Teaching www.ccsenet.org/elt Vol. 5, No. 11; 2012 power of reflection, collaborative learning, self-assessment, noticing their multiple intelligences, critical thinking, accepting the challenges, noticing the power of writing for promoting learning, and gaining responsibility in the process of their learning. power of reflection, collaborative learning, self-assessment, noticing their multiple intelligences, critical thinking, accepting the challenges, noticing the power of writing for promoting learning, and gaining responsibility in the process of their learning. Second, this study gives L2 teachers the insight that incorporating the learning portfolio into language learning activities can provide them with a better understanding of the learners’ preferred learning styles, needs, and difficulties; thus, the teachers can adjust instruction to the students’ individual goals, needs, and learning dispositions. Therefore, learning portfolios can provide the teachers with an opportunity to design future instructional strategies, materials and activities that are more meaningful and valuable to the learners, as well as make curricular decisions and choices, which will improve learners’ motivation and involvement in class. Third, the findings of this study might be applicable to the teaching and learning of the other language skills and language components. However, many investigations need to be done to support these implications. Halliday, M. A. K., & Hasan, R. (1989). Language, context and text:Aspects of language in a social semiotic perspective. 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New Jersey: Center for Applied Linguistics (CAL). National Clearing House on Literacy Education (ED333763). Minjong, S. (1997). The effect of dialogue journal writing on writing quality, reading comprehension, and writing apprehension of EFL college students. Retrieved from http://eric.ed.gov/ERICWebPortal/search/detailmini.jsp... (ED410766) Mirhoseini, S. A. (2009). For our learn of English: Dialogue journal writing in EFL education. TarbiatModares University, Tehran, Iran, 24(1), 40-48. Retrieved from http://www.ameprc.mq.edu.au/docs/...journal/.../SeyyedAbdolhamid.pdf Motallebzadeh, K. (2011, April). Mentoring: Key factor for teacher development in Iranian EFL setting. 45th Annual International IATEFL Conference and Exhibition, Brighton, UK. Retrieved from http://iatefl.britishcouncil.org/.../iatefl/.../mentoring_iatefl_2011_khalil __motallebzadeh.ppt 25 English Language Teaching Vol. 5, No. 11; 2012 www.ccsenet.org/elt Nelson, C. P. Jr. (2002, May). Contradictions in learning to write in a second language classroom: Insights from radical constructivism, activity theory, and complexity theory (Doctoral dissertation, University of Texas at Austin). Retrieved from http://repositories2.lib.utexas.edu/handle/2152/812 Norris, J. M., & Oretgra, L. (2000). Effectiveness of L2 instruction: A research synthesis and quantitative meta-analysis. Language Learning, 50, 417-528. http://dx.doi.org/10.1111/0023-8333.00136 Nunes, A. (2004). Portfolios in the EFL classroom: disclosing an informed practice. ELT Journal, 58(4), 327-335. http://dx.doi.org/10.1093/elt/58.4.327 O’Donoghue, T., & Clarke, S. (2010). Leading Learning: Process, themes and issues in international contexts. Abingdon, Oxon: Routledge. Orland-Barak, L. (2005). Portfolios as evidence of reflective practice: what remains untold? Abstract retrieved from http:// 112.137.138.6/jspui/bitstream/.../726/1/TeacherEducation197.pdf Peyton, J. K. (1993). Dialogue journals: Interactive writing to develop language and literacy. National Clearinghouse on Literacy Education. Retrieved from http://www.cal.org/resources/digest/peyton01.html Peyton, J. K., Staton, J., Richardson, G., & Wolfram, W. (1990). The influence of writing task on ESL students’ written production. Abstract retrieved from Peyton, J. K., Staton, J., Richardson, G., & Wolfram, W. (1990). The influence of writing task on ESL students’ written production. Abstract retrieved from http://www.gallaudet.edu/documents/.../Dialogue-Journal-and-Writing-Abstracts.pdf Razak, R. A., & Asmawi, A. (2004). The use of dialogue journal through e-mail technology in developing writing interest and skills. Malaysian Online Journal of Instructional Technology (MOJIT), 1(2), 14-23. Retrieved from http://pppjj.usm.my/mojit/articles/pdf/1204/TheUseofDialogueJournalThroughEmailTechnology.pdf pppjj.usm.my/mojit/articles/pdf/1204/TheUseofDialogueJournalThroughEmailTechnology.pdf Reid, L. (1997). Exploring the ways that dialogue journaling affects how and why students write: An action research project. Abstract retrieved from http://www.gallaudet.edu/documents/.../Dialogue-Journal-and-Writing-Abstracts.pdf Reid, L. (1997). Exploring the ways that dialogue journaling affects how and why students write: An action research project. References Write at least 250 words. You should spend about 40 minutes on this task. Write about the following topic: Using a computer every day can have more negative than positive effects on the children. Do you agree or disagree? Give reasons for your answer and include any relevant examples from your own knowledge or experience. Write at least 250 words. Appendix A: Pretest Topic You should spend about 40 minutes on this task. Write about the following topic: Using a computer every day can have more negative than positive effects on the children. Do you agree or disagree? Give reasons for your answer and include any relevant examples from your own knowledge or experience. Write at least 250 words. Posttest Topic You should spend about 40 minutes on this task. Write about the following topic: In Britain, when someone gets old they often go to live in a home with other old people where there are nurses to look after them. Sometimes the government has to pay for this care. What’s your idea about this custom? Who do you think should pay for this care, the government or the family? Give reasons for your answer and include any relevant examples from your own knowledge or experience. Write at least 250 words. Taken from http://www.ielts.org/pdf/115030_General_Training_Writing_sample_task_-_Task_2.pdf Taken from http://www.ielts.org/pdf/115030_General_Training_Writing_sample_task_-_Task_2.pdf Appendix B: A Modified Version of Cohen’s (1994) and Jacobs, Zinkgraf, Wormuth, Hartfiel, and Hughey’s (1981) Analytic Scoring Scale Level Criteria 5 Advanced-High 4 Advanced-Low 3 Intermediate-High 2 Intermediate-Low 1 Novice Content * Logical Development of ideas * Main ideas, supporting ideas, and examples Effectively addresses the topic and task, using clearly appropriate explanations, examples, and details Addresses the topic and task with using appropriate explanations, examples, and details Addresses the topic and task using somewhat developed explanations and details Limited development in response to the topic and task using inappropriate explanations, examples and details Questionable responsiveness to the topic and task with using no detail or irrelevant explanations Organization * The sequence of introduction, body, and conclusion Use of cohesive devices Well organized and cohesive devices effectively used Fairly well organized and cohesive devices adequately used Loosely organized and incomplete sequencing; cohesive devices may be absent or misused. References Abstract retrieved from h // ll d d /d / / i l l d i i Ab df Rickards, W. H., Diez, M. E., Ehley, L., Guilbault, L. F., Loacker, G., Hart, J. R., & Smith, P. C. (2008). Learning, reflection, and electronic portfolios: Stepping toward an assessment practice. JGE: the journal of general education, 57(1). Retrieved from http://www its uiowa edu/support/portfolio/pdf/ /learning reflection and eportfolios pdf www.its.uiowa.edu/support/portfolio/pdf/.../learning_reflection_and_eportfolios.pdf Saslow, J., & Ascher, A. (2006). Top Notch 3 B. English for today’s world. NY: Pearson Education. Schmidt, R. (2001). Attention. In P. Robinson (Ed.), Cognition and second language instruction (pp. 3-32). Cambridge, UK: Cambridge University Press. Staton, J. (1987). Dialogue Journals. ERIC Digest. ERIC Educational Reports.ERIC Clearinghouse on Reading and Communication Skills Urbana IL. Retrieved from http:// www.ericdigests.org/pre-926/journals.htm Staton, J. (1991). Creating an attitude of dialogue in adult literacy instruction. In J. K. Peyton, & J. Staton (Eds), Writing our lives: Reflection on dialogue journal writing with adults learning English (pp. xiii-xxvi). New Jersey: Center for Applied Linguistics (CAL). National Clearing House on Literacy Education (ED333763). Voit, M. H. (2009). Do dialogue journals with recasts improve the writing skills for adult learners with limited literacy skills? Retrieved from http:// www.hamline.edu/education/pdf/ESLcapstone_mvoit.pdf Weigle, S. C. (2002). Assessing writing. Cambridge, UK: Cambridge University Press. Yoshihara, R. (2008). The bridge between students and teachers: The effect of dialogue journal writing. Language Teacher, 32(11), 3-7. Retrieved from http://jalt-publications.org/files/pdf-article/32.11art1.pdf Zubizarreta, J. (2008). The learning portfolio: A powerful idea for significant learning. The IDEA Center (Paper No. 44). Retrieved from http://dcccd.edu/Employees/Departments/EA/CoreResources/Documents/CompLit/ File3_ePortfolio_Zubizarreta.pdf Zubizarreta, J. (2009). The learning portfolio: Reflective practice for improving student learning (2nded.). San Francisco, CA: Jossey-Bass A Wiley Imprint. 26 Vol. 5, No. 11; 2012 English Language Teaching www.ccsenet.org/elt Appendix A: Pretest Topic Taken from http://www.ielts-exam.net/IELTS-WritingSamples/IELTS_Sample_Writing_General_Task_2_1.pdf Posttest Topic Taken from http://www.ielts.org/pdf/115030_General_Training_Writing_sample_task_-_Task_2.pdf Appendix B: A Modified Version of Cohen’s (1994) and Jacobs, Zinkgraf, Wormuth, Hartfiel, and Hughey’s (1981) You should spend about 40 minutes on this task. Write about the following topic: In Britain, when someone gets old they often go to live in a home with other old people where there are nurses to look after them. Sometimes the government has to pay for this care. What’s your idea about this custom? Who do you think should pay for this care, the government or the family? Give reasons for your answer and include any relevant examples from your own knowledge or experience. Posttest Topic Ideas are disconnected and lack of logical sequencing; inadequate order of ideas No organization and no use of cohesive devices Language in use choice of vocabulary Register Appropriate choice of words and use of idioms Relatively appropriate choice of words and use of idioms Adequate choice of words but some misuse of vocabulary or idioms Limited range of vocabulary, confused use of words and idioms Very limited vocabulary, very poor knowledge of idioms Grammar Sentence-level structure No errors, full control of syntactic variety Almost no errors, good control of syntactic variety Some errors, fair control of syntactic variety Many errors, poor control of syntactic variety Severe and persistent errors, no control of syntactic variety Mechanics Punctuation Spelling Capitalization Indentation Mastery of spelling and punctuation Few errors in spelling and punctuation Fair number of spelling and punctuation errors Frequent errors in spelling and punctuation No control over spelling and punctuation Appendix B: A Modified Version of Cohen’s (1994) and Jacobs, Zinkgraf, Wormuth, Hughey’s (1981) Appendix B: A Modified Version of Cohen’s (1994) and Jacobs, Zinkgraf, Wormuth, Hughey’s (1981) B: A Modified Version of Cohen’s (1994) and Jacobs, Zinkgraf, Wormuth, Hartfiel, and 27 Vol. 5, No. 11; 2012 English Language Teaching www.ccsenet.org/elt Appendix C: Biodata Information Please complete the following information sheet. The information will be used only as background information for the present research study and will be kept strictly confidential. 1. Name ………………………………… 2. Age …………………………………… 3. Grade ………………………………… 4. Where and how long have you studied English? (Check the following.) Elementary School …………………… years Junior High school …………………… years High school …………………… years University …………………… years Institutions …………………… years 5. Have you ever traveled to or lived in an English-speaking country? a) Where? (Please specify) ……………………………………………………………… …………………………………………………………………………………………… b) For how long? (Please specify) ……………………………………………………… …………………………………………………………………………………………… Thank you for your cooperation Appendix C: Biodata Information Appendix D: RLP Questionnaire Name: Date: Session: ► Don’t worry if your answers to these questions overlap or if you feel one question has already been answered in your response to an earlier question. Do try and write something, however brief, in response to each question. Even noting that nothing surprised you or that there were no high or low emotional moments in your learning tells you something about yourself as a learner and the conditions under which you learn. 1. What have I learned? Posttest Topic (Grammar, Vocabulary, Discourse strategies, etc.) What can I do now? 2. How did I feel in the class? 3. What have I learned about myself as a learner? 4. What activities did I like best, and what activities did I like least? 5. What learning tasks did I respond to most easily? 6. What learning tasks gave me the greatest difficulties? 7. What problems do I still have regarding the learning activities or the skills? Why do these problems exist? What should I do to solve them? 8. What questions do I need to ask my teacher or my classmates? 9. Do I feel satisfied with myself regarding my learning? Why / Why not? Appendix D: RLP Questionnaire Name: Date: Session: ► Don’t worry if your answers to these questions overlap or if you feel one question has already been answered in your response to an earlier question. Do try and write something, however brief, in response to each question. Even noting that nothing surprised you or that there were no high or low emotional moments in your learning tells you something about yourself as a learner and the conditions under which you learn. 1. What have I learned? (Grammar, Vocabulary, Discourse strategies, etc.) What can I do now? 2. How did I feel in the class? 3. What have I learned about myself as a learner? 4. What activities did I like best, and what activities did I like least? 5. What learning tasks did I respond to most easily? 6. What learning tasks gave me the greatest difficulties? 7. What problems do I still have regarding the learning activities or the skills? Why do these problems exist? What should I do to solve them? 8. What questions do I need to ask my teacher or my classmates? 9. Do I feel satisfied with myself regarding my learning? Why / Why not? 28
https://openalex.org/W3030226019	https://research-repository.st-andrews.ac.uk/bitstream/10023/20025/1/Gillespie_2020_PLoSONE_Passiveacoustic_CC.pdf	English	null	Passive acoustic methods for tracking the 3D movements of small cetaceans around marine structures	PloS one	2,020	cc-by	9,188	Passive acoustic methods for tracking the 3D movements of small cetaceans around marine structures Douglas GillespieID1, Laura Palmer1, Jamie Macaulay1, Carol Sparling2, Gordon Hastie1 1 Sea Mammal Research Unit, Scottish Oceans Institute, University of St Andrews, St Andrews, Scotland, 2 SMRU Consulting, Scottish Oceans Institute, University of St Andrews, St Andrews, Scotland Douglas GillespieID1, Laura Palmer1, Jamie Macaulay1, Carol Sparling2, Gordon Hastie1 1 Sea Mammal Research Unit, Scottish Oceans Institute, University of St Andrews, St Andrews, Scotland, 2 SMRU Consulting, Scottish Oceans Institute, University of St Andrews, St Andrews, Scotland * dg50@st-andrews.ac.uk a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Gillespie D, Palmer L, Macaulay J, Sparling C, Hastie G (2020) Passive acoustic methods for tracking the 3D movements of small cetaceans around marine structures. PLoS ONE 15 (5): e0229058. https://doi.org/10.1371/journal. pone.0229058 Editor: William David Halliday, Wildlife Conservation Society Canada, CANADA Received: January 28, 2020 Accepted: May 2, 2020 Published: May 29, 2020 Editor: William David Halliday, Wildlife Conservation Society Canada, CANADA Received: January 28, 2020 Accepted: May 2, 2020 Published: May 29, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0229058 Copyright: © 2020 Gillespie 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 A wide range of anthropogenic structures exist in the marine environment with the extent of these set to increase as the global offshore renewable energy industry grows. Many of these pose acute risks to marine wildlife; for example, tidal energy generators have the potential to injure or kill seals and small cetaceans through collisions with moving turbine parts. Information on fine scale behaviour of animals close to operational turbines is required to understand the likely impact of these new technologies. There are inherent challenges associated with measuring the underwater movements of marine animals which have, so far, limited data collection. Here, we describe the development and application of a system for monitoring the three-dimensional movements of cetaceans in the immediate vicinity of a subsea structure. The system comprises twelve hydrophones and software for the detection and localisation of vocal marine mammals. We present data demonstrating the systems practical performance during a deployment on an operational tidal turbine between October 2017 and October 2019. Three-dimensional locations of cetaceans were derived from the passive acoustic data using time of arrival differences on each hydrophone. Localisation accuracy was assessed with an artificial sound source at known locations and a refined method of error estimation is presented. Calibration trials show that the system can accu- rately localise sounds to 2m accuracy within 20m of the turbine but that localisations become highly inaccurate at distances greater than 35m. The system is currently being used to provide data on rates of encounters between cetaceans and the turbine and to pro- vide high resolution tracking data for animals close to the turbine. These data can be used to inform stakeholders and regulators on the likely impact of tidal turbines on cetaceans. PLOS ONE PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 17630/de341ca6-6754-43ed-b1ac-a55aae6ccfaa. Other data have been included in the manuscript. result of vessel collisions [3] and fisheries bycatch [4]. Many countries are now also looking to generate low carbon electricity through the installation of underwater turbines in areas of high tidal flow. A common design, horizontal-axis turbines, broadly resemble small wind turbines mounted on the sea floor. Just as the prevalence of wind farms has raised concerns about the risk to birdlife, tidal turbines have the potential to injure or kill animals through collisions with moving rotors [5]. Several taxa are considered to be vulnerable to these risks, including diving birds, fish, and marine mammals [6]. Funding: This research was funded through a research grant from the Scottish Government as part of the Marine Mammal Scientific Support Program MMSS/002/15. Representatives of the Scottish government and Marine Scotland sat on the project steering committee, overseeing experiment design, and also provided feedback on early drafts of this paper. To understand interactions between animals and structures, information on under-water movements of animals around them is required. A number of technologies including video, infra-red based detection, and radar have been used to detect and track birds and bats around windfarms to inform estimates of collision risk [7]. However, there are inherent challenges associated with measuring the underwater movements of marine animals, particularly in highly energetic and turbid environments. Movement tags have been used to study the under- water behaviour of marine mammals, particularly in response to underwater sound [8,9]. However, difficulties in deploying and recovering tags, limited deployment periods (days or weeks), and the fact that a tagged animal may never visit a structure of interest, mean that for many species tagging programs are unlikely to yield fine scale data at very specific locations. Competing interests: The authors have declared that no competing interests exist. Small cetaceans are highly vocal, using echolocation clicks to actively sense their environ- ment [10]. Harbour porpoises (Phocoena phocoena) produce narrow-band high-frequency clicks with a centre frequency of ~130kHz and a duration of ~77μs [11,12]. Most delphinid species produce broader band echolocation clicks with energies mostly between ~30 and over 100kHz [10]. Unlike harbour porpoise, dolphins also produce whistles which can be highly variable but are mostly between around 5 and 20kHz with durations of less than a second [13,14]. Materials and methods To collect data on the 3D movements of harbour porpoises and other small odontocetes around anthropogenic structures, we designed and built a hydrophone array, and acoustic acquisition and processing system. We then deployed the system and collected data over a two-year period between October 2017 and October 2019. The system was semi-automatic, with real time detectors reducing amounts of stored data by several orders of magnitude, but operator screening of remaining sounds required to select and confirm detections from the stored data. All procedures and data collection were approved by the University of St Andrews School of Biology Ethics Committee (Reference number SEC18014). 17630/de341ca6-6754-43ed-b1ac-a55aae6ccfaa. Other data have been included in the manuscript. Arrays of hydrophones can be used to detect and locate cetaceans underwater [15,16] and it is possible to track the movements of cetaceans in the vicinity of anthropogenic struc- tures [17]. Here we describe a hydrophone system to detect, classify and localise individual cetaceans with a high degree of spatial and temporal accuracy. We describe both the hardware and soft- ware and discuss the key principles and limitations when using hydrophone arrays to localise cetaceans in three dimensions (3D). We then report on the practical performance of the sys- tem through a study where it was deployed on an operational commercial-scale tidal turbine. Introduction Anthropogenic structures have increased in number in the marine environment over the past several decades with increases in oil and gas exploration and extraction, marine aquaculture, and renewable energy [1]. Many of these activities may pose acute risks to marine wildlife; for example, seabirds can be killed by wind turbines [2] and cetaceans can be injured or killed as a Data Availability Statement: Click detection data and the vessels location from the reported Data Availability Statement: Click detection data and the vessels location from the reported calibration trial are available at https://doi.org/10. 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures 17630/de341ca6-6754-43ed-b1ac-a55aae6ccfaa. Other data have been included in the manuscript. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Hydrophone array In principle, four widely spaced hydrophones can provide 3D locations based on time of arrival differences of signals at the four receivers [15]. However small errors in timing estima- tion can introduce ambiguities and large errors, particularly outside the immediate vicinity of the array. Detecting highly directional sounds on widely spaced hydrophones can also be 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures problematic since if an animal is orientated towards one hydrophone, it will generally not be orientated towards the others. Further, echolocation clicks are often produced with short inter-click-intervals (ICI) which makes matching corresponding clicks between hydrophones challenging with widely spaced hydrophones. Small clusters of hydrophones, where the maxi- mum sound travel time between the hydrophones is small compared to typical inter-click intervals, arranged in a tetrahedral pattern, do not suffer from matching ambiguities but can only measure horizontal and elevation angles to a sound source, not range. For 3D localisation, a practical compromise is to deploy multiple small tetrahedral clusters of four hydrophones. Each cluster can measure angles to sound sources and, when a sound is detected on two or more clusters, the sound can be localised in 3D. Twelve hydrophones (in three clusters), each consisting of a 10mm ceramic sphere with 0.8mm wall thickness (S10 hollow sphere from Yujie Technology Ltd, Qunxing-Square, Hua- qiang North Road, Futian district, Shenzhen, Guangdong, China 518028) were potted in ALH Systems NP1480 water clear polyurethane, shaped to mount in the ends of 10 mm diameter steel tubes epoxied into an acetyl base to provide a 15cm spacing between each hydrophone (Fig 1 inset). Hydrophone sensitivity was calibrated against a Reson TC4013 reference hydrophone and was measured to be -210±1 dBre1V/μPa. Short (<20cm) cables from each hydrophone carried signals inside the mounting tubes to a box containing custom built pre- amplifiers with a gain of 30dB. For robustness, the preamplifiers and twisted pair signal and power cable were solid potted into this box using epoxy. The hydrophone supports were bolted to 1cm x 80cm x 80cm high density polyethylene sheets, which could in turn be bolted to other structures. Fig 1. Photograph of three hydrophone clusters installed on a turbine support structure during installation showing the locations of the three hydrophone clusters (circled) and acquisition junction box (in diamond). Hydrophone array Inset are photographs of a hydrophone cowling and a tetrahedral cluster showing the four hydrophones on their supports and the box containing potted preamplifiers (photo SIMEC Atlantis Energy). Fig 1. Photograph of three hydrophone clusters installed on a turbine support structure during installation showing the locations of the three hydrophone clusters (circled) and acquisition junction box (in diamond). Inset are photographs of a hydrophone cowling and a tetrahedral cluster showing the four hydrophones on their supports and the box containing potted preamplifiers (photo SIMEC Atlantis Energy). https://doi.org/10.1371/journal.pone.0229058.g001 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures To protect the hydrophones from physical damage, each cluster was covered with a 50cm diameter and 50cm high 10mm thick polyethylene cowling. High density polyethylene was chosen as it has an acoustic impedance of 2.33 x105gm/cm2.sec (https://www.ndt.net/links/ proper.htm), similar to sea water (impedance 1.5x105gm/cm2.sec). This means that the reflec- tion coefficient of a sound in water hitting the polyethylene will be approximately 0.21, or -13.3dB. Data acquisition and real time processing Analog signals from hydrophone clusters were brought to an underwater junction box containing the signal conditioning and data acquisition system. Preamplifiers (ETEC 150410AQC, Etec aps. Industrivaenget 8, DK-3300 Frederiksvaerk, Denmark) provided a fur- ther 20dB of gain and had two-pole high and low pass filters at 3.1kHz and 190kHz respec- tively. Outputs of the preamplifiers connected to three NI-9222 four channel simultaneous sampling 16-bit acquisition modules contained in a CRIO-9067 controller (National Instru- ments Corporation, Austin, TX 78759, USA). The CRIO was programmed to acquire data con- tinuously on all channels at a sample rate of 500kS/s per channel. Data were compressed in real time using lossless compression [18] and sent via Ethernet to a shore-side PC for processing. Analog signals from hydrophone clusters were brought to an underwater junction box containing the signal conditioning and data acquisition system. Preamplifiers (ETEC 150410AQC, Etec aps. Industrivaenget 8, DK-3300 Frederiksvaerk, Denmark) provided a fur- ther 20dB of gain and had two-pole high and low pass filters at 3.1kHz and 190kHz respec- tively. Outputs of the preamplifiers connected to three NI-9222 four channel simultaneous sampling 16-bit acquisition modules contained in a CRIO-9067 controller (National Instru- ments Corporation, Austin, TX 78759, USA). The CRIO was programmed to acquire data con- tinuously on all channels at a sample rate of 500kS/s per channel. Data were compressed in real time using lossless compression [18] and sent via Ethernet to a shore-side PC for processing. On shore, real time processing was conducted using the open source PAMGuard software [19]. A bespoke acquisition module was written to control the CRIO system, unpack the com- pressed audio data and insert it into the PAMGuard processing chain. A separate “watchdog” program was written which started automatically if the system rebooted (e.g. after a power out- age) and would start or restart PAMGuard in the event of data processing being interrupted for any reason. On shore, real time processing was conducted using the open source PAMGuard software [19]. A bespoke acquisition module was written to control the CRIO system, unpack the com- pressed audio data and insert it into the PAMGuard processing chain. A separate “watchdog” program was written which started automatically if the system rebooted (e.g. after a power out- age) and would start or restart PAMGuard in the event of data processing being interrupted for any reason. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Offline analysis Click classification and event selection. The click detector in PAMGuard was configured to trigger on any transient sound with energy rising more than 10dB above background noise in the 40 to 150kHz frequency band. It made large numbers of false positive detections as well as cetacean echolocation clicks (true positives). False positives can be caused by a variety of sources including flow noise over the structure and hydrophone mounts, operational noise and passing vessels. Transient signals were classified as porpoise clicks if they were between 20μs and 220μs duration, had a peak frequency between 100 and 150kHz and had total energy in the 100 to 150kHz frequency band at least 6dB higher than both the 40 to 90kHz and the 160 to 190kHz bands. Although harbour porpoise clicks are narrow-band and distinct from the other noises, broadband dolphin clicks can be challenging to distinguish from background transients. Key to identifying echolocation clicks is often their occurrence at regular intervals on a consistent bearing. Data were therefore screened manually post hoc using PAMGuard, primarily viewing displays of bearings to sounds derived from the individual hydrophone clusters plotted against time. This follows the methods described for sperm whales in [20], whereby the analyst assigns clicks to ‘events’ based on their consistency of bearing and other properties (waveform, power spectra, amplitude and inter click intervals). Localisation and associated errors. Three-dimensional click localisation was a multi- stage process which, although largely described in [16], had a series of modifications to the ways in which errors on localisations were calculated. Sounds detected on more than one hydrophone cluster can only originate from the same source if the time of arrival difference between those sounds is less than the distance between the clusters divided by the speed of sound; in this case a maximum of 7.4ms. Although small cetaceans generally produce clicks with inter click intervals (ICI’s) greater than this, ICI’s in porpoises can be as low as 2ms [9] PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 4 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures which can cause ambiguities in matching clicks between hydrophone clusters. To resolve this ‘click match ambiguity’, clicks from the different hydrophone clusters were initially divided into groups consisting of clicks arriving within 7.4ms of a click a different cluster. Offline analysis Hence, clicks within a group might originate from the same sound but it was impossible that any of them matched with clicks in other groups [16]. All possible combinations of clicks within each group were then localised and scored according to the number of clusters used and the quality of the localisation. Combinations using all three hydrophone clusters were given a higher score than combinations on only two hydrophone clusters. Combinations using the same number of clusters were scored according to a log likelihood value from the localisation (see below). The highest scored localisation from the group was selected for further analyses. Clicks detected on multiple hydrophone clusters were localised by measuring the Time of Arrival Difference (TOAD) of the signal on each hydrophone pair [21] and then maximising a likelihood based model comparing expected TOADs for putative locations with measured TOADs. For each possible group of clicks, a TOAD was measured for every possible hydro- phone pair. This means that for a click detected on all three clusters, there would be 66 differ- ent time measurements. Localisation estimates are subject to uncertainties caused by errors in the estimation of the TOADs as well as errors in the hydrophone locations and the speed of sound in water. The contributions of these different errors also vary depending upon which hydrophones are used in each measurement and the position of the localisation relative to each hydrophone pair; for example, a TOAD measured at a point in line with a pair of hydrophones will be directly affected by an error on the sound speed measurement, whereas that error will contribute noth- ing if the sound source is equidistant from the two hydrophones. Estimates of the various error components are shown in Table 1. The total expected timing error σtj for each hydrophone pair is stj ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðvart þ varg þ varcÞ q ð1Þ ð1Þ where vart, varg and varc are the variances due to TOAD timing error, geometry error and speed of sound error. The variance vart due to TOAD estimation is calculated as the square of the estimated tim- ing error from Table 1. The variance contribution for geometry errors, varg is dependent on the relative positions of the hydrophones and sound source. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Offline analysis If p is the position vector of the sound source and hj are the locations of the hydrophones, having estimated errors on each Cartesian coordinate δhji, (using the error within clusters if the hydrophones are in the same cluster or the between clus- ters error if the hydrophones are in different clusters) and ei are unit vectors along the x, y and Table 1. Estimates of the different contributors to localisation errors. Source of Error Magnitude Timing Error Estimate within a hydrophone cluster 1 μs Timing Error Estimate between hydrophone clusters 10 μs (differences in the waveform are likely to be greater between clusters making it likely that there will be a larger error) Location of hydrophones within a cluster 1mm in each dimension Relative location of clusters 5cm in each horizontal dimension, 2cm vertically Speed of sound 10m/s https://doi.org/10.1371/journal.pone.0229058.t001 Table 1. Estimates of the different contributors to localisation errors. Source of Error Magnitude Timing Error Estimate within a hydrophone cluster 1 μs Timing Error Estimate between hydrophone clusters 10 μs (differences in the waveform are likely to be greater between clusters making it likely that there will be a larger error) Location of hydrophones within a cluster 1mm in each dimension Relative location of clusters 5cm in each horizontal dimension, 2cm vertically Speed of sound 10m/s https://doi.org/10.1371/journal.pone.0229058.t001 Table 1. Estimates of the different contributors to localisation errors. 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures z axis, then the variance in the time difference of arrival for a hydrophone pair will be z axis, then the variance in the time difference of arrival for a hydrophone pair will be varg ¼ X2 j¼1 X3 i¼1 dhji d ðhj pÞ ei c !2 ð2Þ ð2Þ where c is the speed of sound. where c is the speed of sound. Finally, if δc is the error in the estimated speed of sound then varc ¼ T dc c 2 ð3Þ varc ¼ T dc c 2 ð3Þ Where T is the expected time delay, i.e. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Offline analysis PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 6 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures Fig 2. Localisation error coordinate frame. The figure shows the localisation and error estimate of a single click from the pinger trial (see below) which was detected on the north and the west hydrophone cluster, but not the south one. The largest error is in the estimation of range e1, so this is aligned with a unit vector from the centre of the array. The other (smaller) errors e2 and e3 are aligned with the horizontal and vertical angle measurements. Fig 2. Localisation error coordinate frame. The figure shows the localisation and error estimate of a single click from the pinger trial (see below) which was detected on the north and the west hydrophone cluster, but not the south one. The largest error is in the estimation of range e1, so this is aligned with a unit vector from the centre of the array. The other (smaller) errors e2 and e3 are aligned with the horizontal and vertical angle measurements. https://doi.org/10.1371/journal.pone.0229058.g002 Localisation filtering. For each localisation a χ2 value was also calculated as w2 ¼ X ðTMj TÞ 2 2st2 j ! ð7Þ ð7Þ Generally, if the clicks are correctly matched between each hydrophone cluster and the SNR of detected clicks is sufficient for accurate timing estimation, the χ2 value should be approxi- mately equal to the number of degrees of freedom (i.e the number of TOAD measurements used minus the number of spatial dimensions, i.e. 3). However, if an incorrect combination of clicks is localised, then it is likely that the χ2 value will be markedly higher. It is therefore possi- ble to reject erroneous localisations based on the magnitude of their χ2 value. Offline analysis T ¼ kp h1k kp h2k c ð4Þ ð4Þ Once the errors for each TOAD measurement for a candidate localisation have been esti- mated, it is possible to calculate a log likelihood estimation for that position, where L x; T ð Þ ¼ X j 1 2 log 2pstj ðTMj TÞ 2 2st2 j ! ð5Þ ð5Þ Where TMi is the measured time delay between the jth hydrophone pair. Note that many Log Likelihood estimators drop the first term in Eq 5 since it is constant. Here however, σtj varies with location, so the term is retained. The log likelihood function for each localisation was maximised using a Simplex algorithm [22]. To avoid the problem of the optimisation function finding local rather than the true max- imum, four random starting points were selected for each localisation, with the first start point being the centre of the array and the subsequent ones being offset by a random distance in each dimension using a number drawn from a Gaussian distribution with width equal to the arrays maximum aperture. Bearings to the sound source remain accurate with distance so long as there is a greater than ~10dB signal to noise ratio (SNR) to determine accurate TOAD’s [21]. However, esti- mates of the range to sound sources become increasingly inaccurate with distance from the array. Localisation errors were therefore calculated as a range error, aligned with a unit vector from the centre of the hydrophones to the localisation, a horizontal direction perpendicular to the first and a third direction perpendicular to the other two (Fig 2), these latter two effectively being the horizontal and vertical bearing accuracies. Errors on localisations were estimated from the curvature of the log likelihood function around its maximum value. The error estimator assumes that the overall log likelihood func- tion follows a normal like distribution of the form Ltot p ð Þ ¼ 1 2 log 2psp þ kp p0k 2 s2 p ! ð6Þ ð6Þ ð6Þ Where p0 is the best estimated position and σp is the error on p. It is therefore straightforward to estimate σp in any direction by slightly varying the position p in each direction to calculate changes in Ltot(p) and then inverting Eq 6 to obtain an estimate of σp. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Deployment on an operational tidal turbine 7m horizontally from the turbine centre, the clusters on the northeast and southeast legs were 3.5m east and 3.5m north or south. The overall array dimension was therefore 10.5m (east- west) by 7m (north-south). Hydrophones were mounted at a mean height of 12.3m below the centre of the turbine axis, i.e. 3.3m below the turbine blades and approximately 3.5m from the sea floor. Cabling to the junction box containing the signal conditioning and acquisition system was terminated with dry mate subsea connectors. After deployment, a 48V DC power and CAT6 Ethernet cable from the junction box was plugged into the turbine’s Connection Management System (CMS) using an ROV wet-mate connector. Both the CMS and the turbine nacelle can be removed for maintenance, but it is intended that the TSS (and hence the monitoring equip- ment) will remain in situ for 25 years. The monitoring equipment received power from the turbine’s auxiliary power supply, so it was only possible to operate the system with the turbine in place. Several different PAMGuard modules were configured for the detection of echolocation clicks, dolphin whistles and diagnostic noise measurements. A schematic of the processing chain is shown in Fig 3. To reduce the CPU load, the click detection trigger was only run on the topmost hydrophone in each cluster, but in the event of a trigger, a short clip of data from all four hydrophones in that cluster would be stored starting 100 samples (equivalent to 0.2ms or 30cm travel distance) prior to the trigger and ending 100 samples after the end of the trigger to ensure signal capture on all hydrophones in that cluster. The high frequency data (500kHz) were also decimated to 48kHz and passed to the PAMGuard whistle detector [23] to detect dolphin whistles. The click detector trigger recorded an instantaneous measure of its background noise mea- surement once per second, which gives a direct assessment of the click detectors absolute threshold in varying noise conditions. In addition, the spectrogram of the 500kHz data was calculated with a Fast Fourier Transform (FFT) length of 1024 samples (2ms) and an advance of 20480 samples (40ms) and the power of the FFT’s averaged and stored every 6 seconds by a Long-Term Spectral Average (LTSA) module. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Deployment on an operational tidal turbine The 12 hydrophone system was deployed on an operational tidal turbine in the Pentland Firth (58˚39’N 3˚08’W), Scotland in 2016. The turbine was part of an array (the MeyGen project) comprised of three Andritz Hydro Hammerfest HS1000 turbines and the one Atlantis Resources AR1500 (https://simecatlantis.com/). The hydrophone system was integrated into the Atlantis AR1500 turbine which has a three-bladed 18m diameter rotor rotating at speeds up to 14rpm. A yaw mechanism maintained the turbine in a position facing the current, which reaches speeds up to 5ms-1 on both the flood and the ebb tides. The deployment was governed by the logistical constraints of installing equipment close to large and complex machinery in a highly energetic environment. Health and safety concerns made it undesirable to use divers during installation so all array hardware was attached to the Turbine Support Structure (TSS), which is a large (25 x 19m) three-legged steel frame that gravity mounts on the sea floor, prior to installation (Fig 1). One hydrophone cluster was installed on the upper surface of each leg (Fig 1), each cluster being as far from the turbine centre as practically possible. The cluster on the westerly leg was PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 7 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures 7m horizontally from the turbine centre, the clusters on the northeast and southeast legs were 3.5m east and 3.5m north or south. The overall array dimension was therefore 10.5m (east- west) by 7m (north-south). Hydrophones were mounted at a mean height of 12.3m below the centre of the turbine axis, i.e. 3.3m below the turbine blades and approximately 3.5m from the sea floor. C bli h j i b i i h i l di i i d i i i Fig 3. Block diagram of PAMGuard processing chain. https://doi.org/10.1371/journal.pone.0229058.g003 Fig 3. Block diagram of PAMGuard processing chain. https://doi.org/10.1371/journal.pone.0229058.g003 https://doi.org/10.1371/journal.pone.0229058.g003 7m horizontally from the turbine centre, the clusters on the northeast and southeast legs were 3.5m east and 3.5m north or south. The overall array dimension was therefore 10.5m (east- west) by 7m (north-south). Hydrophones were mounted at a mean height of 12.3m below the centre of the turbine axis, i.e. 3.3m below the turbine blades and approximately 3.5m from the sea floor. Deployment on an operational tidal turbine Octave band noise measurements in the fre- quency range 1.4kHz to 181kHz were also made from the top hydrophone in each of the three PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 8 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures clusters, averaging and storing data every 10s. Sound recording modules could store either or both of the high frequency 500kHz data or the decimated 48kHz data in wav file format. Recordings could be made either continuously or on a programmed schedule. ecordings could be made either continuously or on a programmed schedule. Operation. Storing raw audio data from the 12 hydrophones in uncompressed files would have required one Terabyte of storage per day. Therefore, high frequency acoustic data were only stored during the first month of operation while detector settings were adjusted. Follow- ing this period, a 10s long, 500kHz sample rate recording was made every hour for diagnostic purposes. The primary data output from the system was from the detectors and noise monitor- ing modules described above. These were stored in files on the onshore computer’s hard drive in a proprietary binary data format. The size of these files varies depending on numbers of detections and levels of operational noise but were typically around 3 Gigabytes per day. Bi-weekly checks of the system were made using remote desktop software. The network connection was insufficient for data transfer, so data were copied to portable hard drives and sent by post to our lab once per month for permanent storage and further analysis. Results The system was deployed on 24th October 2016. Due to problems with the power supply to our equipment, data collection was only able to start on 19th October 2017 and then continued until the turbine was removed for maintenance on 22nd September 2018. During this initial 338 days of operation, the PAM system was operational for 322 days. Twelve days were lost due to power being unavailable either at the turbine or in the sub-station, and four days were lost due to software or computer failures. Continuous PAM data collection resumed on 18th December 2018 and continued until 15th October 2019. After the first month of operation, one hydrophone became notably noisier than the other eleven. We believe that this is electrical noise, probably caused by one half of a differential amplifier input becoming disconnected. Although some louder signals are still visible on this hydrophone, only the other eleven hydro- phones were used in localisation. A typical set of click waveforms recorded on the three hydrophone clusters is shown in Fig 4. Echoes of the signals are visible on several channels, typically occurring between 100 and 200μs after the initial signal. This delay is consistent with echoes from objects between 7.5 and 15cm away so probably came from the hydrophone mounting structures. Localisation trial To measure the localisation and error estimation accuracy of the system, a series of porpoise like sounds were played back to the system from a drifting vessel using the system described in [16]. Localisations from sounds detected on the hydrophones were directly compared with the known vessel position and depth of the sound source. For safety reasons, close approaches to the turbine were made when the turbine was not rotating and the blades were stationary in the ‘Y’ position to minimise the maximum tip height in the water column. Drifts were made with the sound source above the blade tips at a depth of either 10 or 15m. The vessel was located using a GlobalSat BU-353-S4 WAAS enabled Differential GPS with an error of less than 3m 95% of the time. Drifting with the current meant that the sound source should be directly beneath the vessel; however, due to water currents and wind action it was possible that the sound source was offset from the vessel location by several metres. Localisation accuracy Fig 5 shows the horizontal range and vertical localisation accuracy for porpoise-like sounds measured during the calibration trial as a function of horizontal distance from the turbine PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 9 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures Fig 4. A typical harbour porpoise click detected on all three hydrophone clusters. Dotted lines show the regions where no data are recorded for each channel and solid lines show the short sections of stored waveform. Small time delays between signal arrivals within each hydrophone cluster and larger delays between signal arrivals on the different clusters are clearly visible (hydrophone 7 is not working). This group of sounds was localised to a position approximately 15m E and 9m N of the array centre. https://doi.org/10.1371/journal.pone.0229058.g004 Fig 4. A typical harbour porpoise click detected on all three hydrophone clusters. Dotted lines show the regions where no data are recorded for each channel and solid lines show the short sections of stored waveform. Small time delays between signal arrivals within each hydrophone cluster and larger delays between signal arrivals on the different clusters are clearly visible (hydrophone 7 is not working). This group of sounds was localised to a position approximately 15m E and 9m N of the array centre. https://doi.org/10.1371/journal.pone.0229058.g004 https://doi.org/10.1371/journal.pone.0229058.g004 centre. Within 20m of the turbine, horizontal accuracy was within 1 or 2m. Vertical accuracy is poorer than horizontal accuracy with errors of up to 5m even close to the turbine. Beyond a horizontal distance of 35m, both horizontal and vertical localisation errors increased signifi- cantly to 10’s of metres. During the trial the sound source was above the blades, at least 24m above the hydrophones, so it is likely that localisation errors would be smaller within the area swept by the rotors, which is closer to the hydrophones. System noise Noise within the system varied with both the tidal flow and with turbine operations. Fig 6 shows power spectral data over one day of operation along with individual power spectra for a Fig 5. Measured range and depth errors as a function of horizontal distance. https://doi.org/10.1371/journal.pone.0229058.g005 Fig 5. Measured range and depth errors as a function of horizontal distance. https://doi.org/10.1371/journal.pone.0229058.g005 Fig 5. Measured range and depth errors as a function of horizontal distance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 10 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures Fig 6. System noise over a 24 hour period. Panel A shows spectral data over the 24 hours. The four periods of tidal flow are clearly visible. Panel B shows the power spectral density at three different times during the cycle. Panel C shows the noise in the click detector detection band. The turbine operated up until 14:20 at which point generation was paused. Clearly visible are elevated noise levels at high flows over a wide frequency range. When the turbine is operating an additional noise source is evident at 20 kHz. https://doi org/10 1371/journal pone 0229058 g006 Fig 6. System noise over a 24 hour period. Panel A shows spectral data over the 24 hours. The four periods of tidal flow are clearly visible. Panel B shows the power spectral density at three different times during the cycle. Panel C shows the noise in the click detector detection band. The turbine operated up until 14:20 at which point generation was paused. Clearly visible are elevated noise levels at high flows over a wide frequency range. When the turbine is operating an additional noise source is evident at 20 kHz. https://doi org/10 1371/journal pone 0229058 g006 https://doi.org/10.1371/journal.pone.0229058.g006 https://doi.org/10.1371/journal.pone.0229058.g006 time when the turbine was off and a time when it was generating. Also shown is the noise in the 40–150kHz click detection band. There is an increase and decrease in noise over a wide fre- quency range corresponding to the increasing and decreasing tidal flow, with levels being approximately 20dB higher at peak flow compared to slack water. We believe that this noise is caused by water flow over the turbine and hydrophone structures. An additional noise is pres- ent at around 20kHz that differs from the flow noise: it has a sudden start and end and it’s amplitude is constant despite changes in flow speed. System noise It is believed that this noise is caused by electromagnetically excited vibrations (‘coil whine’) in the turbine generating system [24]. From phase differences in the 20kHz signal on different hydrophones, it is apparent that this noise is mechanical and not electrical. Animal detections During the initial 322 days of data collection (October 2017 –September 2018), over 740 mil- lion transient sounds were recorded on the three hydrophone clusters. During offline analysis, 1044 porpoise and 31 dolphin events were marked by the analyst, containing 115380 and 57077 individual clicks respectively. Of these events, 724 porpoise and 26 dolphin events had 10 or more clicks (Table 2). Of all detected transients, only 0.02% were considered to be por- poise or dolphin sounds. Numbers of porpoise clicks per event varied considerably with a mean of 220 (95% CI 31–979). Similarly, durations of events varied from 0.5 to over 2,700 s (95% CI 21–1,200 s). It is likely that some of these events contained more than one animal. Daily encounter rates of both harbour porpoises and dolphins varied markedly by month, with both being low in the summer months and higher in autumn and winter (Fig 7). Many events yielded few localisations, with the similarity of bearings from the different hydrophone clusters indicating that the animals were passing at distances too great for accurate localisation. Fig 8 shows the track of a porpoise passing close to the turbine. Work is ongoing to further analyse the location data in order to understand the fine scale movements of animals close to the turbine. Discussion This study has shown that the PAM system described here can detect and localise small ceta- ceans within tens of metres of anthropogenic structures with a high degree of spatial accuracy. Calibration trials demonstrated that the system was capable of tracking high frequency sounds 11 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Passive acoustic monitoring of marine mammals around anthropogenic structures PLOS ONE Table 2. Porpoise and dolphin encounter rates for the first year of monitoring. Numbers in parenthesis for the numbers of animals per day are the standard error on the mean encounter rate calculated from the variance in the daily counts for each month divided by the number of monitoring days. Month Days operational Porpoise encounters Dolphin encounters Porpoise / day Dolphin / day Oct 2017 12.26 35 5 2.9 (0.9) 0.41 (0.14) Nov 2017 27.03 95 4 3.5 (0.6) 0.15 (0.07) Dec 2017 30.99 126 1 4.1 (0.5) 0.03 (0.03) Jan 2018 28.89 122 1 4.2 (0.5) 0.03 (0.04) Feb 2018 27.79 46 0 1.7 (0.2) 0.00 Mar 2018 25.53 47 0 1.8 (0.4) 0.00 Apr 2018 28.39 39 0 1.4 (0.2) 0.00 May 2018 30.92 19 0 0.6 (0.2) 0.00 Jun 2018 28.78 19 0 0.7 (0.1) 0.00 Jul 2018 30.36 46 1 1.5 (0.2) 0.03 (0.04) Aug 2018 30.62 54 4 1.8 (0.3) 0.13 (0.08) Sep 2018 20.26 76 10 3.8 (0.8) 0.49 (0.16) https://doi org/10 1371/journal pone 0229058 t002 with approximately 1-2m accuracy within 30m of the array. By connecting to power and opti- cal fibres in the turbines export cable, it was possible to operate the system for two years with a high level of reliability and low level of operator intervention (>99% up time when power was available). The longevity of the deployment has led to the collection of 1044 porpoise and 31 dolphin events over a 12 month period. with approximately 1-2m accuracy within 30m of the array. By connecting to power and opti- cal fibres in the turbines export cable, it was possible to operate the system for two years with a high level of reliability and low level of operator intervention (>99% up time when power was available). The longevity of the deployment has led to the collection of 1044 porpoise and 31 dolphin events over a 12 month period. https://doi.org/10.1371/journal.pone.0229058.t002 Discussion While our results show that the system can accurately localise cetacean clicks, few whistles were detected during the deployment and we have made no attempt to localise the few that Fig 7. Dolphin and porpoise detections by month over a one year period. https://doi.org/10.1371/journal.pone.0229058.g007 Fig 7. Dolphin and porpoise detections by month over a one year period. https://doi.org/10.1371/journal.pone.0229058.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 12 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures Fig 8. Example track of a porpoise passing below the disc swept by the turbine rotors during a 160 second encounter. Points are coloured by time. Note the three points that do not lie on the clear animal track, which are probably caused by mismatched clicks on the three hydrophone clusters. At the time of this event, the current speed was .3m/s, the turbine was rotating, but not generating. https://doi.org/10.1371/journal.pone.0229058.g008 Fig 8. Example track of a porpoise passing below the disc swept by the turbine rotors during a 160 second encounter. Points are coloured by time. Note the three points that do not lie on the clear animal track, which are probably caused by mismatched clicks on the three hydrophone clusters. At the time of this event, the current speed was .3m/s, the turbine was rotating, but not generating. https://doi.org/10.1371/journal.pone.0229058.g008 https://doi.org/10.1371/journal.pone.0229058.g008 https://doi.org/10.1371/journal.pone.0229058.g008 were detected. In principle, time of arrival differences between hydrophones for whistles can be extracted [25,26] and could then be used in the same 3D localisation algorithm. However, extracting time delays from these lower frequency sounds to the same level of accuracy as can be achieved for clicks, particularly in higher noise levels at lower frequencies, may not be possi- ble in which case the same levels of 3D localisation accuracy would not be achievable for whis- tles. Detecting and localising whistles during periods of tidal flow and turbine operation, when noise levels are high in the whistle frequency band would be extremely challenging. It is also important to consider the limitations to the range at which cetaceans can be detected. This is a function of background noise, which varies considerably with environmen- tal conditions or anthropogenic activities. Here, noise varied by 20dB over the tidal cycle and with turbine operation, meaning that cetacean detection probability would be lower during peak tidal flows. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Discussion This is important here because high flow periods coincide with turbine rota- tion and hence when there may be risk of collision. We do not know from these data whether the noise is transmitted to our hydrophones through the steel turbine support structure and how much of the noise is present in the water column, which might alert animals to the presence of the turbine. Measured in-water noise levels around the turbine using drifting hydrophones showed that the 20kHz noise present in our data is higher than ambient noise levels out to a range of 200m and that lower frequency sounds generated by the turbine are 5dB above ambient levels over 2km from the turbine [27]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 13 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures The overall false positive rate of the click detection system was very high; only 0.02% of detected transients were retained after manual screening. This is unsurprising since the detec- tors were configured to reduce stored data to a manageable quantity rather than to classify with high precision. The task of extracting events took approximately one or two days manual processing for each week of data. To streamline this process for future studies, the annotated data from this first year of data collection could be used to develop automated encounter detection processes which could either be implemented to run offline in or real-time. Real- time processing has the advantage of further reducing the amount of stored data and could also provide the basis of a real-time detection system if required. For example, an ability to detect porpoises in real time around fish farms would allow for seal deterrents to be turned off when porpoises are present, allaying concerns about the effects of acoustic deterrents on por- poises [28]. The system described here was developed to determine whether or not tidal tur- bines pose a risk to cetaceans or whether animals will naturally avoid them. Should mitigation, such as shutting down turbines when animals approach be required, the time available to shut down a turbine following the localisation of an approaching cetacean might be less than 10s. Discussion Implementing fully automatic detection systems across all turbines in a large array, which may number in the hundreds, and engineering a braking system that could repeatedly stop a tur- bine in that time without regular maintenance would be both challenging and expensive. The development of these methods is important in order to understand the potential risks associated with tidal turbines. Current collision risk models [29] are dependent on both broad-scale avoidance and fine-scale evasion of operating devices. While some studies have shown broad scale avoidance [30], there are currently no published studies describing fine scale evasive behaviour close to moving turbine blades. The techniques described in the cur- rent study should provide the means to address this data gap for harbour porpoises and poten- tially other small cetaceans. Although the study described here deployed the system on a tidal turbine, similar arrays could also be used to study fine scale movement of vocalising animals around a range of other anthropogenic activities such as fish farms, ports, or other energy exploration and extraction devices during both construction and operation. We used three tetrahedral clusters of hydro- phones since it suited the geometry of the available mounting structure and reduced cabling and underwater connectors. However, the localisation methods can be applied to hydrophones in almost any configuration so long as they are distributed about the volume of interest and they are spatially close enough to each other that sounds are likely to be received on a sufficient number of receivers. Optimal spacing may also vary for different cetacean species given differ- ences in vocal behaviour and sound characteristics. Passive acoustic monitoring alone is unable to localise animals which cease vocalising and is less likely to detect animals orientated away from the structure. Classification and tracking accuracy may be improved through the integration of other sensor systems on the platform. For example, high frequency multi-beam sonar has proven to be highly effective for the detec- tion and classification of small marine mammals; integrating this would potentially provide a means of detecting and tracking species that vocalise infrequently or not at all [31]. In conclusion, the results presented here show that arrays of hydrophones are an effective means of detecting and tracking small cetaceans out to ranges of tens of metres from anthro- pogenic structures over extended time periods. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 Discussion The system provides an efficient means of reducing data volumes to manageable sizes and provides the basis of an effective long-term monitoring tool for identifying and tracking individual animals in discrete locations. From a conservation and management perspective, the approach can be used for monitoring cetacean movements around potentially high-risk anthropogenic activities or structures such as tidal turbines. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 14 / 16 PLOS ONE Passive acoustic monitoring of marine mammals around anthropogenic structures Author Contributions Conceptualization: Douglas Gillespie, Carol Sparling, Gordon Hastie. Data curation: Laura Palmer. Formal analysis: Douglas Gillespie, Laura Palmer, Jamie Macaulay. Funding acquisition: Carol Sparling, Gordon Hastie. Investigation: Douglas Gillespie, Laura Palmer, Jamie Macaulay. Methodology: Douglas Gillespie, Laura Palmer, Jamie Macaulay, Gordon Hastie. Project administration: Carol Sparling Gordon Hastie Conceptualization: Douglas Gillespie, Carol Sparling, Gordon Hastie. Project administration: Carol Sparling, Gordon Hastie. Project administration: Carol Sparling, Gordon Hastie. Software: Douglas Gillespie, Jamie Macaulay. Software: Douglas Gillespie, Jamie Macaulay. Supervision: Douglas Gillespie, Gordon Hastie. Supervision: Douglas Gillespie, Gordon Hastie. Writing – original draft: Douglas Gillespie. Writing – original draft: Douglas Gillespie. Writing – review & editing: Laura Palmer, Jamie Macaulay, Carol Sparling, Gordon Hastie. Writing – review & editing: Laura Palmer, Jamie Macaulay, Carol Sparling, Gordon Hastie. Software availability Both the PAMGuard data analysis software and the CRIO data acquisition software are open source and are available at https://sourceforge.net/p/pamguard/svn/HEAD/tree/ PamguardJava/ and https://sourceforge.net/p/plabuoy/svn-code/HEAD/tree/ respectively. Acknowledgments The work would also not have been possible without the extensive cooperation of the engineer- ing team at Meygen Atlantis, particularly Lorna Slater, Fraser Johnson and Bruce Mackay. Pre- amplifiers were designed by Mark Johnson. We are also grateful to members of the project steering group for comments on early drafts of this manuscript. References 1. Stojanovic TA, Farmer CJQ. The development of world oceans & coasts and concepts of sustainability. Mar Policy. 2013; 42: 157–165. 2. Dierschke V, Furness RW, Garthe S. Seabirds and offshore wind farms in European waters: Avoidance and attraction. Biol Conserv. 2016; 202: 59–68. 3. Vanderlaan AS, Taggart CT. Vessel collisions with whales: the probability of lethal injury based on ves- sel speed. Mar Mammal Sci. 2007; 23: 144–156. 4. Read AJ, Drinker P, Northridge S. Bycatch of Marine Mammals in U. S. and Global Fisheries. Conserv Biol. 2006; 20: 163–169. 5. Onoufriou J, Brownlow A, Moss S, Hastie G, Thompson D. Empirical determination of severe trauma in seals from collisions with tidal turbine blades. J Appl Ecol. 2019; 56: 1712–1724. 6. Wilson B, Batty RS, Daunt F, Carter C. Collision risks between marine renewable energy devices and mammals, fish and diving birds. Rep Scott Exec Scott Assoc Mar Sci Oban Scotl PA37 1QA. 2006. 7. Desholm M, Fox AD, Beasley PDL, Kahlert J. Remote techniques for counting and estimating the num- ber of bird–wind turbine collisions at sea: a review. Ibis. 2006; 148: 76–89. 8. Johnson MP, Tyack PL. A digital acoustic recording tag for measuring the response of wild marine mammals to sound. Ocean Eng IEEE J Of. 2003; 28: 3–12. 9. 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A comparison of bottlenose dolphin whistles in the Atlantic Ocean: factors promoting whistle variation. J Mammal. 2008; 89: 1229–1240. 14. Quick NJ, Janik VM. Whistle rates of wild bottlenose dolphins (Tursiops truncatus): influences of group size and behavior. J Comp Psychol. 2008; 122: 305. 15. References Watkins WA, Schevill WE. Sound source location by arrival-times on a non-rigid three-dimensional hydrophone array. Deep Sea Research and Oceanographic Abstracts. Elsevier; 1972. pp. 691–706. 16. Macaulay J, Gordon J, Gillespie D, Malinka C, Northridge S. Passive acoustic methods for fine-scale tracking of harbour porpoises in tidal rapids. J Acoust Soc Am. 2017; 141: 1120–1132. 17. Malinka C, Gillespie D, Macaulay J, Joy R, Sparling C. First in-situ passive acoustic monitoring for marine mammals during operation of a tidal turbine in Ramsey Sound, Wales. Mar Ecol Prog Ser. 2018; 590: 247–266. https://doi.org/10.3354/meps12467 18. Johnson M, Partan J, Hurst T. Low complexity lossless compression of underwater sound recordings. J Acoust Soc Am. 2013; 133: 1387–1398. 19. Gillespie D, Gordon J, Mchugh R, Mclaren D, Mellinger D, Redmond P, et al. PAMGUARD: Semiauto- mated, open source software for real-time acoustic detection and localisation of cetaceans. J Acoust Soc Am. 2008; 30: 54–62. https://doi.org/10.1121/1.4808713 20. Lewis T, Boisseau O, Danbolt M, Gillespie D, Lacey C, Leaper R, et al. Abundance estimates for sperm whales in the Mediterranean Sea from acoustic line-transect surveys. J Cetacean Res Manage. 2018; 18: 103–117. 21. Gillespie D, Macaulay J. Time of arrival difference estimation for narrow band high frequency echoloca- tion clicks. J Acoust Soc Am. 2019; 146: EL387–EL392. 22. Nelder JA, Mead R. A simplex method for function minimization. Comput J. 1965; 7: 308–313. 23. Gillespie D, Caillat M, Gordon J, White P. Automatic detection and classification of odontocete whistles. J Acoust Soc Am. 2013; 134: 2427–2437. 24. Le Besnerais J, Lanfranchi V, Hecquet M, Brochet P. Characterization and reduction of audible mag- netic noise due to PWM supply in induction machines. IEEE Trans Ind Electron. 2009; 57: 1288–1295. 25. Janik VM, Parijs SM, Thompson PM. A two-dimensional acoustic localization system for marine mam- mals. Mar Mammal Sci. 2000; 16: 437–447. 26. Quick NJ, Rendell LE, Janik VM. A mobile acoustic localization system for the study of free-ranging dol- phins during focal follows. Mar Mammal Sci. 2008; 24: 979–989. 27. Risch D, van Geel N, Gillespie D, Wilson B. Characterisation of underwater operational sound of a tidal stream turbine. J Acoust Soc Am. 2020; 147. 28. Findlay CR, Ripple HD, Coomber F, Froud K, Harries O, van Geel NCF, et al. Mapping widespread and increasing underwater noise pollution from acoustic deterrent devices. Mar Pollut Bull. 2018; 135: 1042–1050. 29. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229058 May 29, 2020 References Schmitt P, Culloch R, Lieber L, Molander S, Hammar L, Kregting L. A tool for simulating collision proba- bilities of animals with marine renewable energy devices. PloS One. 2017; 12: e0188780. 30. Joy R, Wood JD, Sparling CE, Tollit DJ, Copping AE, McConnell BJ. Empirical measures of harbor seal behavior and avoidance of an operational tidal turbine. Mar Pollut Bull. 2018; 136: 92–106. 31. Hastie GD, Wu G-M, Moss S, Jepp P, MacAulay J, Lee A, et al. Automated detection and tracking of marine mammals: A novel sonar tool for monitoring effects of marine industry. Aquat Conserv Mar Freshw Ecosyst. 2019; 29: 119–130. 16 / 16
https://openalex.org/W4310338872	https://zenodo.org/record/7352279/files/REVIS%C3%83O%20NARRATIVA%20DAS%20PESQUISAS%20EPIDEMIOL%C3%93GICAS%20DE%20LES%C3%95ES%20ESPORTIVAS%20NO%20VOLEIBOL%20FEMININO%20%E2%80%93%20ISSN%201678-0817.pdf	Portuguese	null	REVISÃO NARRATIVA DAS PESQUISAS EPIDEMIOLÓGICAS DE LESÕES ESPORTIVAS NO VOLEIBOL FEMININO	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	3,138	REVISÃO NARRATIVA DAS PESQUISAS EPIDEMIOLÓGICAS DE LESÕES ESPORTIVAS NO VOLEIBOL FEMININO Ciê i d S úd Edi ã 116 NOV/22 / 20/11/2022 Andressa Luísa Teixeira Gomes¹ Bruna Araújo Trindade¹ Cezar Augusto Costa Fernandes¹ Jaíne Dias Leite¹ Tatiane Beatriz Patrocínio da Silva¹ 1 INTRODUÇÃO As lesões no vôlei tiveram um aumento exponencial partindo da década de 1980 devido ao alto volume de novos praticantes. Normalmente podem ocorrer por saltos repetitivos, afetando membros inferiores ou decorrentes de rebatida de bola acima da cabeça afetando membros superiores e constantemente ombros, devido a movimentos de exercícios articulares excessivos. (Eerkes; 2012) Por não ser um esporte que não tem por dinâmica o contato físico entre adversários, o vôlei acaba sendo um esporte, onde lesões ocorrem por quedas mal ajustadas pelas atletas, já que se trata de um esporte que se destaca por saltos repetitivos, seja para atacar ou defender sua quadra. (Gerberich, et at; 2016) O Brasil é uma das potências esportivas no quesito seleções de vôlei feminino no mundo, de acordo com o Ranking da Federação Internacional de Voleibol, classificado na segunda posição do ranking, atualizado em 20 de setembro de 2021, com 366 pontos. Atrás apenas da primeira colocada, a Seleção Norte Americana, que possui 379 pontos. A Seleção Brasileira já foi bicampeã olímpica em 2008 e 2012 nos jogos olímpicos de Pequim e Londres, respectivamente. (FIVB; 2022) O Brasil é uma das potências esportivas no quesito seleções de vôlei feminino no mundo, de acordo com o Ranking da Federação Internacional de Voleibol, classificado na segunda posição do ranking, atualizado em 20 de setembro de 2021, com 366 pontos. Atrás apenas da primeira colocada, a Seleção Norte Americana, que possui 379 pontos. A Seleção Brasileira já foi bicampeã olímpica em 2008 e 2012 nos jogos olímpicos de Pequim e Londres, respectivamente. (FIVB; 2022) As lesões esportivas são mais incidentes em esportes de equipe do que em esportes individuais, sendo lesões traumáticas as de maior incidência, seguidas de perto por lesões ocasionadas por uso excessivo. Sendo assim, se fazem necessários maiores estudos a fim de prevenir tais lesões. (Theisen, et al; 2013) As lesões no vôlei podem ocorrer de diversas formas, dependendo da modalidade, como, por exemplo vôlei de praia ou quadra e, de acordo com o tipo, isso pode alterar o mecanismo de lesão, devido ao tipo de solo onde o esporte é praticado. Por exemplo, as lesões por entorse de tornozelo possuem incidência menor no vôlei de praia, devido ao solo propiciar uma aterrissagem mais macia e, por ter menos atletas em quadra, diminui a possibilidade de realizar a aterrissagem em cima do pé de outra pessoa com resultado de uma entorse de tornozelo. 1 INTRODUÇÃO (Eerkes; 2012) A incidência de casos no voleibol é muito recorrente devido a sobrecarga excessiva causada por fatores intrínsecos e extrínsecos nos MMSS e MMII, porém essas lesões ocorrem com mais frequência nos MMII, mas especificamente no joelho e tornozelo. Esses grupos musculares tendem a sofrer sobrecargas maiores nas articulações, devido ao impacto causado por arremessos, saltos e movimentos repetitivos durante o jogo. (Antônio e Santos; 2012) O voleibol é uma atividade imensamente dinâmica, sendo necessária uma capacitação e preparação especial que busquem potencializar o musculoesquelético, com o intuito de diminuir prováveis lesões no decorrer da disputa e/ou treino prático. As lesões musculoesqueléticas são sintomas crônicos que tem grande potencial de acometer atletas de voleibol, com grande probabilidade de ocorrer por treinos constantes, movimentos repetitivos, e/ou exercícios articulares excessivos. (Stefanello; 2007) Devido à falta de pesquisas relacionadas com o tema, se fez necessária essa revisão narrativa a fim de identificar as lesões mais incidentes no esporte Voleibol e quais seus mecanismos de lesão para servir de parâmetro para profissionais da saúde no que diz respeito a criação de protocolos preventivos de tais lesões dentro do esporte supracitado. Sendo assim, o objetivo do estudo é realizar um levantamento das pesquisas epidemiológicas de lesões no vôlei feminino e por meio de uma revisão narrativa, determinar as lesões e seus segmentos acometidos de acordo com a posição de quadra. 2 OBJETIVOS GERAIS Realizar um levantamento das pesquisas epidemiológicas de lesões no vôlei feminino e por meio de uma revisão narrativa, determinar as lesões e seus segmentos acometidos de acordo com a posição de quadra. 2.1 Objetivos específicos Identificar os mecanismos de lesão mais incidentes a fim de servir de parâmetro para a criação de protocolos de prevenção e discutir os mecanismos de lesões mais comuns para o desenvolvimento de protocolos de prevenção. 3 METODOLOGIA Com o objetivo de obter maiores informações inerentes às lesões do voleibol feminino, por meio de uma revisão narrativa, selecionamos artigos relacionados com pesquisas epidemiológicas de lesões esportivas no voleibol feminino. Para a composição desta revisão narrativa, foram realizadas buscas nas bases de dados EbscoHost, Sciello, Pubmed, Medline e Lilacs utilizando as palavras chave Athletic Injuries – Traumatismos em Atletas, Volleyball – Voleibol, Women or Female – Mulher ou feminino e Epidemiology – Epidemiologia. Os critérios de inclusão consistem em artigos de revisão sistemática e epidemiológica datados de 2012 até 2022 (alguns artigos mais antigos foram inseridos devido a sua relevância perante o tema), atletas femininas de voleibol amadoras e profissionais com idade de 16 a 40 anos. Os critérios de exclusão consistem em artigos anteriores a 2011, atletas masculinos de qualquer idade (com ressalva para artigos que comparam voleibol masculino com feminino) e atletas femininas com menos de 15 anos e maior de 41 anos, artigos de estudo de caso também foram desconsiderados devido ao seu foco um único tipo de lesão. Nas bases de dados EbscoHost, Sciello, PUBMED e Lilacs, foram encontrados 98 artigos e após aplicação dos critérios de exclusão, foram selecionados 13 artigos relacionados com o tema. A base de dados Medline Bireme não apresentou artigos relacionados com o objetivo dessa revisão narrativa. A figura 1 detalha resumidamente o fluxo da pesquisa. Resumindo, ao todo, nas bases de dados propostas, encontramos 98 artigos com as palavras chave propostas e após a aplicação dos critérios de exclusão, finalizamos com 13 artigos selecionados após a retirada dos artigos repetidos. Dos 13 artigos, 3 deles utilizamos apenas para complemento do desenvolvimento da introdução, para análise de dados dos 10 artigos, três foram publicados em português (30%) e sete foram publicados em inglês (70%). Figura 1 – Fluxograma com distribuição de artigos por base de dados. Figura 1 – Fluxograma com distribuição de artigos por base de dados. De acordo com a tabela 1, entre os 10 artigos selecionados, sete apresentaram incidência maior de lesões relacionadas à entorse de tornozelo e três artigos apresentaram incidência em lesões de joelho, sendo por entorse de joelho, disfunção patelofemoral e uso excessivo. Referente às entorses de tornozelos, infelizmente os artigos não descrevem quais os mecanismos de lesão e somente um artigo relaciona a lesão com a posição da atleta em quadra. 3 METODOLOGIA Segundo Antônio e Santos (2012), as entorses de tornozelo são mais frequentes em atletas de ataque, devido aos movimentos de alto impacto (saltos corridas e impactos com solo após aterrissagem). Foram sintetizados os artigos de 2012 a 2021 e os resultados apontaram os mecanismos de lesão com 90% sendo entorse e membros acometidos com prevalência de 70% tornozelo e 30% joelho, porém não existem informações suficientes para determinar entorse em inversão ou eversão. Assim como não há dados o suficiente para determinar posicionamento em quadra de cada atleta lesionado, conforme detalhamento de dados na tabela 1. Sabe-se que o objetivo central do trabalho é realizar um levantamento das pesquisas epidemiológicas de lesões no vôlei feminino e por meio de uma revisão narrativa para determinar as lesões, seus segmentos de acordo com a posição de quadra. Pretende-se com isso desenvolver um protocolo de prevenção de tais lesões no Voleibol. Sabe-se que o objetivo central do trabalho é realizar um levantamento das pesquisas epidemiológicas de lesões no vôlei feminino e por meio de uma revisão narrativa para determinar as lesões, seus segmentos de acordo com a posição de quadra. Pretende-se com isso desenvolver um protocolo de prevenção de tais lesões no Voleibol. 3.1 Tabela ARTIGO AUTORES ANO MEMBRO ACOMETIDO LESÃO P E Q PREVALÊNCIA DE LESÕES EM ATLETAS DE VOLEIBOL FEMININO E POSSÍVEIS RELAÇÕES COM REINAMENTO INADEQUADO E ESTRESSE Antônio, V S; Santos, M A G N. 2012 Tornozelo Entorse S m in n p d a PREVALÊNCIA DE LESÕES MUSCULOESQUELÉTICAS EM ATLETAS ADOLESCENTES Tomazoni, S S; Zanetto, B G S; Leal, E C P J. 2011 Joelho Entorse N s p e q EPIDEMIOLOGY OF SPORTS INJURIES ON COLLEGIATE ATHLETES AT A SINGLE CENTER Rosa, B B; et al. 2014 Tornozelo Entorse N s p e q SPORTS-RELATED INJURIES IN THE YOUNG FEMALE ATHLETE Colvin, A C; Lynn, A. 2010 Tornozelo Entorse N s p ARTIGO e q CHARACTERISTICS AND CONTRIBUTING FACTORS RELATED TO SPORTS INJURIES IN YOUNG VOLLEYBALL PLAYERS Vanderlei, F M; et al. 2013 Tornozelo Entorse N s p e q EPIDEMIOLOGY OF BASKETBALL, SOCCER, AND VOLLEYBALL INJURIES IN MIDDLE- SCHOOL FEMALE ATHLETES Foss, K D B; Myer, G D; Hewett, T E. 2014 Joelho Disfunção patelofemoral N s p e q INCIDENCE, AETIOLOGY AND PREVENTION OF MUSCULOSKELETAL INJURIES IN VOLLEYBALL: A SYSTEMATIC REVIEW OF THE LITERATURE Kilic, O; et al. 2017 Tornozelo Entorse N s p e q DESCRIPTIVE EPIDEMIOLOGY OF INJURIES SUSTAINED IN NATIONAL COLLEGIATE ATHLETIC ASSOCIATION MEN’S AND WOMEN’S VOLLEYBALL, 2013-2014 TO 2014-2015 Baugh, C M; et al. 2018 Tornozelo Entorse N s p e q THE FIRST DECADE OF WEB-BASED SPORTS INJURY SURVEILLANCE: Kerr, Z Y; et al. 2018 Tornozelo Entorse N s p DESCRIPTIVE EPIDEMIOLOGY OF INJURIES IN US HIGH SCHOOL GIRLS’ VOLLEYBALL (2005-2006 THROUGH 2013-2014) AND NATIONAL COLLEGIATE ATHLETIC ASSOCIATION WOMEN’S VOLLEYBALL (2004-2005 THROUGH 2013-2014) e q INJURY EPIDEMIOLOGY AND TIME LOST FROM PARTICIPATION IN WOMEN’S NCAA DIVISION I INDOOR VERSUS BEACH VOLLEYBALL PLAYERS Juhan, T; et al. 2021 Joelho Uso excessivo N s p e q e q Tabela 1 – Artigos selecionados separados por nome, autores, ano, membro acometido e lesão. 4 DISCUSSÃO O presente estudo tem o objetivo de realizar um levantamento das pesquisas epidemiológicas de lesões no vôlei feminino e por meio de uma revisão narrativa determinar as lesões, seus segmentos acometidos de acordo com a posição de quadra, porém, nos resultados apresentados dos artigos selecionados somente Antônio e Santos (2012) indica que a posição mais afetada por lesões seriam atletas da posição de ataque (ponteiras ou opostas). Nos resultados apresentados, oito artigos apresentaram lesões por entorse sendo o tornozelo o membro mais acometido (sete artigos) seguido por Joelho (um artigo). Das demais lesões, dois artigos (Overuse e disfunção patelofemoral), somente o joelho teve incidência levantada. artigo). Das demais lesões, dois artigos (Overuse e disfunção patelofemoral), somente o joelho teve incidência levantada. Kilic et al (2017) alega que existem poucas evidências a respeito de incidências para fatores de risco dentro do vôlei, haja visto que são poucos os estudos que as indicam, porém, faz uma ressalva de que a maioria das lesões costumam ocorrer dentro dos treinamentos. Essa informação vai ao encontro do que é citado por Foss, Myer e Hewett (2014), Baugh et al (2018), Tomazoni, Zanetto e Leal (2011) e Vanderlei et al (2013). Antônio e Santos (2012) também informa no seu artigo de que a incidência de lesão é maior nos treinos (em seu artigo que das 9 atletas lesionadas, 60% se machucaram em treinos e 40% lesionaram-se em jogos), e ressalta que essas lesões podem ter ocorrido por excesso de treino ou treinamento de forma errônea. Nesse estudo, os treinos eram realizados 3 vezes por semana com uma carga horária de 4 horas cada treino, totalizando 12 horas semanais de treinamento. Foi realizado com o Time de Voleibol Feminino da Escola Estadual José Pavan, da cidade de Jacarezinho–PR, com atletas com idade de 12 a 18 anos. Colvin e Lynn (2010) alegam em seu artigo que atletas do voleibol apresentam taxa de lesão de joelho menor do que basquete e futebol, pois as atletas possuem a aterrissarem de maneira mais simétrica, dividindo a descarga de peso nos membros inferiores de forma mais igualitária. Os autores ressaltam ainda que, atletas de voleibol estão mais suscetíveis a lesões de ombro devido a atividade frequente de elevação dos braços acima da cabeça. 4 DISCUSSÃO Importante ressaltar que o artigo não cita estatisticamente a incidência de lesões em tornozelo, mas citam que a utilização de órteses profiláticas para tornozelos (tornozeleiras dupla bilateral acolchoada) podem ser usadas com intuito de diminuir a incidência dessa lesão. Das lesões citadas por essa revisão, a maior incidência foi em tornozelo e joelho com o entorse de tornozelo em destaque, porém o artigo de Tomazoni, Zanetto e Leal (2011) cita entorses de joelho como lesão mais incidente. Nesse estudo, foi realizado um comparativo com outros esportes (futebol, futsal, handebol, atletismo e taekwondo) e o voleibol apresentou índice de lesão maior dentre todos os esportes testados (28%). O estudo cita ainda que, lesões no treinamento do voleibol sao mais incidentes, pois as atletas são expostas a situações de repetitividade constante para aperfeiçoar as técnicas de ataque e defesa e que, por ser um esporte sem contato com o adversário, a taxa deveria ser menor, porem os movimentos sao realizados de maneiras repetitivas, vigorosas e rápidas o que acaba se tornando um dentre os fatores de risco de lesão. Referente ao retorno das atletas a prática esportiva, Juhan et al (2021) fez um comparativo entre voleibol de quadra e de praia e no comparativo a lesão mais incidente foi a de Joelho por overuse no vôlei de quadra, porém as atletas do vôlei de praia precisam de um tempo maior para se recuperar e apresentaram incidência de lesão maior em abdômen e ombro. Baught et al (2018) fez um comparativo em seu artigo entre lesões do vôlei masculino (6 equipes) e feminino (33 equipes) dividindo os resultados em lesões que resultaram períodos maiores e menores do que 24 horas para retorno à prática do esporte. Das 510 lesões relatadas no artigo, 24,3% foram tornozelo e dessas lesões, 31,2% foram entorses. Além disso, foi relatado que as mulheres apresentaram índice maior de lesão por Overuse quando comparado com os homens. Dado importante relatado é de que, das atletas que tiveram entorse de tornozelo e retornaram antes de 24h sofreram a lesão devido a contato com outras jogadoras na aterrissagem. Quanto a posição em quadra, apenas Antônio e Santos (2012) sugeriram a maior incidência de lesão em posições de ataque, porém Kerr et al (2018) informou que seu estudo apresentou lesões por concussão mais incidentes em líberos. 5 CONCLUSÃO De acordo com os artigos selecionados dos anos de 2012 a 2022, esta revisão sugere que a lesão por entorse de tornozelo é a mais incidente no voleibol feminino, sendo mais frequente em treinamentos do que em jogos. Vale ressaltar que a maioria dos artigos cita lesões no voleibol amador, que normalmente não possuem uma equipe multidisciplinar para acompanhar a equipe nos treinamentos, deixando todos os cuidados ao treinador, que pode não dar conta de tantas funções simultâneas. devido a esse fato, se faz necessário a criação de protocolos de acompanhamento multidisciplinar para acompanhar e, caso seja preciso, intervir com o intuito de evitar lesões, principalmente no voleibol amador. Devido ao fato de não identificar todos os fatores de risco, esse estudo sugere que se fazem necessários maiores estudos a respeito do tema, principalmente separando as lesões por posição das atletas na quadra, tendo em vista que as funções são diferentes e os mecanismos de trauma/lesão serão diferentes também. 4 DISCUSSÃO A lesão citada não foi a mais incidente do artigo (entorse de tornozelo foi a primeira colocada), porém relata uma posição defensiva que normalmente está sujeita a recepção de ataques do time adversário. É importante salientar que a maior parte dos estudos selecionados estão ligados ao vôlei amador, e é muito provável que eles não possuam um time multidisciplinar para prevenção dessas lesões (educador físico, fisioterapeuta, nutricionista). Muitas vezes é apenas o técnico quem determina a carga de treinamento e descanso de cada equipe e infelizmente não vai possuir conhecimento necessário ou não vai dar conta de orientar todas as atletas quanto aos riscos e/ou formas de prevenção. Existe também o fato que atletas profissionais normalmente possuírem todo um preparo físico dentro e fora de quadra e muitas das vezes a equipe multidisciplinar está presente reduzindo ou prevenindo fatores de risco para lesões. REFERÊNCIAS Farina, E C R; Mansoldo, A C. INCIDÊNCIA DAS LESÕES EM ATLETAS FEDERADAS NAS CATEGORIAS DE BASE DO VOLEIBOL NO ESTADO DE SÃO PAULO. 2006. Revista digital Buenos Aires. Antonio, V S; Santos, M A G N S. PREVALÊNCIA DE LESÕES EM ATLETAS DE VOLEIBOL FEMININO E POSSÍVEIS RELAÇÕES COM TREINAMENTO Antonio, V S; Santos, M A G N S. PREVALÊNCIA DE LESÕES EM ATLETAS DE VOLEIBOL FEMININO E POSSÍVEIS RELAÇÕES COM TREINAMENTO INADEQUADO E ESTRESSE. 2012. Revista Hórus. Eerkes, K. LESÕES NO VOLEIBOL. 2012. Sports medicine. Stefanello, J. SITUAÇÕES DE ESTRESSE NO VÔLEI DE PRAIA DE ALTO RENDIMENTO: UM ESTUDO DE CASO COM UMA DUPLA OLÍMPICA. 2007. Revista Portuguesa de Ciência do Desporto. ¹Autor(a) Post seguinte → Post seguinte → Post seguinte → ← Post anterior ← Post anterior Gerberich, S G; at al. ANÁLISE DE LESÕES GRAVES ASSOCIADAS A ATIVIDADES DE VOLEIBOL. 2016 Gerberich, S G; at al. ANÁLISE DE LESÕES GRAVES ASSOCIADAS A ATIVIDADES DE VOLEIBOL. 2016 Theisen, D; at al. O RISCO DE LESÃO É DIFERENTE NO ESPORTE JUVENIL INDIVIDUAL E COLETIVO. 2012. Revista de ciências e medicina no esporte. Tomazoni, S S; Zanetto, B G S; Leal, E C P J. PREVALÊNCIA DE LESÕES MUSCULOESQUELÉTICAS EM ATLETAS ADOLESCENTES. 2011. ConScientiaeSaúde. Colvin, A C; Lynn, A. SPORTS-RELATED INJURIES IN THE YOUNG FEMALE ATHLETE. 2010. Monte Sinai J Med. Vanderlei, F M; et al. CHARACTERISTICS AND CONTRIBUTING FACTORS RELATED TO SPORTS INJURIES IN YOUNG VOLLEYBALL PLAYERS. 2013. Notas de Res. BMC. Kilic, O; et al. INCIDENCE, AETIOLOGY AND PREVENTION OF MUSCULOSKELETAL INJURIES IN VOLLEYBALL: A SYSTEMATIC REVIEW OF THE LITERATURE. 2017. Taylor&Francis. Baugh, C M; et al. DESCRIPTIVE EPIDEMIOLOGY OF INJURIES SUSTAINED IN NATIONAL COLLEGIATE ATHLETIC ASSOCIATION MEN’S AND WOMEN’S VOLLEYBALL, 2013-2014 TO 2014-2015. 2018. Saúde esportiva. Kerr, Z Y; et al. THE FIRST DECADE OF WEB-BASED SPORTS INJURY SURVEILLANCE: DESCRIPTIVE EPIDEMIOLOGY OF INJURIES IN US HIGH SCHOOL GIRLS’ VOLLEYBALL (2005-2006 THROUGH 2013-2014) AND NATIONAL COLLEGIATE ATHLETIC ASSOCIATION WOMEN’S VOLLEYBALL (2004-2005 THROUGH 2013-2014). 2018. J Athl Trem. RevistaFT A RevistaFT é uma Revista Científica Eletrônica Multidisciplinar Indexada de Alto Impacto e Qualis “B”. Periodicidade mensal e de acesso livre. Leia gratuitamente todos os artigos e publique o seu também clicando aqui. Contato Queremos te ouvir. WhatsApp: 11 98597-3405 e-Mail: contato@revistaft.com.br ISSN: 1678-0817 CNPJ: 45.773.558/0001-48 Copyright © Editora Oston Ltda. 1996 - 2022 Rua José Linhares, 134 - Leblon \| Rio de Janeiro-RJ \| Brasil Copyright © Editora Oston Ltda. 1996 - 2022 Rua José Linhares, 134 - Leblon \| Rio de Janeiro-RJ \| Brasil
https://openalex.org/W2949562329	http://en.hpchsu.ru/publikatsii/the-journal-of-regional-history-v-2-no-4/political-cooperation-in-the-revolutionary-process-of-1917-1920-on-the-materials-of-voronezh-provinc.pdf	Latin	null	Political Cooperation in the Revolutionary Process of 1917–1920 (on the Materials of Voronezh Province)	Historia provinciae - žurnal regionalʹnoj istorii	2,018	cc-by	17,902	1* Для цитирования: Разиньков М.Е. Политическое сотрудничество в революционном процессе 1917–1920 годов (по материалам Воронежской губернии) // Historia Provinciae – Журнал региональной истории. 2018. Т. 2. № 4. С. 116–148. DOI: 10.23859/2587-8344-2018-2- 4-5 For citation: Razin’kov, M. “Political Cooperation in the Revolutionary Process of 1917–1920 (on the Materials of Voronezh Province)”. Historia Provinciae – The Journal of Regional History, vol. 2, no. 4 (2018): 116–148, http://doi.org/ 10.23859/2587-8344-2018-2-4-5 © Разиньков М.Е., 2018 © Razin’kov, M., 2018 1* Для цитирования: Разиньков М.Е. Политическое сотрудничество в революционном процессе 1917–1920 годов (по материалам Воронежской губернии) // Historia Provinciae – Журнал региональной истории. 2018. Т. 2. № 4. С. 116–148. DOI: 10.23859/2587-8344-2018-2- 4-5 Журнал региональной истории. 2018. Т. 2. № 4. С. 116 148. DOI: 10.23859/2587 8344 2018 2 4-5 For citation: Razin’kov, M. “Political Cooperation in the Revolutionary Process of 1917–1920 (on the Materials of Voronezh Province)”. Historia Provinciae – The Journal of Regional History, vol. 2, no. 4 (2018): 116–148, http://doi.org/ 10.23859/2587-8344-2018-2-4-5 1 Дронов В.Т. Согласие как цивилизационный архетип российского общества // Социс. 2016. № 9. С. 157–160. Обзоры см.: Кармадонов О.А. Солидарность, интеграция, конъюнкция // Социс. 2015. № 2. С. 3–12; Бондалетов В.В. Эволюция идей самоорганизации // Социс. 2015. № 3. С. 132–139. 2 Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 1986. 373 p.; Raleigh D.J. Revolutionary Politics in Provincial Russia: The Tsaritsyn “Re- public” in 1917 // Slavic Review. 1981. Vol. 40. № 2. P. 194–209; Raleigh D.J. The Revolution of 1917 and the Establishment of Soviet Power in Saratov // Politics and Society in Provincial Russia: Saratov, 1590 – 1917 / Edited by Rex A. Wade and Scott Seregny. Columbus, Ohio: Ohio State University Press, 1989. P. 277–306. http://hpchsu.ru http://hpchsu.ru RESEARCH DOI 10.23859/2587-8344-2018-2-4-5 УДК 94(47).084.3 Razin’kov Mikhail Candidate of Historical Sciences, Associate Professor, Voronezh State University of Forestry and Technologies named after G.F. Morozov (Voronezh, Russia) razinkov_mihail@mail.ru Политическое сотрудничество в революционном процессе 1917–1920 годов (по материалам Воронежской губернии)1* Political Cooperation in the Revolutionary Process of 1917–1920 (on the Materials of Voronezh Province) Аннотация. Революционный процесс 1917–1922 гг. демонстрирует разнообразие форм сотрудничества на протяжении всего изучаемого периода. Февральский режим, режимы «демократической контрреволюции» основывались на представлениях о политической соли- дарности как фундаменте для построения нового российского государства. Октябрьский и Белые режимы предлагали различные варианты политического сотрудничества. Автор пред- лагает рассмотреть указанный процесс не только в масштабах России, но и в рамках отдель- ной Воронежской губернии. 4 5 For citation: Razin’kov, M. “Political Cooperation in the Revolutionary Process of 1917–1920 (on the Materials of Voronezh Province)”. Historia Provinciae – The Journal of Regional History, vol. 2, no. 4 (2018): 116–148, http://doi.org/ 10.23859/2587-8344-2018-2-4-5 © Разиньков М.Е., 2018 © Razin’kov, M., 2018 2018 ∙ Vol. 2 ∙ № 4 116 http://hpchsu.ru RESEARCH Ключевые слова: Революция 1917 г., Гражданская война в России, политический про- цесс, политическое сотрудничество, политическая консолидация, солидарность, Воронеж- ская губерния, партии в 1917–1920 гг., органы самоуправления. Abstract. The revolutionary process of 1917–22 demonstrates the variety of instruments for cooperation throughout the period under the study. The February Regime and the regimes of ‘dem- ocratic counter-revolution’ were based on the ideas of political solidarity as the foundation for building a new Russian state. The October (the Bolshevik) regime and the White Movement offered various options for political cooperation. The author proposes to consider this process not only on a country scale, however also within the framework of Voronezh province. Keywords: the Russian Revolution of 1917, the Russian Civil War, political process, political cooperation, political consolidation, solidarity, Voronezh province, parties in 1917–20, self- governing authorities 1 Дронов В.Т. Согласие как цивилизационный архетип российского общества // Социс. 2016. № 9. С. 157–160. Обзоры см.: Кармадонов О.А. Солидарность, интеграция, конъюнкция // Социс. 2015. № 2. С. 3–12; Бондалетов В.В. Эволюция идей самоорганизации // Социс. 2015. № 3. С. 132–139. 2 2015. № 3. С. 132–139. 2 Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 1986. 373 p.; Raleigh D.J. Revolutionary Politics in Provincial Russia: The Tsaritsyn “Re- public” in 1917 // Slavic Review. 1981. Vol. 40. № 2. P. 194–209; Raleigh D.J. The Revolution of 1917 and the Establishment of Soviet Power in Saratov // Politics and Society in Provincial Russia: Saratov, 1590 – 1917 / Edited by Rex A. Wade and Scott Seregny. Columbus, Ohio: Ohio State University Press, 1989. P. 277–306. Основная часть Февральский режим дал большие возможности для демократизации россий- ского общества. В этих условиях активизируются процессы, связанные с поли- тическим сотрудничеством3. Очевидно, что различные участники революцион- ного процесса становились союзниками, вступали в организации, где должны были взаимодействовать друг с другом в рамках политического поля4. Про- граммные документы различных политических сил обнаруживали существен- ное сходство, позволяя обосновывать блокирование идеологически5. Изначаль- но либеральное Временное правительство очень быстро полевело и к лету 1917 г. туда входили социалисты. При этом основная либеральная партия – кадетов – участвовала во всех трех коалиционных правительствах и в 3-м его составе су- мела даже усилить свои позиции с 4-х министерских кресел до 6-ти. Площад- кой для сотрудничества праволиберальных сил стало Государственное совеща- ние в Москве (август 1917 г.). В сентябре 1917 г. состоялось Демократическое совещание в Петрограде, на котором присутствовали делегаты от Советов, ор- ганов местного самоуправления, профсоюзов, кооперативов, армейских коллек- тивов, национальных учреждений. В избранном на совещании Предпарламенте состояло 135 эсеров, 92 меньшевика, 75 кадетов, 58 большевиков, 30 энесов. Коалиционные идеи были характерны для исполнительной власти на местах – губернских комиссаров, губернских и уездных исполнительных комитетов. 3 Кружинов В.М., Сокова З.Н. Органы многопартийной социалистической власти на Урале осенью 1917 г. // Российская история. 2017. № 5. С. 44–61; Фельдман М.А. Промыш- ленные рабочие России в 1917 г.: подходы к «социалистической» революции // Российская история. 2017. № 5. С. 44–61; Шацилло М.К. Российские предприниматели в поисках контак- тов с советской властью в 1917–1918 гг. // Российская история. 2016. № 1. С. 28–43; Морозо- ва О.М. Дон в 1917 году: мирная прелюдия революции // 1917 год в судьбах региона, страны и мира. Взгляд из XXI века / под общ. ред. В.И. Голдина. Архангельск: САФУ, 2017. С. 132– 136; Rosenberg W.G. Liberals in the Russian Revolution: the Constitutional Democratic Party, 1917 – 1921. Princeton, N.J.: Prinston University Press, 1974; Mawdsley E. The Russian Civil War. Edinburgh: Printed and bound by Grafica Veneta, 2000; Smele J.D. The “Russian” Civil Wars, 1916–1926. Ten years that shook the world. London: Hurst & Company, 2015. 4 4 Поскольку приводимые факты сотрудничества (но не их систематизация в данном клю- че) являются общеизвестными, обзор ситуации в масштабах бывшей Российской империи основан на анализе информации специализированной энциклопедической литературы: Рево- люция и Гражданская война в России: 1917–1923 гг.: Энциклопедия. В 4 т. М.: ТЕРРА, 2008; Политические партии России. Конец XIX – первая треть ХХ века. Энциклопедия /отв. ред. В.В. Шелохаев. Введение Рассматривать революцию сугубо как конфликт, тем более системный кри- зис, верно по существу, но мало что дает для понимания ее преобразовательно- го потенциала, следовательно, продуктивность такого подхода ограничена. В то же время социологические концепции обращают внимание на важнейшую роль социальной интеграции, солидарности, сотрудничества, консолидации при вы- работке идеи общественного согласия1. Эти концепции, а также мысль о том, что революции делаются не только ради разрушения, дали повод для написания этого исследования. В данной статье я предлагаю посмотреть на революцию как на разнообразие моделей сотрудничества, осуществлявшихся не только в столичных центрах, но и в провинции. В качестве кейс-примера ситуации в русской провинции приводится Воронежская губерния. Отметим, что в зару- бежной историографии развитие революционной ситуации, включая и элемен- ты политического сотрудничества, в провинции на материалах Саратова и ряда других поволжских городов рассмотрел известный специалист по русской ис- тории Д.Дж. Рейли2. 2 Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 1986. 373 p.; Raleigh D.J. Revolutionary Politics in Provincial Russia: The Tsaritsyn “Re- public” in 1917 // Slavic Review. 1981. Vol. 40. № 2. P. 194–209; Raleigh D.J. The Revolution of 1917 and the Establishment of Soviet Power in Saratov // Politics and Society in Provincial Russia: Saratov, 1590 – 1917 / Edited by Rex A. Wade and Scott Seregny. Columbus, Ohio: Ohio State University Press, 1989. P. 277–306. 2018 ∙ Vol. 2 ∙ № 4 117 http://hpchsu.ru 3 Кружинов В.М., Сокова З.Н. Органы многопартийной социалистической власти на Урале осенью 1917 г. // Российская история. 2017. № 5. С. 44–61; Фельдман М.А. Промыш- ленные рабочие России в 1917 г.: подходы к «социалистической» революции // Российская история. 2017. № 5. С. 44–61; Шацилло М.К. Российские предприниматели в поисках контак- тов с советской властью в 1917–1918 гг. // Российская история. 2016. № 1. С. 28–43; Морозо- ва О.М. Дон в 1917 году: мирная прелюдия революции // 1917 год в судьбах региона, страны и мира. Взгляд из XXI века / под общ. ред. В.И. Голдина. Архангельск: САФУ, 2017. С. 132– 136; Rosenberg W.G. Liberals in the Russian Revolution: the Constitutional Democratic Party, 1917 – 1921. Princeton, N.J.: Prinston University Press, 1974; Mawdsley E. The Russian Civil War. Edinburgh: Printed and bound by Grafica Veneta, 2000; Smele J.D. The “Russian” Civil Wars, 1916–1926. Ten years that shook the world. London: Hurst & Company, 2015. Основная часть М.: РОССПЭН, 1996; Гражданская война и военная интервенция в СССР. Энциклопедия / гл. ред. С. С. Хромов. М.: Советская энциклопедия, 1983. р р 5 Модели общественного переустройства России. ХХ век / отв. ред. В.В. Шелохаев. М.: РОССПЭН, 2004; Разиньков М.Е. Трудовая народно-социалистическая партия и Российская коммунистическая партия (большевиков): сравнительный анализ программных установок // История и историография правого народничества: Сб. статей / под ред. Г.Н. Мокшина. Воро- неж: Истоки, 2014. С. 141–152. 2018 ∙ Vol. 2 ∙ № 4 118 http://hpchsu.ru RESEARCH Другой территорией сотрудничества стали органы революционного само- управления. В Советах ожидаемое большинство составляли социалистические партии – ПСР, РСДРП, народные социалисты. ВЦИК, созданный на I съезде Советов в июне 1917 г., включал 107 меньшевиков, 101 эсера, 35 большевиков, 8 объединенных социал-демократов, 4 трудовика и народных социалиста, 1 члена Еврейской социалистической партии. Советы делегировали своих пред- ставителей в городские Думы – место сосредоточения либералов. В результате перевыборов уже летом городское самоуправление носило партийно- коалиционный характер. Социальное представительство в нем обеспечивали не только Советы, но и профсоюзы, в которых также руководящую роль играли представители различных партий. В рамках одного крупного города различны- ми профсоюзами могли руководить и большевики, и меньшевики, и эсеры, и представители национальных партий, которые просто вынуждены были всту- пать друг с другом в диалог. На 3-й Всероссийской конференции профсоюзов (июнь 1917 г.) был избран временный управляющий орган, состоявший из 16 большевиков, 16 меньшевиков, 3 эсеров. Комитеты общественных организаций, возглавлявшиеся кадетами, меньшевиками и эсерами, до июля 1917 г. оказыва- ли значительное влияние на организацию революционной власти и в дальней- шем продолжали отстаивать свое право на существование именно через идею представительства различных политических сил. Социалистические партии, включая большевиков, на протяжении всего 1917 г. рассматривали возможность создания единого фронта. В.М. Чернов вы- ступал с идеей объединения социалистов в единую партию; меньшевики в мае – августе 1917 г. пытались воссоздать единство РСДРП с помощью созыва кон- ференции и съезда. На 2-м съезде Трудовой народно-социалистической партии (22–28 сентября 1917 г.) подчеркивалось, что для консолидации общественных и государственных сил партия будет сотрудничать с правыми эсерами, меньше- виками-интернационалистами, группой «Единство» и кадетами. Большевики рассматривали себя как часть общего политического поля с другими социалистическими партиями, по меньшей мере, до сентября 1917 г., а в ряде случаев и до созыва Учредительного собрания. Говоря о возможности мирного развития пролетарской революции, В.И. 6 Ленин В.И. Полн. соб. соч. В 55 т. М.: Госполитиздат, 1977. Т. 34. С. 222–223. 7 Там же. 8 Кружинов В.М., Сокова З.Н. Указ. соч. С. 44–61; Протасов Л.Г. Всероссийское Учре- дительное собрание: история рождения и гибели. М.: РОССПЭН, 1997. С. 271. 9 Революция и Гражданская война в России. Т. 1. С. 361–362. Основная часть Ленин приводил пример кор- ниловских событий, когда даже кратковременный союз большевиков, меньше- виков и эсеров дал «полнейшую, с невиданной ни в одной революции легкости достигнутую победу над контрреволюцией, он дал такое сокрушительное по- давление буржуазной, помещичьей и капиталистической, союзно- империалистической и кадетской контрреволюции, что гражданская война с этой стороны развалилась в прах, превратилась в ничто в самом начале, распа- 2018 ∙ Vol. 2 ∙ № 4 119 http://hpchsu.ru RESEARCH лась до какого бы то ни было “боя”»6. По мысли большевистского вождя, такой союз позволил бы революции перейти из военной в политическую плоскость, поскольку «внутри Советов борьба партий за власть может идти мирно, при полном демократизме Советов…»7. В процессе октябрьского переворота боль- шевики продолжали искать компромиссы и не рушить за собой мосты оконча- тельно, вступая в различные коалиционные управляющие органы, как это было, например, на Урале или в Курске8. Отказ В.И. Ленина и Л.Д. Троцкого сформи- ровать «однородное социалистическое правительство» вызвал первый кризис в захватившей власть партии. лась до какого бы то ни было “боя”»6. По мысли большевистского вождя, такой союз позволил бы революции перейти из военной в политическую плоскость, поскольку «внутри Советов борьба партий за власть может идти мирно, при полном демократизме Советов…»7. В процессе октябрьского переворота боль- шевики продолжали искать компромиссы и не рушить за собой мосты оконча- тельно, вступая в различные коалиционные управляющие органы, как это было, например, на Урале или в Курске8. Отказ В.И. Ленина и Л.Д. Троцкого сформи- ровать «однородное социалистическое правительство» вызвал первый кризис в захватившей власть партии. Третьим аспектом сотрудничества стало слияние близких политических группировок. В июне 1917 г. объединились «трудовики» и народные социали- сты, в июле в состав РСДРП(б) влились «межрайонцы», к августу – большинст- во членов группы «Вперед». В сентябре объединились небольшие Либерально- республиканская и Радикально-демократическая партии. Такое сближение за- трагивало и национальные партии. Например, летом 1917 г. объединились Со- циалистическая еврейская и Сионистско-социалистическая рабочие партии. Наконец, появлялись внепартийные общественные объединения политиче- ского характера. С февраля по декабрь 1917 г. действовал Временный комитет Государственной Думы, в который входили октябристы, кадеты, меньшевики, представители большинства фракций Думы. С августа начал работу Совет об- щественных деятелей в Москве, объединявший членов Госдумы, кадетов, про- мышленников, цензовых земцев, представителей профессуры. О намерениях по поводу политического блокирования с социалистами заявлялось на втором Со- вещании общественных деятелей 12–14 октября 1917, которое «высказалось за поддержку на выборах в Учредительное собрание групп, стоящих на государст- венной платформе, за соединение с Трудовой народно-социалистической пар- тией и группой “Единство”»9. Основная часть После большевистского переворота возможности для политического со- трудничества сохранились. Конец 1917–1918 гг. стали временем поиска демо- кратической альтернативы большевистской диктатуре. Целый спектр межпар- тийного и политико-социального блокирования был представлен на выборах в Учредительное собрание, где наряду с предсказуемыми блоками, например, эсеров и меньшевиков, можно было встретить соглашения кадетов и плеханов- ского «Единства», религиозных объединений с энесами, большевиков с мень- 2018 ∙ Vol. 2 ∙ № 4 120 http://hpchsu.ru RESEARCH шевиками и т.д.10 При этом большевистский режим взаимодействовал с други- ми политическими силами. Собственные проекты сотрудничества предлагали организаторы Белого движения и национальные правительства. Центром притяжения социалистических и либеральных политических сил стали антибольшевистские правительства, такие как КОМУЧ, Временное Си- бирское правительство, Временное правительство Северной области, Уфимская директория. У истоков Добровольческой армии стоял Гражданский совет, в ко- торый входили 4 социалиста и 7 либералов (в т.ч. М.М. Федоров, Б.В. Савин- ков, П.Б. Струве). Верховную власть осуществлял триумвират: А.М. Каледин управлял Донской областью, Л.Г. Корнилов командовал армией, М.В. Алексеев сосредоточил у себя гражданское управление, внешние связи и финансы. Не- смотря на то, что в дальнейшем победила идея военной диктатуры, основа Бе- лого движения воспринималась как различного рода конфедерации и федера- ции: Вооруженные силы Юга России, союз белых областей во главе с Верхов- ным правителем А.В. Колчаком, федеративный проект Доно-Кавказского союза П.Н. Краснова. Показательно, что в конце 1917–1918 гг. Советская Россия так- же воспринималась как конфедерация революционных республик (Донецко- Криворожская, Терская, Кубано-Черноморская и т.п.), что институционально сближало ее с антибольшевистскими конфедерациями. Самым заметным союзом в рамках Октябрьского режима был, конечно, со- юз большевиков с ПЛСР (ноябрь 1917 – март 1918 гг.). Левые эсеры участвова- ли в работе всех отделов ВЦИК, занимая там половину мест, пользовались зна- чительным влиянием в Московском областном СНК, Совете комиссаров Союза коммун Северной области, Центрально-Черноземном регионе, СНК Сибири, играли доминирующую роль в СНК Туркестана и в ТурЦИК. Вытесненная ле- том 1918 г. из российского политического поля, ПЛСР успешно действовала как левацкий центр на Украине. Впрочем, вытеснение это оказалось в некото- рой степени условным, поскольку левые эсеры, наряду с анархистами, участво- вали во Втором Всероссийском съезде профсоюзов (16–25 января 1919 г.). ПЛСР (объединенная) была допущена на 8-й Всероссийский съезд Советов в декабре 1920 г. Сохранялись контакты большевиков с социалистическими партиями, при- чем стремление к таким контактам было обоюдным. Часть социалистов пред- почитала взаимодействовать с большевиками, а не с белыми режимами. Боль- шевики, в свою очередь, оказались заинтересованы в контактах с социалистами, когда к концу 1918 г. стало ясно, что Гражданская война затягивается. 10 См. схемы блокирования: Протасов Л.Г. Всероссийское Учредительное собрание: ис- тория рождения и гибели. М.: РОССПЭН, 1997. С. 134, 137. Основная часть Несмот- ря на фактический разрыв отношений РСДРП (объединенной) с большевиками в июне – октябре 1918 г., она вернулась к политике институционального со- 10 См. схемы блокирования: Протасов Л.Г. Всероссийское Учредительное собрание: ис- тория рождения и гибели. М.: РОССПЭН, 1997. С. 134, 137. 2018 ∙ Vol. 2 ∙ № 4 121 http://hpchsu.ru RESEARCH трудничества против сил контрреволюции, выступая как оппозиция во ВЦИК, Советах, профсоюзах. Доклады «О задачах профсоюзов» на Втором Всероссий- ском съезде профсоюзов читали не только В.И. Ленин, но и Ю.О. Мартов, и С.А. Лозовский (РСДРП (интернационалистов)). В более сложной ситуации оказалась ПСР, объявившая в мае 1918 г. войну большевикам, но потерпевшая поражение от Белого движения. Уже в декабре 1918 г. на совещании в Уфе видными эсеровскими деятелями было принято решение о прекращении войны с большевиками. ЦК долгое время не признавал действия Уфимской делегации легитимными, но, в конечном итоге, и он решился пойти на переговоры с РКП(б). Вместе с тем, если в антибольшевистских правительствах эсеры и меньше- вики могли занимать ведущие позиции, то в рамках советской системы им была уготовлена роль безуспешного критика. Поэтому вполне понятен проявивший- ся к концу Гражданской войны еще один вид сотрудничества в форме ассими- ляции, т.е. перехода многих деятелей соцпартий, а порой и партий целиком, в ряды большевиков. В 1920 – 1921 г. в состав РКП(б) вошли Бунд и Российская социалистическая рабочая партия интернационалистов (возникшая в 1919 г. при слиянии РСДРП(и) и Российской партии независимых с.-д. левых интерна- ционалистов). Происходило и слияние с РКП(б) альтернативных коммунисти- ческих группировок: Партии народников-коммунистов (октябрь – ноябрь 1918 г.), Партии революционного коммунизма (сентябрь 1920 г.). Сходные процессы наблюдались и на национальных территориях распавшейся империи. Напри- мер, в Польше Коммунистическая рабочая партия, созданная в 1918 г. в резуль- тате объединения Социал-Демократии королевства Польши и Литвы с Поль- ской социалистической партией – левицей, стала в 1919–1920 гг. центром мощ- ного притяжения отделов национальных партий коммунистической и социали- стической направленности. В партию вошли польские отделения Бунда, По- алей-Цион, Украинской социал-демократической рабочей партии, Белорусской социалистической партии. Еще одной формой сотрудничества, получившей большое распространение в послеоктябрьский период, стали внепартийные объединения, такие как Коми- тет спасения Родины и революции, Союз защиты Учредительного собрания, Союз возрождения России, Правый, Национальный, Тактический, Политиче- ский центры. Из приведенных фактов видно, что разнообразие форм сотрудничества яв- лялось частью революционной политической культуры не только в период Февральского режима, но и в послеоктябрьское время, рассматриваемое обычно как период установления однопартийной диктатуры. 11 Государственный архив Воронежской области (ГАВО). Ф. Р-2393. Оп. 1. Д. 12. Л. 7; Д. 13. Л. 7–8. 12 ГАВО. Ф. Р-2393. Оп. 1. Д. 3. Л. 111 об.; Д. 10. Л. 89. 13 ГАВО. Ф. Р-2393. Оп. 1. Д. 2. Л. 27, 53, 59; Государственный архив общественно- политической истории Воронежской области (ГАОПИ ВО). Ф. 5. Оп. 1. Д. 320. Л. 23; Собо- лев П.Н. Подготовка социалистической революции и установление Советской власти в Воро- нежской губернии. Воронеж: Воронежское книжное издательство, 1955. С. 56. 14 Филипцева С.В. Завершение деятельности губернского земства в Воронеже в 1918 г. // Из истории Воронежского края. Сб. ст. / отв. ред. А.Н. Акиньшин. Воронеж: ВГУ, 2011. Вып. 18. С. 218–221. 15 ГАВО. Р-2393. Оп. 1. Д. 3. Л. 114, 117–118. 16 ГАОПИ ВО. Ф. 5. Оп. 1. Д. 459. Л. 21–23. Основная часть Важнейшими формами сотрудничества были: взаимодействие партий и общественных организаций в революционных государственных и муниципальных органах, сотрудничество 2018 ∙ Vol. 2 ∙ № 4 122 http://hpchsu.ru RESEARCH партий вплоть до их слияния, создание внепартийных общественно- политических объединений. На мой взгляд, подобное взаимодействие, имевшее значительный демократический потенциал, создавало мощную и разнообраз- ную основу для демократического компонента развернувшегося процесса мо- дернизации Российского государства. Для Февральского режима сотрудничест- во было основой новой власти, организовывавшейся из партий, органов госу- дарственного управления и самоуправления. Эту линию пытались продолжить демократические правительства, сформировавшиеся в Сибири, Поволжье, Се- верной области в 1918 г. Октябрьский режим рассматривал сотрудничество не только как вынужденную меру, но и как источник пополнения рядов правящей партии, позиционировавшей себя как единственный центр притяжения всех здоровых сил страны. Белые режимы, тяготея, как и большевики, к диктатуре, идентифицировали себя с западными ценностями, сотрудничая со всеми анти- большевистскими силами, но предпочитая, однако, их праволиберальный ком- понент. Для уяснения нюансов развития революционного процесса немаловажно посмотреть на ситуацию в русской провинции, характерным примером которой может стать аграрная Воронежская губерния с великоросским большинством. Можно с уверенностью сказать, что, как и Временное правительство, мест- ные активисты стремились создать новую власть на основе широкого сотруд- ничества государственных учреждений и общественных организаций, выра- жающейся в коллегиальности решений по большинству вопросов. В образован- ный 11 марта 1917 г. Воронежский Комитет общественного спокойствия на па- ритетных началах вошли представители Городской Думы, Совета Рабочих Де- путатов и Комитета объединенных общественных организаций и учреждений, а также делегаты от солдат и офицеров. Одновременно в комитет объединенных общественных организаций входили представители 101 учреждения и учебных заведений. 17 марта в состав городской Думы вошли 26 делегатов от Советов11. Несмотря на то, что к лету 1917 г. взаимоотношения воронежского Совета с Комитетом общественного спокойствия заметно ухудшились, взаимодействие с органами власти, напрямую подчинявшимися Временному правительству, со- хранялось и даже продолжало расширяться. Спектр точек соприкосновения был достаточно велик: культурно-просветительская работа, совещания по продо- вольствию, топливу, социальная помощь, борьба с преступностью и нейтрали- зация крестьянского движения. На проводившихся в Губернском исполнитель- ном комитете заседаниях и в различных совещаниях неизменно присутствовали представители от государственных органов, ответственных комиссий и комите- 11 Государственный архив Воронежской области (ГАВО). Ф. Р-2393. Оп. 1. Д. 12. Л. 7; Д 13 Л 7–8 осуд р Д. 13. Л. 7–8. 2018 ∙ Vol. 2 ∙ № 4 123 http://hpchsu.ru RESEARCH тов, органов земского и городского самоуправления, армейского командова- ния12. тов, органов земского и городского самоуправления, армейского командова- ния12. 17 Соболев П.Н. Подготовка социалистической революции и установление Советской власти в Воронежской губернии. Воронеж: Воронежское книжное издательство, 1955. С. 56. 18 Воронежский телеграф. 1917. 12 сентября. С. 1. 19 ГАВО. Р-2393. Оп. 1. Д. 3. Л. 20, 34 об., 58 об., 73, 77, 79; Д. 4. Л. 85, 90. 20 Воронежский областной краеведческий музей. Фонд участников революции и граж- данской войны. Папка № 655/I. Врачев И.Я. 21 Путь жизни. 1917. № 1–7. 17 Соболев П.Н. Подготовка социалистической революции и установление Советской власти в Воронежской губернии. Воронеж: Воронежское книжное издательство, 1955. С. 56. 18 Воронежский телеграф. 1917. 12 сентября. С. 1. 19 ГАВО Р 2393 О 1 Д 3 Л 20 34 б 58 б 73 77 79 Д 4 Л 85 90 Основная часть После Октябрьского переворота взаимоотношения между Советом, общест- венными организациями и институциями, созданными в ходе Февральского ре- жима или даже до него, не были разорваны сразу. Губернская продовольствен- ная управа, земства, воронежская городская Дума, хотя и высказывали недо- вольство новой властью, однако периодически выносили постановления о не- обходимости продолжать работу в новых условиях. До декабря 1917 г. работал губернский комиссар Б.А. Келлер, назначенный на эту должность еще в июле 1917 г. До января 1918 заседали дооктябрьский состав окружного суда, проку- ратуры и губернская земская управа13. Решение Губисполкома о ликвидации учреждений губернского земства последовало только в августе 1918 г., причем земские учреждения, вместе со старыми служащими, инкорпорировались в гу- бернский совнархоз. Показательно, что власть большевиков не «требовала кро- ви» даже бывших очевидных противников. Так, первый губернский комиссар Временного правительства и председатель губернской земской управы В.Н. Томановский еще в декабре 1918 г. занимал должность заведующего админист- ративно-фискальным подотделом финансового отдела Воронежского губиспол- кома14. Продолжил работу в качестве профессора Воронежского сельскохозяй- ственного института, а затем академика АН СССР и ВАСХНИЛ Б.А. Келлер, привлекавшийся в 1920-е гг. к работе в воронежском облисполкоме. р р р Сотрудничество на протяжении 1917 – первой половины 1918 гг. сегментов российской политической системы объясняется присутствием в них представи- телей одних и тех же партий. Так, в участниках заседания Губернского испол- нительного комитета 20 мая 1917 г. значились эсеры, энесы, социал-демократы и анархист. Председателем тогда был назначен эсер В.П. Кабытченко, товари- щем председателя – энес (бывший кадет) П.Я. Ростовцев15. После июльских выборов в воронежскую городскую Думу была сформирована управа с равным представительством эсеров, меньшевиков и кадетов16. Сходная ситуация была в уездных центрах. Из 22 депутатов Валуйской городской думы 17 человек были эсерами, 2 – большевиками, 1 был избран от кадетов и народных социалистов. 2018 ∙ Vol. 2 ∙ № 4 124 http://hpchsu.ru RESEARCH В Острогожской городской думе кадеты получили 3 места, большевики – 617. Избранная в июле воронежская городская дума дала коалицию эсеров, меньше- виков и кадетов18. 19 ГАВО. Р-2393. Оп. 1. Д. 3. Л. 20, 34 об., 58 об., 73, 77, 79; Д. 4. Л. 85, 90. 20 Воронежский областной краеведческий музей. Фонд участников революции и граж- данской войны. Папка № 655/I. Врачев И.Я. 21 Путь жизни. 1917. № 1–7. р р ф р 19 ГАВО. Р-2393. Оп. 1. Д. 3. Л. 20, 34 об., 58 об., 73, 77, 79; Д. 4. Л. 85, 90. В Острогожской городской думе кадеты получили 3 места, большевики – 617. Избранная в июле воронежская городская дума дала коалицию эсеров, меньше- виков и кадетов18. Одновременно социалистические партии взаимодействовали в рамках Сове- та, где к лету 1917 г. присутствовали эсеры, большевики, меньшевики, предста- вители Бунда. В августе 1917 г. было принято решение о предоставлении места в Совете Еврейской социалистической партии, в сентябре – Трудовой народно- социалистической партии. Летом 1917 г. при Совете функционировала объеди- ненная библиотека эсеров и социал-демократов. По крайней мере, в августе – сентябре 1917 г. существовал Социалистический клуб, куда входили предста- вители эсеров и большевиков19. Иначе говоря, противоречия между партиями не представлялись фатальными и точки соприкосновения находили организа- ционное оформление. Сотрудничество также осуществлялось на уровне коми- тетов партий, комитета Юго-Восточных железных дорог, чрезвычайных орга- нов: Революционного распорядительного комитета, созданного для борьбы с корниловским выступлением, Комитета общественной безопасности и Времен- ного революционного комитета (ВРК), существовавших во время октябрьско- ноябрьских событий 1917 г. Имеющаяся информация доказывает, что у воронежских большевиков были серьезные разногласия по поводу необходимости разрыва с социалистическим движением и непосредственно переворота. Губком большевиков был создан только в октябре 1917 г., т.е. контакты с меньшевиками не порывались оконча- тельно на протяжении почти всего февральского режима. Председатель губко- ма, старый большевик Н.Н. Кардашев, не принимал прямого участия в органи- зации переворота в Воронеже. Последний был осуществлен под руководством группы «молодых» во главе с приехавшим из Петрограда после июльских со- бытий «межрайонцем» А.С. Моисеевым. Член большевистского губкома С.Д. Турчанинов в начале декабря 1917 г. вышел из партии в знак протеста против Октябрьского переворота20. Показательно, что публикации воронежской боль- шевистской газеты «Путь жизни» за август – сентябрь содержат как заявления о необходимости гражданской войны и выпады против социал-шовинистов, так и объявления о работе социалистического клуба с выступлениями эсеровских ру- ководителей21. Перед самым переворотом большевики и левые эсеры предлага- 2018 ∙ Vol. 2 ∙ № 4 125 http://hpchsu.ru ли создать бюро для организации перевыборов в Совет, куда бы входили не только они, но и социалисты-революционеры, меньшевики, беспартийные22. Переход власти к ВРК в Воронеже не означал исключения социалистиче- ских партий из политической жизни. Надо сказать, что представители Военно- революционного комитета не стремились выступать откровенными диктатора- ми и рассматривали себя как орган, попытавшийся предотвратить в Воронеже контрреволюционное выступление. Высылаемые в милицию, банк, к городско- му голове и в думу делегаты и комиссары резко отстаивали свои взгляды, но не стремились занять управляющие посты в указанных организациях насильно. Это даже вызывало удивление у отстраненных от власти деятелей и порождало надежды на возможный пересмотр ситуации. Всю первую половину ноября Со- вет пытался функционировать в дооктябрьском составе. 22 Путь жизни. 1917. № 12. С. 3–4. 23 Воронежский телеграф. 1917. 12 ноября. С. 2–3. 24 Воронежский телеграф. 1917. 9–11 ноября. 25 Воронежский телеграф. 1917. 18 ноября. С. 1. р р ф р 25 Воронежский телеграф. 1917. 18 ноября. С. 1. 26 ГАВО. Ф. Р-458. Оп. 1. Д. 50. Л. 19 об.–20, 43; Д. 258. Л. 1 об. 27 ГАВО. Р-2393. Оп. 1. Д. 8а. Л. 4–4 об.; Соболев П.Н. Указ. соч. С. 106, 142–143; За власть Советов. Сборник воспоминаний участников революционных событий в Воронеж- ской губернии в 1917 – 1918 годах / под ред. И.Г. Воронкова, Т.М. Севастьяновой. Воронеж: Воронежское книжное издательство, 1957. С. 86, 116–122. 28 За власть Советов. С. 26, 46, 50–53; ГАВО. Ф. Р-2393. Оп. 1. Д. 8. Л. 117. 29 ГАВО. Ф. Р-905. Оп. 1. Д. 20. Л. 107–108 об. 30 Соболев П.Н. Указ. соч. С. 111. 31 ГАВО. Ф. Р-2393. Оп. 1. Д. 2. Л. 13–14, 17, 21–21 об., 36–37, 38 об., 40, 42, 48, 51об., 58 об., 62 об., 64 об., 81–82, 84, 95–96, 105. В Острогожской городской думе кадеты получили 3 места, большевики – 617. Избранная в июле воронежская городская дума дала коалицию эсеров, меньше- виков и кадетов18. Фракции эсеров, меньшевиков и беспартийных вышли из Совета только 10 ноября23. В ноябре 1917 г. эсерам удалось собрать крестьянский съезд в Воронеже, вплоть до вес- ны 1918 г. они заседали в продовольственных комиссиях. Сотрудничество пар- тий и общественных организаций на базе протеста против большевистско- левоэсеровской власти было заметно в ходе выборов в Учредительное собра- ние. Прошедшие 12–14 ноября выборы сопровождались не только объявления- ми профсоюзов о нежелании сотрудничать с узурпаторами, но и попытками консолидировать «здоровые силы общества». Так, воронежская организация Трудовой народно-социалистической партии создала целый общественный фронт, куда входили продолжавший функционировать Комитет объединенных общественных организаций, Союз интеллигенции, союз инженеров и техников, а также объединения кооператоров24. Партии и профсоюзные организации не- однократно заявляли о необходимости сотрудничать с органами управления, созданными «февральским режимом» (думой, управами, комитетами), призы- вали налаживать отношения между ними на межгубернском уровне25. В марте 1918 г., после подписания Брест-Литовского мира, фракция эсеров заявила о своем протесте, но осталась в Совете. Эсеры и меньшевики вплоть до весны 1918 г. занимали важные должности в комитете служащих Юго- Восточных железных дорог, Союзе служащих-чиновников правительственных учреждений, Союзе инженеров и техников, имели доступ к средствам Государ- ственного банка. По крайней мере, до июня 1918 г. в губернии легально издава- лись газеты меньшевиков и эсеров. Председатель свергнутого большевиками Совета А.В. Михайлов фигурирует в документах как один из организаторов гу- бернского съезда профсоюзов в 1919 г., а меньшевистское влияние в профсою- 2018 ∙ Vol. 2 ∙ № 4 126 http://hpchsu.ru RESEARCH зах прослеживается, по крайней мере, до 1920 г.26 При выборах Совета в уезд- ном Павловске в декабре 1917 г. председателем был избран большевик, а его товарищем – эсер. Более того, до конца лета 1918 г. здесь действовали два Со- вета – городской, находившийся под контролем эсеров, и уездный – контроли- руемый большевиками27. зах прослеживается, по крайней мере, до 1920 г.26 При выборах Совета в уезд- ном Павловске в декабре 1917 г. председателем был избран большевик, а его товарищем – эсер. Более того, до конца лета 1918 г. здесь действовали два Со- вета – городской, находившийся под контролем эсеров, и уездный – контроли- руемый большевиками27. В 1918 г. продолжалось сотрудничество большевиков с партией левых эсе- ров. В октябре 1917 г. регулярно созывались «пятерки» представителей этих партий, где обсуждались все тактические вопросы. Во время переворота члена- ми Военно-революционного комитета были, наряду с большевиками А.С. Мои- сеевым, В.Н. Губановым, И.А. Чуевым и И.Я. Врачевым, левые эсеры Е.Ф. Му- равьев, И.С. Пляпис и Н.И. Григорьев. Н.И. 32 ГАОПИ ВО. Ф. 1. Оп. 1. Д. 13. Л. 1–1 об., 8–9. 33 ГАОПИ ВО. Ф. 5. Оп. 1. Д. 342. Л. 1. 34 ГАОПИ ВО. Ф. 1. Оп. 1. Д. 12. Л. 2. 35 Воронежский областной краеведческий музей. Фонд участников революции и граж- данской войны. Папка 655/I. Врачев И.Я. В Острогожской городской думе кадеты получили 3 места, большевики – 617. Избранная в июле воронежская городская дума дала коалицию эсеров, меньше- виков и кадетов18. Григорьев в декабре 1917 г. на ко- роткое время стал председателем ВРК28. Дружины, возглавляемые левыми эсе- рами Е.Ф. Муравьевым и М.А. Чернышевым, сыграли одну из ключевых ролей во время переворота 30 октября 1917 г. Небольшой отряд во главе с левым эсе- ром Титковым выполнял в конце 1917 – начале 1918 гг., по сути, функции ЧК, арестовывая подозрительные собрания и участвуя в подавлении вспышек недо- вольства в городе29. На III Всероссийский съезд Советов были делегированы депутаты от обеих фракций. 28 декабря 1917 г. большевиками и левыми эсера- ми был организован губернский крестьянский съезд, где из 169 делегатов было 92 большевика, 61 левый эсер и 16 беспартийных30. В мае 1918 г. левые эсеры руководили продовольственным и земельным отделами губкома Советов, за- нимали пост председателя революционного трибунала, участвовали в национа- лизации воронежских банков. В июне 1918 г. был поставлен вопрос о передаче под контроль левых эсеров финансового отдела, участии их в чрезвычайной комиссии по борьбе с контрреволюцией, спекуляцией и преступностью, изда- нии газеты, в том числе перебазировавшегося в Воронеж ЦК левых эсеров Ук- раины. Сотрудничество фракций осуществлялось и в профсоюзах (например, почтово-телеграфных служащих) и при регулировании социальных конфлик- тов31. Даже после того, как отношения с ПЛСР начали охлаждаться (в Вороне- же уже с середины июня 1918 г.), Воронежский комитет РКП(б) 17 июня пред- лагал левым эсерам занять вместо ключевых продовольственного и финансово- 2018 ∙ Vol. 2 ∙ № 4 127 RESEARCH RESEARCH http://hpchsu.ru го комиссариата земельный, земский, призрения, юридический, хозяйственный и медико-санитарный. 36 ГАОПИ ВО. Ф. 5. Оп. 1. Д. 323. Л. 144–146. 37 Коротун С.Н. Секции национальных меньшинств в Воронежской губернии (1918– 1921) // Из истории Воронежского края. Сб. ст. / отв. ред. А.Н. Акиньшин. Воронеж: Цен- трально-Черноземное книжное издательство, 2011. Вып. 18. С. 224–229; Коротун С.Н., Тол- качева С.П., Шевченко Е.А. Национальные меньшинства Воронежского края в 1917 – 1941 гг. Воронеж: Воронежский государственный педагогический университет, 2012. С. 64, 66–68, 217–218, 228, 242; Документы Краснянского восстания / публ. Я. В. Леонтьева // Крестьян- ский фронт 1918–1922 гг. Сб. статей и материалов / под ред. А. В. Посадского. М.: АИРО– XXI, 2013. С. 308; ГАОПИ ВО. Ф. 5. Оп. 1. Д. 320. Л. 23; ГАВО. Ф. Р-2393. Оп. 1. Д. 2. Л. 21. 38 Воронежский телеграф. 1917. 14 декабря. С. 3. го комиссариата земельный, земский, призрения, юридический, хозяйственный и медико-санитарный. го комиссариата земельный, земский, призрения, юридический, хозяйственный и медико-санитарный. го комиссариата земельный, земский, призрения, юридический, хозяйственный и медико-санитарный. Несмотря на разрыв, произошедший после июльского мятежа левых эсеров, повальных арестов в Воронеже не произошло. 24 июля губернский комитет ПЛСР счел возможным обратиться к комитету РКП(б) с просьбой снять арест с его газеты «Знамя трудовой борьбы». В уездном Задонске еще в конце августа 1918 г. ставился вопрос о недопущении левых эсеров в комиссии и президиум местного Совета, причем от ПЛСР поступали заявления о необходимости заня- тия членами партии мест в Совете32. Известно, что в Землянске левый эсер на- ходился в исполкоме даже в конце сентября 1918 г.33. Нежелание окончательно порывать с большевиками привело к тому, что часть воронежских левых эсеров уже в октябре 1918 г. приняла участие в соз- дании партии народников-коммунистов, а часть вообще заявила о необходимо- сти слияния с РКП(б)34. Следует вспомнить, что в 1920–1921 гг. бывшие руко- водители воронежских левых эсеров Е.Ф. Муравьев и М.Ф. Цепляева пошли на сотрудничество с органами ЧК и участвовали в качестве подставных лиц несу- ществующей эсеровской подпольной организации при ликвидации антоновско- го восстания в Тамбовской губернии. В целом, пожелавшие сотрудничать с большевиками левые эсеры пошли по пути ассимиляции. Это позволило им ос- таться в постреволюционной субкультуре «заслуженных революционеров», но лишило возможности политической деятельности. Так, в 1957 г. на встрече уча- стников революции 1917 г. почетными гостями были Е.Ф. Муравьев, М.А. Чер- нышев35. Те же, кто (как один из крупных деятелей ПЛСР в России и на Украи- не Я.С. Базарный) попытался пойти на примирение с властью, но лелеял наде- жду на возможное восстановление политической деятельности, жестоко попла- тился в 1930-х гг. Сотрудничество проявлялось и на уровне национальных партий и комите- тов. Проживание на территории Воронежской губернии беженцев из западных регионов бывшей империи, наличие сегмента еврейского и немецкого населе- ния привело к активности здесь национальных партий и организаций. Во время Февральского режима проявили себя Бунд, сионистская организация, Поалей- Цион, Еврейская и Польская социалистические партии, причем некоторые из них имели доступ к работе Совета. Известно, что польские социалисты примы- кали к левым эсерам и были настроены наиболее комплиментарно к Октябрь- скому режиму. 5 декабря 1917 г. в мандатную комиссию воронежского Совета 2018 ∙ Vol. 2 ∙ № 4 128 http://hpchsu.ru RESEARCH обратились польские делегаты, сообщавшие, что Воронеже образовалась груп- па Польской партии социалистов-революционеров, на собрании 3 декабря 1917 г. тридцати трех членов которой в Совет был делегирован К. Будогоский. Воронежский телеграф. 1917. 15 декабря. С. 3. 40 Два архивных документа / сост. С.Н. Третьяков. М.: Издательство Олега Пахмутова, 2014. С. 91–95. го комиссариата земельный, земский, призрения, юридический, хозяйственный и медико-санитарный. Поляки выступили с полной поддержкой Октябрьского переворота: «ибо вторая Российская революция пережила великий праздник, тот день объединения съезда советов с центральным комитетом советов раб. и солд. депутатов. Рево- люционная демократия только теперь углубит революцию, объединяясь вокруг совета народных комиссаров». В том же обращении поляки приветствовали декреты СНК и Польскую Демократическую народную республику36. Один из лидеров польских социалистов С.И. Данилькевич в конце 1917 г. занимал пост товарища председателя Воронежского Совета. В дальнейшем сотрудничество осуществлялось в форме ассимиляции на- циональных организаций в структуры РКП(б) и культурно-просветительных органов. В 1918 г. существовал Губернский комиссариат по еврейским нацио- нальным делам. Еврейская секция работала не только при губкоме РКП(б), но и в местной организации РКСМ. К августу 1918 г. при губкоме РКП(б) работали литовская, латвийская, немецкая секции. До осени 1918 г. в Воронеже присут- ствовала украинская «Громада», Краевой комитет украинской ПЛСР, эвакуиро- вавшийся из Харькова. В 1922 г. в Воронеже существовал Украинский клуб имени Т.Г. Шевченко, при отделе нацменьшинств губернского отдела народно- го образования было создано Украинское губернское бюро37. В дальнейшем сотрудничество осуществлялось в форме ассимиляции на- циональных организаций в структуры РКП(б) и культурно-просветительных органов. В 1918 г. существовал Губернский комиссариат по еврейским нацио- нальным делам. Еврейская секция работала не только при губкоме РКП(б), но и в местной организации РКСМ. К августу 1918 г. при губкоме РКП(б) работали литовская, латвийская, немецкая секции. До осени 1918 г. в Воронеже присут- ствовала украинская «Громада», Краевой комитет украинской ПЛСР, эвакуиро- вавшийся из Харькова. В 1922 г. в Воронеже существовал Украинский клуб имени Т.Г. Шевченко, при отделе нацменьшинств губернского отдела народно- го образования было создано Украинское губернское бюро37. р р у р р В Воронежской губернии на рубеже 1917–1918 гг. проявили себя и межпар- тийные организации, такие как комитет защиты родины и революции и Союз защиты Учредительного собрания. Так, на заседании организационного собра- ния Союза защиты Учредительного собрания 13 декабря 1917 г. в Воронеже участвовали представители от 24 организаций, в том числе от партий, проф- союзов, рабочих комитетов, военных 59-го полка под председательством энеса Н.В. Чехова. Правда, собрание было разогнано Военно-революционным коми- тетом38. В уездных Валуйках в заседании комитета спасения родины и револю- ции 5 декабря 1917 г. участвовали кадеты, эсеры, энесы, социал-демократы, представители местного Совета рабочих и солдатских депутатов, уездной укра- 2018 ∙ Vol. 2 ∙ № 4 129 http://hpchsu.ru RESEARCH инской рады и председателя уездной комиссии по выборам в Учредительное собрание39. Есть сведения о деятельности подобного же рода организаций в уездных Боброве, Острогожске. го комиссариата земельный, земский, призрения, юридический, хозяйственный и медико-санитарный. В то же время большевистско-левоэсеровская власть обнаружила способность заключать союзы с военизированными форми- рованиями неустойчивой политической ориентации. В апреле 1918 г. в Воро- неже на несколько дней возникла «федерация» из представителей красноармей- ских отрядов, занявших город и позиционировавших себя как «анархисты», во- ронежского руководства большевиков и левых эсеров. На несколько дней Ис- полком Совета был распущен. Вероятнее всего, с «анархистами», которых под- держала большая часть воронежского гарнизона, пошли на сотрудничество именно левые эсеры, тем более, что вошедшие в город части находились под общим руководством левого эсера Г.К. Петрова40. 39 Воронежский телеграф. 1917. 15 декабря. С. 3. 40 39 Воронежский телеграф. 1917. 15 декабря. С. 3. 40 Два архивных документа / сост. С.Н. Третьяков. М.: Издательство Олега Пахмутова, 2014 С 91–95 2 D.J. Raleigh, Revolution on the Volga: 1917 in Saratov (Ithaca , N.Y.: Cornell University Press, 1986); D.J. Raleigh, “Revolutionary Politics in Provincial Russia: The Tsaritsyn ‘Republic’ in 1917”, Slavic Review, vol. 40. no. 2 (1981), 194–209; D.J. Raleigh, “The Revolution of 1917 and the Establishment of Soviet Power in Saratov”, in Politics and Society in Provincial Russia: Sara- tov, 1590 – 1917; edited by A. Rex Wade and Scott Seregny, 277–306 (Columbus, Ohio: Ohio State University Press, 1989). 1 V.T. Dronov, “Consent as a civilizational archetype of Russian society”, Sotsis, no. 9 (2016): 157–60. See reviews: O.A. Karmadonov, “Solidarity, integration, conjunction”, Sotsis, no. 2 (2015): 3–12; V.V. Bondaletov, “Evolution of self-organization ideas”, Sotsis, no. 3 (2015): 132– 39. Заключение Сказанное выше, конечно, не отменяет наличия в стране мощного социаль- ного, политического конфликта. Вместе с тем, мы фиксируем существенное развитие политического режима в центре и на местах в аспекте сотрудничества. Ритмы этого развития могли не совпадать, однако формы, в которых оно проте- кало, демонстрируют общность. В феврале – октябре 1917 г. сотрудничество осуществлялось на уровне Временного правительства и восходящих к нему управленческих структурах на местах, в органах революционного самоуправ- ления, создавались межпартийные блоки, происходило слияние близких по идеологии партий. Отличительной чертой этого периода стало как раз многооб- разие субъектов политического сотрудничества, искавших разнообразные фор- мы взаимодействия. Полагаю, что переломным моментом в смене режимов стал не только Октябрьский переворот, но и неудавшееся Учредительное соб- рание, после краха которого начался демонтаж Февральского режима, держав- шегося до этого времени именно на структурах общественной солидарности. Впрочем, в 1918 г. подобные тенденции продолжали существовать в рамках режимов «демократической контрреволюции», действовавших под знаменем солидарности абсолютно всех антибольшевистских политических сил. Октябрьский режим демонстрировал возможности для компромиссов на по- литическом, социальном, национальном уровнях, отрицая, однако, плюрализм как основу культурно-идеологической составляющей политической системы. В качестве форм политического сотрудничества продолжали существовать кон- такты между социалистическими партиями, в новую политическую систему ин- тегрировались не только органы «демократического» самоуправления (Советы, 2018 ∙ Vol. 2 ∙ № 4 130 http://hpchsu.ru RESEARCH профсоюзные объединения), но и рабочие структуры земств, городских дум, национальные организации. Предпочтительной формой государственно- территориального устройства представлялась федерация. Одновременно Белые режимы предлагали сходную авторитарную модель политического взаимодей- ствия, сохраняя, однако, внешние атрибуты демократического взаимодействия: межпартийные и внепартийные объединения, сотрудничество партий в прави- тельствах, наличие местного самоуправления. профсоюзные объединения), но и рабочие структуры земств, городских дум, национальные организации. Предпочтительной формой государственно- территориального устройства представлялась федерация. Одновременно Белые режимы предлагали сходную авторитарную модель политического взаимодей- ствия, сохраняя, однако, внешние атрибуты демократического взаимодействия: межпартийные и внепартийные объединения, сотрудничество партий в прави- тельствах, наличие местного самоуправления. 1 V.T. Dronov, “Consent as a civilizational archetype of Russian society”, Sotsis, no. 9 (2016): 157–60. See reviews: O.A. Karmadonov, “Solidarity, integration, conjunction”, Sotsis, no. 2 (2015): 3–12; V.V. Bondaletov, “Evolution of self-organization ideas”, Sotsis, no. 3 (2015): 132– 39. 2 D.J. Raleigh, Revolution on the Volga: 1917 in Saratov (Ithaca , N.Y.: Cornell University Press, 1986); D.J. Raleigh, “Revolutionary Politics in Provincial Russia: The Tsaritsyn ‘Republic’ in 1917”, Slavic Review, vol. 40. no. 2 (1981), 194–209; D.J. Raleigh, “The Revolution of 1917 and the Establishment of Soviet Power in Saratov”, in Politics and Society in Provincial Russia: Sara- tov, 1590 – 1917; edited by A. Rex Wade and Scott Seregny, 277–306 (Columbus, Ohio: Ohio State University Press, 1989). 3 V.M. Kruzhinov and Z.N. Sokova, “The bodies of multi-party socialist power in the Urals in the autumn of 1917”, Russian History, no. 5 (2017): 44–61; M.A. Fel’dman, "Industrial workers of Russia in 1917: Approaches to the ‘socialist’ revolution", Russian History, no. 5 (2017): 44–61; M.K. Shatsillo, "Russian entrepreneurs in search of contacts with the Soviet power in 1917–1918", Russian History, no. 1 (2016); 28–43; O.M. Morozova, "Don in 1917: A peaceful prelude to the revolution:, in 1917 in the Destinies of the Region, Country and the World. A glance from the 21st century; ed. V.I. Goldin (Arkhangelsk: NArFU, 2017), 132–36; W.G. Rosenberg, Liberals in the Russian Revolution: The Constitutional Democratic Party, 1917–1921 (Princeton, N.J.: Prinston University Press, 1974); E. Mawdsley, The Russian Civil War (Edinburgh: , 2000); J.D. Smele, The ‘Russian’ Civil Wars, 1916–1926. Ten years that shook the world. London: Hurst & Company, 2015. Introduction To view the revolution purely as a conflict, even more as a systemic crisis, would be true, however does little to understand its transformative potential, hence the productivity of this approach is limited. At the same time, sociological concepts pay attention to the crucial role of social integration, solidarity, cooperation and consoli- dation in the development of the idea of social harmony1. These concepts, as well as the idea that revolutions are made not only for the sake of destruction, provided the grounds for this study. In this article, I suggest considering revolution as a variety of cooperative models applied not only to the capital cities, however also to the provinc- es. The Voronezh province is given as a case study to consider the situation in the Russian provinces in general, outside of large cities. We shall note that in the foreign historiography, the development of the revolutionary situation in the provinces, in- cluding elements of political cooperation, was reviewed on the materials of Saratov and several other biggest cities on the shore of Volga by the well-known expert on Russian history, D.J. Raleigh2. 2018 ∙ Vol. 2 ∙ № 4 131 http://hpchsu.ru Main body In the Soviets, the expected majority was made up of socialist parties – the SRs, the RSDLP and the Popular Socialists. The All-Russian Central Executive Committee set up at the First All-Russia Congress of Soviets in June 1917 included 107 Mensheviks, 101 Socialist-Revolutionaries, 35 Bolsheviks, eight ‘united’ Social- Democrats, four Trudoviks and Popular Socialists and one member of the Jewish So- cialist Workers Party. The Soviets delegated their representatives to the city Dumas – the liberals’ assembly points. Because of re-elections, in summer, the city govern- ment was party-coalitional in nature. The social representation in it was provided not only by the Soviets, but also by the trade unions, which were headed by representa- tives of various parties too. Within the scope of one large city, various trade unions could be headed by the Bolsheviks, Mensheviks, Socialist-Revolutionaries and repre- sentatives of national parties who simply had to enter a dialogue with each other. At the Third All-Russian Conference of Trade Unions (June 1917), a provisional gov- erning body was elected consisting of 16 Bolsheviks, 16 Mensheviks and three So- cialist-Revolutionaries. The committees of civic organizations headed by the Cadets, Mensheviks and Socialist-Revolutionaries had a significant influence on shaping rev- olutionary power until July 1917 and from then onwards continued to assert their right for existence through the idea of representing various political forces. Throughout 1917, the socialist parties, including the Bolsheviks, considered the possibility of creating a united front. V.M. Chernov advanced the idea of uniting so- cialists into a single party; the Mensheviks tried to recreate the unity of the RSDLP in May–August 1917 by convening a conference and a congress. At the second congress of the Popular Socialist Party (22–28th September 1917), it was stressed that to con- solidate the social and governmental forces, the party would cooperate with the Right Socialist-Revolutionaries, the Menshevik internationalists, the Edinstvo (‘Unity’) group and Cadets. The Bolsheviks regarded themselves as part of a common political framework, along with other socialist parties, at least until September 1917, and in some cases even before the convocation of the Constituent Assembly. Speaking about the possi- bility of peaceful development of the proletarian revolution, V.I. Main body The February Regime gave great opportunities for the democratization of Russian society. Under these circumstances, the processes associated with political coopera- tion became more intense.3 It is obvious that various participants of the revolutionary process became allies and joined organizations where they had to interact with each other within the political framework.4 The policy papers of various political forces showed significant similarity, allowing justification of the blocking from the ideolog- ical point of view.5 Initially, the liberal Provisional Government veered to the left very quickly, and by the summer of 1917 it was comprised of socialists. In addition to the above, the main liberal party, Cadets, participated in all three coalition govern- ments, and it even managed to strengthen its positions in its third composition, in- creasing the number of seats from four to six. The platform for cooperation between the right-wing liberal forces was the Moscow State Conference (held in August 1917). In September 1917, the All-Russia Democratic Conference took place in Pet- rograd attended by delegates from the Soviets, local self-governing authorities, trade unions, cooperatives, army corps and national institutions. In the pre-parliament elected at the meeting, there were 135 Socialist-Revolutionaries, 92 Mensheviks, 75 Cadets, 58 Bolsheviks, and 30 Popular Socialists. The coalitional ideas were charac- teristic for the executive power at the local level – governorate commissioners, pro- vincial and district executive committees. 4 As the given facts of cooperation (but not their systematization in this context) are well known, a review of the situation on the scale of the former Russian Empire is based on the analysis of information from specialized encyclopedic literature: Revolution and the Civil War in Russia: 1917–1923; Chief editor S.A. Kondratov (Moscow: TERRA, 2008); Political parties of Russia. End of 19th – the first third of the 20th century; chief editor V.V. Shelokhaev (Moscow: ROSSPEN, 1996); Civil War and Military Intervention in the USSR / chief editor S.S. Khromov (Moscow: Sovetskaya entsiklopediya, 1983). 5 Models of public restructuring in Russia. The 20th century; chief editor V.V. Shelokhaev (Moscow: ROSSPEN, 2004); M.E. Razinkov, "The Popular Socialist Party and the Russian Com- munist Party (the Bolsheviks): A comparative analysis of program instructions", in History and His- toriography of Right-wing Narodnichestvo: Collection of articles; ed. G.N. Mokshin [and others]] (Voronezh: Istoki, 2014), 141–52. 2018 ∙ Vol. 2 ∙ № 4 132 http://hpchsu.ru RESEARCH Another ground for cooperation belonged to the authorities of revolutionary self- government. 6 V.I. Lenin, Lenin Collected Works, vol. 34 (Moscow: Gospolitizdat, 1977), 222–23. 7 V.M. Kruzhinov and Z.N. Sokova, The bodies of multi-party socialist, 44–61; L.G. Protasov, All-Russian Constituent Assembly: The History of Birth and ‘Death’ (Moscow: ROSSPEN, 1997), 271. 8 Revolution and the Civil War in Russia, vol. 1, 361–62. 9 See bloc-founding charts: L.G. Protasov, All-Russian Constituent Assembly: The History of Birth and ‘Death’ (Moscow: ROSSPEN, 1997), 134, 137. Main body Lenin cited the ex- ample of the Kornilov affair, when even a short-term alliance of the Bolsheviks, Mensheviks and Socialist-Revolutionaries gave the “complete victory over the coun- ter-revolution achieved with an ease unprecedented in any revolution; it gave such a crushing suppression of bourgeois, landlord and capitalist, allied imperialist and Ca- det counter-revolution, that Civil War from crumbled to dust on this side, turned into nothing at the very beginning, disintegrated to any kind of ‘a battle’.” It was the Bol- shevik leader’s idea that such an alliance would allow to view the revolution not from the military perspective but from a political angle, because “within the Soviets, the struggle of the parties for power could proceed peacefully, with the full democracy of 2018 ∙ Vol. 2 ∙ № 4 133 http://hpchsu.ru RESEARCH the Soviets...”6 In the course of the October coup, the Bolsheviks kept looking for compromises and avoided burning bridges, whilst joining various coalition governing authorities, as it was, for example, in the Urals or in Kursk7. The refusal of V.I. Lenin and L.D. Trotsky to form a ‘homogeneous socialist government’ caused the first cri- sis in the party that seized power. The third aspect of cooperation was the merger of close political groups. In June 1917, the Trudoviks (Labor Group) and the Popular Socialists united, in July the Mezhraiontsy (‘Interdistrictites’) merged into the RSDLP(b), by August – the same happened to most members of the Vperyod (‘Forward’) group. In September, the small Liberal-Republican and Radical-Democratic parties merged too. This rap- prochement also affected the national parties. As an example, in the summer of 1917, the Jewish Socialist and Zionist-Socialist Workers Parties united as well. Finally, there emerged public associations of a political nature however with no party affiliations. From February to December 1917, the Provisional Committee of the State Duma was functioning, which included the Octobrist Party, Cadets, Men- sheviks and representatives of most Duma factions. Since August, the council of community leaders in Moscow commenced its activities, which united members of the State Duma, Cadets, industrialists, members of zemstvos with electoral qualifica- tions and representatives of the academia. Main body It was announced on the intentions of the political alliance (blocs) with the Socialists at the second meeting of community leaders on 12–14th October 1917, which “advocated support of the groups using the state platform as their ground during the elections to the Constituent Assembly and merger with the Popular Socialist Party and the Edinstvo ‘Unity’ group8. After the Bolshevik coup, the possibilities for political cooperation remained. The end of 1917–18 was the time to search for a democratic alternative to the Bolshevik dictatorship. The whole spectrum of inter-party and political-social formation of blocs was presented at the elections to the Constituent Assembly, where, along with predictable blocs, for example, the Socialist-Revolutionaries and Mensheviks, one could come across the agreements of Cadets and Plekhanov’s ‘Unity’, religious as- sociations together with the Popular Socialists, the Bolsheviks with the Mensheviks, etc.9 At the same time, the Bolshevik regime interacted with other political forces. The leaders of the White Movement and national governments offered their own co- operation projects. 2018 ∙ Vol. 2 ∙ № 4 134 http://hpchsu.ru RESEARCH The anti-Bolshevik governments became center of attraction for socialist and lib- eral political forces, such as the Committee of Members of the Constituent Assembly, the Provisional Siberian Government, the Provisional Government of the Northern Region and the Ufa Directory. The Civil Council stood at the origins of the Volunteer Army, and the said council included four socialists and seven liberals (including M.M. Fedorov, B.V. Savinkov and P.B. Struve). The supreme power was exercised by the triumvirate: A.M. Kaledin ruled the Don region; L.G. Kornilov commanded the army; M.V. Alexeyev concentrated in his hands the civil administration, foreign contacts and finances. Even though in the time following, the idea of a military dicta- torship triumphed, the basis of the White Movement was perceived as confederations and federations of various sorts: The Armed Forces of South Russia, the union of the regions headed by the white Supreme Ruler A.V. Kolchak, the federative project of the Don-Caucasus union suggested by P.N. Krasnov. It is significant that in the late 1917–18, Soviet Russia was also perceived as a confederation of revolutionary repub- lics (Donetsk-Krivorozhskaya, Terskaya, Kuban-Chernomorskaya, etc.), which insti- tutionally brought it together with the anti-Bolshevik confederations. y g g The most notable union within the scope of the October regime without doubt was the alliance of the Bolsheviks with Left SRs (November 1917 – March 1918). Main body ( ) At the same time, whilst in the anti-Bolshevik governments, the Socialist- Revolutionaries and Mensheviks could occupy leading positions, within the frame- work of the Soviet system they were given the role of an unsuccessful critic. There- fore, it is quite understandable that another type of cooperation appeared at the end of the Civil War in the form of assimilation, i.e. transition of many socialist party fig- ures and sometimes even parties themselves to the ranks of the Bolsheviks. In 1920– 21, the Bund and the Russian Social Democratic Labor Party (Internationalists) merged with the RSDLP(i) in 1919, and the Russian party of independent social- democratic left-wing Internationalists joined the All-Russian Communist Party (bol’shevikov). There was also another merge of alternative communist groups with the RCP(b): The Party of the Narodnik Communists (October–November 1918) and the Party of Revolutionary Communism (September 1920). Similar processes were observed in the national territories of the disintegrated empire. As an example, the communist Polish Labor Party established in 1918 because of unification of the So- cial Democracy of the Kingdom of Poland and Lithuania with the Polish Socialist Party – the Levite became a powerful center to attract the communist and socialist orientated branches of the national parties in 1919–20. The party included the Polish local branches of the Bund, Poalei Zion, the Ukrainian Social-Democratic Labor Par- ty and the Belarusian Socialist Party. Another form of cooperation that was widely spread in the post-October period were the associations without party affiliation, such as the Committee for Salvation of Motherland and the Revolution, the Union for Constituent Assembly Protection, the Union of the New Russian Renaissance and the center-Right, as well as national, tac- tical and political centers (parties). It can be seen from the above-mentioned facts that the diversity of cooperative forms was part of the revolutionary political culture, not only during the February Regime, but also in the post-October period, which is usually regarded as the period of one-party dictatorship. The most important forms of cooperation were as follows: interaction of the parties and public organizations in the revolutionary state and mu- nicipal authorities, cooperation of the parties up to their merging and creation of non- party socio-political associations. In my opinion, this interaction, which had a signifi- cant democratic potential, created a powerful and diverse basis for the democratic component in the expanded modernization process of the Russian state. Main body The Left SRs participated in the work of all the collegiate of the All-Russian Central Executive Committee occupying half of the seats there, used considerable influence in the Moscow Regional Council of People’s Commissars, the Council of Commis- sioners of the Northern Region Commune Union, in the Central Chernozem Region and the Council of People’s Commissars in Siberia and played a dominant role in the Council of People’s Commissars (Turkestan) and in the Turkestan Central Executive Committee. Driven out of the Russian political framework in the summer of 1918, the Left SRs operated successfully as a leftist center in Ukraine. However, this forcing out was to some extent conditional, since the Left SRs, along with the anarchists, par- ticipated in the Second All-Russia Congress of Trade Unions (16–25th January 1919). The party of the Left SRs (the United Socialist Party of Russia) could participate in the Eighth All-Russian Congress of Soviets in December 1920. The contacts of the Bolsheviks with the socialist parties remained, and the striv- ing for such cooperation was mutual. Some of the socialists preferred to interact with the Bolsheviks and not with the White Movement regimes. The Bolsheviks, in turn, were interested in contacts with the socialists, when by the end of 1918 it became clear that the Civil War became stalled. Despite the factual break in relations between the RSDLP (United) and the Bolsheviks in June-October 1918, the former returned to the policy of institutional cooperation against the forces of the counter-revolution, acting as opposition in the Central Executive Committee, the Soviets and the trade unions. The reports “On the Tasks of Trade Unions” at the Second All-Russian Con- gress of Trade Unions were read not only by V.I. Lenin but also by Yu.O. Martov 2018 ∙ Vol. 2 ∙ № 4 135 http://hpchsu.ru RESEARCH and S.A. Lozovsky (the RSDLP (Internationalist). The SRs found themselves in a more difficult situation declaring a war against the Bolsheviks in May 1918, however being defeated by the White Movement. As early as in December 1918, at a meeting in Ufa, the prominent Socialist Revolutionaries decided to end the war with the Bol- sheviks. The Central Committee did not recognize the actions of the Ufa delegation as legitimate for a long time but ultimately decided to agree to the talks with the RSDLP(b). Main body For the Feb- 136 2018 ∙ Vol. 2 ∙ № 4 http://hpchsu.ru RESEARCH ruary regime, the cooperation was the basis of a new government formed from par- ties, state and self-government authorities. The democratic governments formed in Siberia, the Volga region and the Northern region in 1918 tried to extend this line. The October regime viewed this cooperation not only as a forced measure but also as a source of replenishment for the ruling party that positioned itself as the only center of attraction for all ‘healthy’ forces in the country. The White regimes gravitating to- wards a dictatorship like the Bolsheviks, identified themselves in line with the West- ern values, whilst cooperating with all the anti-Bolshevik forces, but preferring, how- ever, their right-liberal component. To understand the nuances of the revolutionary process development, it is im- portant to look at the situation in the Russian province, a typical example of which may be the agrarian Voronezh province, where mostly Russians resided. It may be safely said that like the Provisional Government, the local activists sought to create a new government based on a broad cooperation between the state institutions and public organizations, which was expressed in the collegiality of deci- sions on most issues. The Voronezh Committee of Public Safety formed on 11th March 1917 on an equal footing included representatives of the City Duma, the Sovi- et of Workers’ Deputies and the committee of joint public organizations and institu- tions, as well as delegated soldiers and officers. Simultaneously, the committee of joint public organizations included representatives of 101 establishments and educa- tional institutions. On 17th March, the City Duma included 26 delegates from the So- viets10. Even though by the summer of 1917, the relationship between the Voronezh So- viet and the Committee of Public Safety had deteriorated noticeably, the interaction with the authorities that were directly subordinated to the Provisional Government remained and even continued to expand. The range of common points was quite im- pressive: cultural and educational work, meetings regarding food, fuel, social assis- tance, combatting crime and neutralizing the peasant movement. At the meetings that were held by the Governorate Executive Committee and various consultations, the state authorities, dedicated commissions and committees, zemstvo and city self- government authorities and army commanders were permanently represented11. 2018 ∙ Vol. 2 ∙ № 4 137 10 Gosudarstvennyi arkhiv Voronezhskoi oblasti (GAVO) f. р-2393, op. 1, d. 12, l. 7; d. 13, l. 7–8. 11 GAVO f. Р-2393, op. 1, d. 3, l. 111 ob.; d. 10, l. 89. 12 GAVO f. Р-2393, op. 1, d. 2, l. 27, 53, 59; Gosudarstvennyi arkhiv obshchestvenno- politicheskoi istorii Voronezhskoi oblasti (GAOPI VO) f. 5, op. 1, d. 320, l. 23; P.N. Sobolev, Preparation of the socialist revolution and establishment of the Soviet power in Voronezh province (Voronezh: Voronezhskoe knizhnoe izdatel’stvo, 1955), 56. 13 S.V. Filiptseva, “End of the provincial zemstvo functioning in Voronezh in 1918”, in From the history of the Voronezh Region, issue 18, 218–21 (Voronezh: VGU, 2011). 14 GAVO р-2393, op. 1, d. 3, l. 114, 117–18. 15 GAOPI VO f. 5, op. 1, d. 459, l. 21–23. 16 P.N. Sobolev, Preparation of the socialist revolution and establishment of the Soviet power in Voronezh province (Voronezh: Voronezhskoe knizhnoe izdatel’stvo, 1955), 56. 17 Voronezhskii telegraf [Voronezh Telegraph], 12th September, 1917, 1. Main body After the October coup, the relationship between the Soviet, public organizations and institutions created during the February Regime or even prior to it, was not bro- ken at once. The Governorate Food Administration, zemstvos and Voronezh City Duma expressed dissatisfaction with the new authorities; however, they periodically issued resolutions with regards to the need to continue operating under the new con- ditions. The provincial commissar B.A. Keller appointed to this post as far back as 2018 ∙ Vol. 2 ∙ № 4 137 http://hpchsu.ru RESEARCH July 1917 worked until December 1917. The pre-October staff of the district court, the prosecutor’s office and the provincial zemstvo council held meetings until Janu- ary 191812. The decision of the Provincial Committee on the liquidation of provincial zemstvo institutions was not made until August 1918, and the zemstvo institutions, together with the old employees, were incorporated into the regional economic Soviet (sovnarkhoz). It is significant that the power of the Bolsheviks did not ‘demand blood’ even of former obvious opponents. Thus, the first provincial commissar of the Provisional Government and chairman of the provincial zemstvo council V.N. Tomanovsky as early as in December 1918 held the post of head of the administrative and fiscal sub-department of the financial department in Voronezh Governorate Ex- ecutive Committee13. B.A. Keller, who was involved in the performance of work in the Voronezh Oblast Executive Committee in the 1920-s continued his work as pro- fessor at the Voronezh Agricultural Institute and later as an academician of the USSR Academy of Sciences and VASKhNIL. July 1917 worked until December 1917. The pre-October staff of the district court, the prosecutor’s office and the provincial zemstvo council held meetings until Janu- ary 191812. The decision of the Provincial Committee on the liquidation of provincial zemstvo institutions was not made until August 1918, and the zemstvo institutions, together with the old employees, were incorporated into the regional economic Soviet (sovnarkhoz). It is significant that the power of the Bolsheviks did not ‘demand blood’ even of former obvious opponents. Thus, the first provincial commissar of the Provisional Government and chairman of the provincial zemstvo council V.N. Tomanovsky as early as in December 1918 held the post of head of the administrative and fiscal sub-department of the financial department in Voronezh Governorate Ex- ecutive Committee13. B.A. 12 GAVO f. Р-2393, op. 1, d. 2, l. 27, 53, 59; Gosudarstvennyi arkhiv obshchestvenno- politicheskoi istorii Voronezhskoi oblasti (GAOPI VO) f. 5, op. 1, d. 320, l. 23; P.N. Sobolev, Preparation of the socialist revolution and establishment of the Soviet power in Voronezh province (Voronezh: Voronezhskoe knizhnoe izdatel’stvo, 1955), 56. (Voronezh: Voronezhskoe knizhnoe izdatel stvo, 1955), 56. 13 S.V. Filiptseva, “End of the provincial zemstvo functioning in Voronezh in 1918”, in From the history of the Voronezh Region, issue 18, 218–21 (Voronezh: VGU, 2011). 14 GAVO р-2393, op. 1, d. 3, l. 114, 117–18. 15 GAOPI VO f. 5, op. 1, d. 459, l. 21–23. 16 P.N. Sobolev, Preparation of the socialist revolution and establishment of the Soviet power in Voronezh province (Voronezh: Voronezhskoe knizhnoe izdatel’stvo, 1955), 56. 17 Voronezhskii telegraf [Voronezh Telegraph], 12th September, 1917, 1. 18 GAVO р-2393, op. 1, d. 3, l. 20, 34 ob., 58 ob., 73, 77, 79; d. 4, l. 85, 90. 19 Voronezh Regional Museum of Local Lore. Foundation of participants in the revolution and civil war. Folder No. 655/I. Vrachev I.Ya. 20 Put’ Zhizni [The Way of Life]. 1917. No. 1–7. 21 Put’ Zhizni [The Way of Life]. 1917. No. 12. Pp. 3–4. Main body Keller, who was involved in the performance of work in the Voronezh Oblast Executive Committee in the 1920-s continued his work as pro- fessor at the Voronezh Agricultural Institute and later as an academician of the USSR Academy of Sciences and VASKhNIL. The cooperation of various segments in the Russian political system during 1917 – the first half of 1918 was explained by the presence of representatives of the same parties in those segments. Thus, the SRs, the Popular Socialists, the Social Democrats and an anarchist were listed as the participants of the meeting of the Governorate Ex- ecutive Committee on 20th May 1917. At that time, the socialist-revolutionary V.P. Kabytchenko was appointed chairman, and the popular socialist (former Cadet) P.Ya. Rostovtsev – as deputy chairman14. After the July elections in the Voronezh City Duma, a council with an equal representation of the Socialist-Revolutionaries, Men- sheviks and Cadets was formed15. There was a similar situation in the uyezd (district) centers. Out of the 22 deputies of the Valuyki Town Council, 17 were socialist- revolutionaries, two deputies were Bolsheviks, and one person was elected from Ca- dets and the Popular Socialists. In the Ostrogozhsk City Duma, Cadets received three seats, whereas the Bolsheviks won six seats16. The Voronezh City Duma elected in July provided a coalition of the Socialist-Revolutionaries, the Mensheviks and Ca- dets17. At the same time, the socialist parties cooperated within the Soviet, where by the summer of 1917 there were the Socialist-Revolutionaries, the Bolsheviks, the Men- , p , , 16 P.N. Sobolev, Preparation of the socialist revolution and establishment of the Soviet power in Voronezh province (Voronezh: Voronezhskoe knizhnoe izdatel’stvo, 1955), 56. 17 Voronezhskii telegraf [Voronezh Telegraph], 12th September, 1917, 1. 138 2018 ∙ Vol. 2 ∙ № 4 http://hpchsu.ru RESEARCH sheviks and representatives of the Bund. In August 1917, a decision was made to grant a seat in the Council to the Jewish Socialist Workers Party, and in September – to the Popular Socialist Party. In the summer of 1917, the united library of the Social- ist-Revolutionaries and Social Democrats functioned in the Soviet (Council). At least in August–September 1917, there existed a socialist club, which included representa- tives of the Socialist-Revolutionaries and the Bolsheviks18. In other words, the con- tradictions between the parties were not fatal, and the points of contact were institu- tionalized. 22 Voronezhskii telegraf [Voronezh Telegraph], 12th November, 1917, 2–3. 23 Voronezhskii telegraf [Voronezh Telegraph], 9–11th November, 1917. 24 Voronezhskii telegraf [Voronezh Telegraph], 18th November, 1917, 1. 25 GAVO f. р-458, op. 1, d. 50, l. 19 ob.–20, 43; d. 258, l. 1ob. Main body The cooperation was also carried out at the level of party committees, the Committee of South-Eastern Railway and emergency bodies: The Revolutionary Rules Committee established to curtail Kornilov’s speeches, the Committee for Pub- lic Safety and the Provisional Revolutionary Committee (the VRK) that existed dur- ing the events of October–November 1917. The available information proves that the Voronezh Bolsheviks had serious disa- greements regarding the need for a break with the socialist movement and the coup itself. The Bolsheviks’ Provincial Committee was created only in October 1917; that means that the contacts with the Mensheviks were not cut off completely during al- most the entire of February Regime. The chairman of the Provincial Committee, the member of the old Bolsheviks N.N. Kardashev did not take a direct part in the organ- ization of the coup in Voronezh. The latter was carried out under the leadership of a ‘young’ group led by the member of Mezhraiontsy (‘Interdistrictites’) A.S. Moiseyev who came from Petrograd after the events of July. The member of the Bolshevik Pro- vincial Committee S.D. Turchaninov in early December 1917 left the party in protest against the October coup19. It is significant that the publications of the Voronezh Bol- shevik newspaper Put’ Zhizni (‘The Way of Life’) in August–September contained both a statement about the need for a civil war and attacks on the social-chauvinists, as well as announcements of the work of the Socialist club accompanied by the speeches of the Socialist-Revolutionary leaders20. Before the coup, the Bolsheviks and the Left SRs proposed creation of a bureau to arrange re-elections to the Council, which included not only them but also Socialist-Revolutionaries, Mensheviks and po- litically uncommitted people21. The succession of power to the Military Revolution Committee in Voronezh did not mean that the socialist parties were excluded from the political life. It must be said that representatives of the Military Revolutionary Committee did not intend to position themselves as explicit dictators and regarded themselves as an authority that tried to prevent the counter-revolutionary actions in Voronezh. The delegates and the 2018 ∙ Vol. 2 ∙ № 4 139 http://hpchsu.ru RESEARCH commissars sent forward to the militia and banks, to the mayor of the city and to the Duma, advocated their interests sharply, however did not seek to take up managerial posts against someone’s will in these organizations. Main body This led to the figures that were removed from power raising their eyebrows in surprise and gave rise to hopes for a possible revision of the situation. Throughout the first half of November, the Council tried to function with the help of the pre-October staff. The factions of the Socialist- Revolutionaries, Mensheviks and non-party people withdrew from the Council only on 10th November22. In November 1917, the Socialist-Revolutionaries managed to gather a peasant congress in Voronezh, and until the spring of 1918 they held meet- ings in the food commissions. The cooperation of the parties and public organizations based on protesting the Bolshevik – left-wing socialist revolutionist power was nota- ble during the elections to the Constituent Assembly. The elections that took place on 12–14th November were accompanied not only by the declarations of trade union about unwillingness to cooperate with usurpers but also an attempt to consolidate the ‘sound forces of society’. Thus, the Voronezh organization of the Popular Socialist Party created a whole social front, where the Committee of united social organiza- tions continued its work, as well as the Union of Intelligentsia, the Union of Engi- neers and Technicians as well as cooperators’ associations23. The parties and trade union organizations have repeatedly stated the need to cooperate with the governing authorities created by the ‘February regime’ (the Duma, councils and committees) and called for opening up relations with them at the inter-provincial level24. commissars sent forward to the militia and banks, to the mayor of the city and to the Duma, advocated their interests sharply, however did not seek to take up managerial posts against someone’s will in these organizations. This led to the figures that were removed from power raising their eyebrows in surprise and gave rise to hopes for a possible revision of the situation. Throughout the first half of November, the Council tried to function with the help of the pre-October staff. The factions of the Socialist- Revolutionaries, Mensheviks and non-party people withdrew from the Council only on 10th November22. In November 1917, the Socialist-Revolutionaries managed to gather a peasant congress in Voronezh, and until the spring of 1918 they held meet- ings in the food commissions. The cooperation of the parties and public organizations based on protesting the Bolshevik – left-wing socialist revolutionist power was nota- ble during the elections to the Constituent Assembly. 26 GAVO р-2393, op. 1, d. 8а, l. 4–4 ob.; P.N. Sobolev, Preparation of the socialist revolution, 106, 142–43; For the Power of the Soviets. Collection of memoirs of participants in the revolution- ary events in Voronezh province in 1917 – 1918; ed. I.G. Voronkov, T.M. Sevast’yanova (Voro- nezh: Voronezhskoe knizhnoe izdatel’stvo, 1957), 86, 116–122. 27 For the Power of the Soviets, 26, 46, 50–53; GAVO f. р-2393, op. 1, d. 8, l. 117. 28 GAVO f. р-905, op. 1, d. 20, l. 107–08ob. 29 P.N. Sobolev, Preparation of the socialist revolution, 111. 30 GAVO f. р-2393, op. 1, d. 2, l. 13-14, 17, 21–21 ob., 36–37, 38 ob., 40, 42, 48, 51 ob., 58 ob., 62 ob., 64 ob., 81–82, 84, 95–96, 105. 26 GAVO р-2393, op. 1, d. 8а, l. 4–4 ob.; P.N. Sobolev, Preparation of the socialist revolution, 106, 142–43; For the Power of the Soviets. Collection of memoirs of participants in the revolution- ary events in Voronezh province in 1917 – 1918; ed. I.G. Voronkov, T.M. Sevast’yanova (Voro- nezh: Voronezhskoe knizhnoe izdatel’stvo, 1957), 86, 116–122. Main body The elections that took place on 12–14th November were accompanied not only by the declarations of trade union about unwillingness to cooperate with usurpers but also an attempt to consolidate the ‘sound forces of society’. Thus, the Voronezh organization of the Popular Socialist Party created a whole social front, where the Committee of united social organiza- tions continued its work, as well as the Union of Intelligentsia, the Union of Engi- neers and Technicians as well as cooperators’ associations23. The parties and trade union organizations have repeatedly stated the need to cooperate with the governing authorities created by the ‘February regime’ (the Duma, councils and committees) and called for opening up relations with them at the inter-provincial level24. p g p p In March 1918, after signing of the Brest-Litovsk Peace Treaty, the Socialist Revolutionary faction declared its protest but remained in the Council. Up to the spring of 1918, the Socialist-Revolutionaries and the Mensheviks occupied important positions in the committee of servicemen of the South-East Railway, the Union of public servants at governmental institutions, and the Union of Engineers and Techni- cians and had access to the resources of the State Bank. At least until June 1918, the newspapers of the Mensheviks and Socialist-Revolutionaries were legally published in the province. A.V. Mikhailov, Chairman of the Council overthrown by the Bolshe- viks, appears in the documents as one of the organizers of the provincial congress of trade unions in 1919, and the Menshevik’s influence in the trade unions can be traced at least until 192025. During the election of the Council in the uyezd town of Pavlovsk in December 1917, a Bolshevik was elected as chairman, and a Socialist- Revolutionary – as his deputy. Moreover, before the end of the summer of 1918, 2018 ∙ Vol. 2 ∙ № 4 140 http://hpchsu.ru RESEARCH there were two Councils functioning there – the City Council under the control of the Socialist-Revolutionaries and the Uyezd Council controlled by the Bolsheviks26. In 1918, the Bolsheviks continued their cooperation with the Left SRs. In October 1917, the representatives of these parties were regularly convened in ‘fives’, where all the tactical issues were discussed. During the coup, the members of the Military Revolutionary Committee, along with the Bolsheviks A.S. Moiseyev, V.N. Gubanov, I.A. Chuyev and I.Ya. Vrachev, there were the Left SRs E.F. Muravyov, I.S. Plyapis and N.I. Grigoryev. In December 1917, N.I. ary events in Voronezh province in 1917 – 1918; ed. I.G. Voronkov, T.M. Sevast’yanova (Voro- nezh: Voronezhskoe knizhnoe izdatel’stvo, 1957), 86, 116–122. 27 For the Power of the Soviets, 26, 46, 50–53; GAVO f. р-2393, op. 1, d. 8, l. 117. 28 GAVO f. р-905, op. 1, d. 20, l. 107–08ob. 29 P.N. Sobolev, Preparation of the socialist revolution, 111. 30 GAVO f. р-2393, op. 1, d. 2, l. 13-14, 17, 21–21 ob., 36–37, 38 ob., 40, 42, 48, 51 ob., 58 ob., 62 ob., 64 ob., 81–82, 84, 95–96, 105. V , ), , 27 For the Power of the Soviets, 26, 46, 50–53; GAVO f. р-2393, op. 1, d. 8, l. 117. 28 P.N. Sobolev, Preparation of the socialist revolution, 111. 30 GAVO f. р-2393, op. 1, d. 2, l. 13-14, 17, 21–21 ob., 36–37, 38 ob., 40, 42, 48, 51 ob., 58 ob., 62 ob., 64 ob., 81–82, 84, 95–96, 105. G VO . р 905, op. , d. 0, . 07 08ob. 29 P.N. Sobolev, Preparation of the socialist revolution, 111. 31 GAOPI VO f. 1, op. 1, d. 13, l. 1–1ob., 8–9. 32 GAOPI VO f. 5, op. 1, d. 342, l. 1. 33 GAOPI VO f. 1, op. 1, d. 12, l. 2. 34 Voronezh Regional Museum of Local Lore. Foundation of participants in the revolution and civil war f 655/I. Vrachev I.Ya. Main body Grigoryev became Chairman of the VRK for a short time27. The comrades led by the Left SRs E.F. Muravyov and M.A. Chernyshev played one of the key roles during the coup of 30th October 1917. A small detachment led by the Left SR Titkov as a matter of fact performed in late 1917 – early 1918 the functions of the Cheka by arresting suspicious meetings and partici- pating in suppressing outbreaks of discontent in the city28. Deputies from both fac- tions were delegated to the Third All-Russian Congress of Soviets. On 28th December 1917, the Bolsheviks and the Left SRs organized the provincial peasant congress, where out of 169 delegates, there were 92 Bolsheviks, 61 Left Socialist- Revolutionaries and 16 non-party people29. In May 1918, the Left Socialist- Revolutionaries headed the food and land divisions of the Soviets’ Provincial Com- mittee, held the post of chairman of the revolutionary tribunal and participated in the nationalization of the Voronezh banks. In June 1918, the question was raised about the transfer of the financial department to the control of the Left Socialist- Revolutionaries, their participation in the Emergency Commission for Combating Counterrevolution, speculation and crime (sabotage) and publication of the newspa- per, including newspaper of the Ukrainian Left Socialist-Revolutionary Central Committee relocated to Voronezh. The cooperation between factions was also prac- ticed in the trade unions (for example, amongst postal and telegraph employees) and whilst regulating social conflicts30. Even after the relations with the Left SRs started to be cooled (in Voronezh, it happened as early as in mid-June 1918), on 17th June, the Voronezh Committee of the RSDLP(b) proposed to the Left SRs to take the land, zemstvo’s, charity, legal, economic and health care commissariats instead of the key food and financial ones 141 2018 ∙ Vol. 2 ∙ № 4 http://hpchsu.ru Despite the gap that occurred after the Left SRs uprising in July 1918, there were no widespread arrests in Voronezh. On 24th July, the Provincial Committee of the Left SRs considered it possible to ask the Committee of the RCP (b) to lift an attach- ment of their newspaper, Znamya Trudovoi Bor’by (‘The Banner of Labor Struggle’). Main body In the uyezd chief town of Zadonsk, at the end of August 1918, the question was raised about prevention of the Left Socialist-Revolutionaries to be in the commission and in the presidium of the local council, and the Left SRs provided petitions that it was necessary for the party members to take seats in the council31. It is known that in Zemlyansk, one Left SRs was present in the Executive Committee even at the end of September 191832. The unwillingness to finally break away from the Bolsheviks led to the fact that part of Voronezh Left Socialist-Revolutionaries as early as in October 1918 took part in establishing the Party of Narodnik Communists, while still others in general de- clared the need to merge with the RCP (b)33. It will be remembered that in 1920–21, the former leaders of the Left Socialist Revolutionaries in Voronezh E.F. Muravyov and M.F. Tseplyaeva agreed to the cooperation with the Cheka authorities and partic- ipated as assumed names of the non-existent Socialist-Revolutionary underground organization in the liquidation of ‘Antonov’s mutinity’ or the Tambov Rebellion. In general, the Left Socialist-Revolutionaries wishing to cooperate with the Bolsheviks opted for assimilation. This allowed them to remain in the post-revolutionary subcul- ture of the ‘merited revolutionaries’, however deprived them of the possibility to car- ry out political activities. Thus, in 1957, at the meeting of the participants in the Rev- olution of 1917, the honored guests were E.F. Muravyov and M.A. Chernyshev34. Those same people who tried to reconcile with the authorities, however cherished the hope of a possible restoration of political activity, like one of the major figures of the Left SRs in Russia and Ukraine Ya.S. Bazarny, paid heavy price for it in the 1930-s. y p y p The cooperation manifested itself at the level of national parties and committees as well. The accommodation of refugees from the Western regions of the former em- pire on the territory of Voronezh province and presence of a segment with the Jewish and German population led to the activity of national parties and organizations there. During the February regime, the Bund, the Zionist organization, Poalei Zion, the Jew- ish and Polish socialist parties showed themselves, and some of them had access to the Council’s work. It is known that the Polish Socialists adjoined the Left SRs and were most inclined towards the October regime. 35 GAOPI VO f. 5, op. 1, d. 323. l. 144–46. 36 S.N. Korotun, "The sections of national minorities in the Voronezh Governorate in 1917– 1941", in From the history of Voronezh territory. Collection of articles; Responsible ed. A.N. Akinshin (Voronezh: Tsentral'no-Chernozemnoe knizhnoe izdatel'stvo, 2011), vol. 18, 224–229; S.N. Korotun, S.P. Tolkacheva and E.A. Shevchenko, The national minorities in Voronezh Region in 1917–1941 (Voronezh: Voronezhskii gosudarstvennyi pedagogicheskii universitet, 2012), 64, 66–68, 217–18, 228, 242; “Documents of Krasnyansk uprising; publication of Ya. V. Leont’ev”, in Peasants’ front 1918–1922. Collection of articles and materials; edited by A.V. Posadskii (Mos- cow: AIRO-XXI, 2013), 308; GAOPI VO f. 5, op. 1, d. 320, l. 23; GAVO f. р-2393, op. 1, d. 2, l. 21 . 37 Voronezhskii telegraf [Voronezh Telegraph], 14th December, 1917, 3. 38 Voronezhskii telegraf [Voronezh Telegraph], 15th December, 1917, 3. 39 Two Archival Documents; compiled by S.N. Tret’yakov (Moscow: Izdatel'stvo Olega Pakhmutova, 2014), 91–95. Main body On 5th December 1917, the Polish delegates appealed to the credentials committee of Voronezh Council, informing 2018 ∙ Vol. 2 ∙ № 4 142 http://hpchsu.ru RESEARCH them that Voronezh had formed a group of the Polish Socialist Revolutionary Party, and at its meeting on 3rd December 1917 held by 33 members, K. Budogosky had been delegated to the Council. The Poles came out with full support of the October coup: “since the second Russian revolution was perceived as a great holiday, the day of unification of the Congress of Soviets with the Central Executive Committee of Soviets of Worker and Soldier Deputies. The revolutionary democracy would only deepen the revolution now by uniting around the Council of People’s Commissars”. In the same address, the Poles welcomed the decrees of the Council of People’s Commissars and the Polish Democratic People’s Republic35. One of the leaders of Polish socialists S.I. Danil’kevich served as comrade chairman of Voronezh Council at the end of 1917. Later, the cooperation was carried out by assimilating national organizations into the structures of the RCP(b) and cultural and educational authorities. In 1918, there existed the Provincial Commissariat for Jewish National Affairs. The Jewish section worked not only under the Provincial Committee of the RCP(b) but also in the local organization of the Revolutionary Communist Youth League. By August 1918, the Lithuanian, Latvian and German sections worked under the Provincial Committee of the RCP(b). Until the autumn of 1918, in Voronezh the Ukrainian ‘Gromada’ was present, along with the Regional Committee of the Ukrainian Left SRs that was evac- uated from Kharkov. In 1922, in Voronezh, there existed the Ukrainian Club named after T.G. Shevchenko; the Ukrainian Provincial Bureau was created within the de- partment of national minorities in the provincial department of public education36. In Voronezh province, the inter-party organizations, such as the Committee for the Salvation of Motherland and Revolution and the Union for Constituent Assembly Protection, were also very well represented at the turn of 1917–18. Thus, on 13th De- cember, at the coordination meeting of the Union for Constituent Assembly Protec- tion, representatives of 24 organizations participated in Voronezh, including those from the parties, trade unions, workers’ committees and 59th regiment under the command of N.N. Chekhov. However, the meeting was dispersed by the Military Revolutionary Committee37. The Cadets, Socialist-Revolutionaries, Popular Social- 2018 ∙ Vol. Main body 2 ∙ № 4 143 http://hpchsu.ru RESEARCH ists, Social Democrats, representatives of the local Council of Workers’ and Soldiers’ Deputies, the uyezd Ukrainian Rada and the chairman of the uyezd commission for the Constituent Assembly elections participated in the meeting of the Committee for the Salvation of Motherland and Revolution on 5th December 1917 in the uyezd cen- ter of Valuyki38. There are indications about the activities of similar organizations in the uyezd chief towns of Bobrov and Ostrogozhsk. At the same time, the Bolshevik – Left Socialist-Revolutionary authorities found the ability to conclude alliances with militant formations of unstable political orientation. In April 1918, in Voronezh, there emerged for a few days a ‘federation’ of representatives of the Red Army detach- ments who occupied the city and positioned themselves as ‘anarchists’ and Voronezh leadership of the Bolsheviks and Left Socialist-Revolutionaries. The Executive Committee of the Council was dissolved for a few days. Most likely, the ‘anarchists’ supported by the greater part of Voronezh garrison, were approached for cooperation by the Left Socialist-Revolutionaries, even more so that the units which entered the city were under the general leadership of the Left SR, G.K. Petrov39. Список литературы Два архивных документа / сост. С.Н. Третьяков. М.: Издательство Олега Пахмутова, 2014. 126 с. Дронов В.Т. Согласие как цивилизационный архетип российского общества // Социс. 2016. № 9. С. 157–160. Дронов В.Т. Согласие как цивилизационный архетип российского общества // Социс. 2016. № 9. С. 157–160. 2016. № 9. С. 157 160. Кармадонов О.А. Солидарность, интеграция, конъюнкция // Социс. 2015. № 2. С. 3–12. Коротун С.Н. Секции национальных меньшинств в Воронежской губернии (1918–1921) // Из истории Воронежского края. Сб. ст. / отв. ред. А.Н. Акиньшин. Воронеж: Центрально- Черноземное книжное издательство, 2011. Вып. 18. С. 224–229. Коротун С.Н., Толкачева С.П., Шевченко Е.А. Национальные меньшинства Воронежско- го края в 1917–1941 гг. Воронеж: Воронежский государственный педагогический универси- тет, 2012. 328 с. Крестьянский фронт 1918–1922 гг. Сб. статей и материалов / под ред. А.В. Посадского. Кармадонов О.А. Солидарность, интеграция, конъюнкция // Социс. 2015. № 2. С. 3–12. Коротун С.Н. Секции национальных меньшинств в Воронежской губернии (1918–1921) // Из истории Воронежского края. Сб. ст. / отв. ред. А.Н. Акиньшин. Воронеж: Центрально- Черноземное книжное издательство, 2011. Вып. 18. С. 224–229. Коротун С.Н., Толкачева С.П., Шевченко Е.А. Национальные меньшинства Воронежско- го края в 1917–1941 гг. Воронеж: Воронежский государственный педагогический универси- тет, 2012. 328 с. Коротун С.Н., Толкачева С.П., Шевченко Е.А. Национальные меньшинства Воронежско- го края в 1917–1941 гг. Воронеж: Воронежский государственный педагогический универси- тет, 2012. 328 с. Крестьянский фронт 1918–1922 гг. Сб. статей и материалов / под ред. А.В. Посадского. М.: АИРО-ХХI, 2013. 740 с. Крестьянский фронт 1918–1922 гг. Сб. статей и материалов / под ред. А.В. Посадского. М.: АИРО-ХХI, 2013. 740 с. Кружинов В.М., Сокова З.Н. Органы многопартийной социалистической власти на Урале осенью 1917 г. // Российская история. 2017. № 5. С. 44–61. Кружинов В.М., Сокова З.Н. Органы многопартийной социалистической власти на Урале осенью 1917 г. // Российская история. 2017. № 5. С. 44–61. Ленин В.И. Полное собрание сочинений: В 55 т. М.: Госполитиздат, 1977. Т. 34. Ленин В.И. Полное собрание сочинений: В 55 т. М.: Госполитиздат, 1977. Т. 34. Модели общественного переустройства России. ХХ век / отв. ред. В.В. Шелохаев. М.: РОССПЭН, 2004. 608 с. р Модели общественного переустройства России. ХХ век / отв. ред. В.В. Шелохаев. М.: РОССПЭН, 2004. 608 с. Модели общественного переустройства России. ХХ век / отв. ред. В.В. Шелохаев. М.: РОССПЭН, 2004. 608 с. Морозова О.М. Дон в 1917 году: мирная прелюдия революции // 1917 год в судьбах ре- гиона, страны и мира. Взгляд из XXI века / под общ. ред. В.И. Голдина. Conclusion It goes without saying that the above-mentioned facts do not void the presence of a powerful social and political conflict in the country. At the same time, we record the availability and significant evolution of political regime with regards to coopera- tion, both in the center and at the local level. Its rhythms might not have coincided; however, the forms in which it progressed demonstrated similarities. In February– October 1917, the cooperation was carried out at the level of the Provisional Gov- ernment and the local administrative structures ascending to it; in revolutionary self- government authorities, inter-party blocs were formed, and the ideological parties merged. A distinctive feature of this period was the various parties to the political co- operation, which were after different forms of interaction. We believe not only the October coup became the turning point in the regime change, but also the failed Con- stituent Assembly, after the collapse of which the ‘demolition’ of the February re- gime began, which until that time rested upon the very structures of social solidarity. However, in 1918 such tendencies continued to exist within framework of ‘democrat- ic counter-revolution’ regimes operating under the banner of solidarity of absolutely all anti-Bolshevik political forces. The October regime demonstrated opportunities for compromises at the political, social and national levels; however, denying pluralism as the basis for cultural and ideological components in the political system. As forms of political cooperation, the contacts between the socialist parties continued to exist; not only ‘democratic’ self- 144 2018 ∙ Vol. 2 ∙ № 4 http://hpchsu.ru RESEARCH government authorities (the Soviets and trade union associations) were integrated into the new political system, but also the working arrangements of zemstvos, city dumas and national organizations. The federation was a preferable form of state-territorial organization. At the same time, the White regimes offered a similar authoritarian model of political interaction, whilst preserving, however, the external attributes of democratic interaction: inter-party and non-party associations, cooperation of parties in governments and the existence of local self-government. Список литературы Edinburgh: Printed and bound by Grafica Veneta, 2000. 362 p. 362 p. Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 362 p. Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 362 p. Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 1986 373 p p Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 1986. 373 p. Raleigh D.J. Revolution on the Volga: 1917 in Saratov. Ithaca, N.Y.: Cornell University Press, 1986. 373 p. 1986. 373 p. Raleigh D.J. Revolutionary Politics in Provincial Russia: The Tsaritsyn “Republic” in 1917 // Slavic Review 1981 Vol 40 №2 P 194 209 Raleigh D.J. Revolutionary Politics in Provincial Russia: The Tsaritsyn “Republic” in 1917 // Slavic Review, 1981. Vol. 40. № 2. P. 194–209. Raleigh D.J. 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https://openalex.org/W4391217161	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0298008&type=printable	English	null	Retraction: Inhibitory potentials of Streptomyces exfoliatus strain ‘MUJA10’ against bacterial pathogens isolated from rural areas in Riyadh, Saudi Arabia	PloS one	2,024	cc-by	252	1. Alahadeb JI (2022) Inhibitory potentials of Streptomyces exfoliatus strain ‘MUJA10’ against bacterial pathogens isolated from rural areas in Riyadh, Saudi Arabia. PLoS ONE 17(4): e0266297. https://doi. org/10.1371/journal.pone.0266297 Retraction: Inhibitory potentials of Streptomyces exfoliatus strain ‘MUJA10’ against bacterial pathogens isolated from rural areas in Riyadh, Saudi Arabia The PLOS ONE Editors The PLOS ONE Editors retract this article [1] because it was identified as one of a series of sub- missions for which we have data concerns and concerns about authorship, peer review, and data availability. We regret that the issues were not addressed prior to the article’s publication. The author either did not respond directly or could not be reached. The author either did not respond directly or could not be reached. Reference 1. Alahadeb JI (2022) Inhibitory potentials of Streptomyces exfoliatus strain ‘MUJA10’ against bacterial pathogens isolated from rural areas in Riyadh, Saudi Arabia. PLoS ONE 17(4): e0266297. https://doi. org/10.1371/journal.pone.0266297 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: The PLOS ONE Editors (2024) Retraction: Inhibitory potentials of Streptomyces exfoliatus strain ‘MUJA10’ against bacterial pathogens isolated from rural areas in Riyadh, Saudi Arabia. PLoS ONE 19(1): e0298008. https://doi.org/ Published: January 25, 2024 PLOS ONE PLOS ONE Published: January 25, 2024 Copyright: © 2024 The PLOS ONE Editors. 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. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0298008 January 25, 2024
https://openalex.org/W2059867779	https://www.epj-conferences.org/articles/epjconf/pdf/2010/05/epjconf_ICEM14_15002.pdf	English	null	Experimental and numerical analysis of clamped joints in front motorbike suspensions	EPJ web of conferences	2,010	cc-by	3,902	D. Croccolo1,a, M. De Agostinis1, N. Vincenzi1 D. Croccolo1,a, M. De Agostinis1, N. Vincenzi1 1DIEM, Faculty of Engineering, University of Bologna, V.le Risorgimento 2, 40136 Bologna, Italy aculty of Engineering, University of Bologna, V.le Risorgimento 2, 40136 Bologna, Italy 1DIEM, Faculty of Engineering, University of Bologna, V.le Risorgimento 2, 40136 Bol Abstract. Clamped joints are shaft-hub connections used, as an instance, in front motorbike suspensions to lock the steering plates with the legs and the legs with the wheel pin, by means of one or two bolts. The preloading force, produced during the tightening process, should be evaluated accurately, since it must lock safely the shaft, without overcoming the yielding point of the hub. Firstly, friction coefficients have been evaluated on “ad-hoc designed” specimens, by applying the Design of Experiment approach: the applied tightening torque has been precisely related to the imposed preloading force. Then, the tensile state of clamps have been evaluated both via FEM and by leveraging some design formulae proposed by the Authors as function of the preloading force and of the clamp geometry. Finally, the results have been compared to those given by some strain gauges applied on the tested clamps: the discrepancies between numerical analyses, the design formulae and the experimental results remains under a threshold of 10%. a e-mail : dario.croccolo@unibo.it 1 Introduction Clamped joints are shaft-hub connections used, as an instance, in front motorbike suspensions to lock the steering plates with the legs (fork clamp) and the legs with the wheel pin (wheel clamp) by means of one or two bolts. The fundamental design parameter for this type of couplings is the preloading force, produced during the tightening process: it should be accurately evaluated since it must lock safely the shaft (legs or wheel pin) without overcoming the yielding point of the hub (fork or wheel clamp). Bolts are normally tightened by means of a calibrated torque wrench so that the preloading force results as a function of the total applied torque, of the screw geometry and of the friction coefficients (underhead friction and thread friction). Friction coefficients, which can typically assume values included in the range 0.05–0.5, are known to be strongly influenced by several parameters, such as type of contact, surface finishing, lubrication, wear and spoiling. As a matter of fact the same tightening torque produces a high locking force if friction coefficients are low; conversely, high friction coefficients could provide an insufficient locking force. Therefore an incorrect evaluation of friction parameters may lead to dangerous failures [1], especially in applications where human safety is involved, such as front motorbike suspensions. In previous works [2, 3], Croccolo et al. investigated the tensile state of clamped joints in front motorbike suspensions, via FEM: they developed and proposed some engineering design formulae, in order to estimate the maximum stress on the clamp and the mean coupling pressure as functions of the bolt preloading force and of the clamp geometry. The present work deals with the extension of © Owned by the authors, published by EDP Sciences, 2010 DOI:10.1051/epjconf/20100615002 EPJ Web of Conferences 6, 15002 (2010) 6 © Owned by the authors, published by EDP Sciences, 2010 DOI:10.1051/epjconf/20100615002 EPJ Web of Conferences 6, 15002 (2010) 6 Experimental and numerical analysis of clamped joints in front motorbike suspensions D. Croccolo1,a, M. De Agostinis1, N. Vincenzi1 2 Methodology Firstly the well known relationships (Eq.(1a,b) [4]) between the applied tightening torque T [Nmm] and the imposed preloading force FV [N] have been studied, by applying the Design of Experiment (DOE) approach [5, 6] in order to evaluate the friction parameters accurately. A full factorial plane, characterized by 4 variables with 2 levels each, has been designed. Three replicas have been carried out, in order to reduce the influence of noise (experimental error) and of any non-investigated factors: a total of 3!24=48 experimental tests have been run. The levels include cast versus forged aluminium alloy, anodized versus spray-painted surfaces, lubricated versus dry screws and first tightening (fresh unspoiled surfaces) versus sixth tightening (spoiled surfaces) [5]. In Tab.1 the DOE parameters are summarized. a( ) T = FV " 0.16" p+ 0.58"µm "d2 + 0.5"µm "du [ ] b( ) T = K "FV "d (1) (1) p [mm] is the thread pitch, d [mm] is the nominal thread diameter, µm is the overall friction coefficient according to [4, 7], d2 [mm] is the mean thread diameter (d2=d-0,6495!p), du [mm] is the underhead mean diameter and K is the nut factor [8, 9]. p [mm] is the thread pitch, d [mm] is the nominal thread diameter, µm is the overall friction coefficient according to [4, 7], d2 [mm] is the mean thread diameter (d2=d-0,6495!p), du [mm] is the underhead mean diameter and K is the nut factor [8, 9]. Table 1. The Design of Experiment (DOE) parameters: variables and levels. Variable Low Level (0) High level (1) A. Lubrication Dry Lubricated B. Process Cast Forged C. Surface finishing Spray-painted Anodized D. Tightening First tightening (unspoiled surfaces) Sixth tightening (spoiled surfaces) Table 1. The Design of Experiment (DOE) parameters: variables and levels. Some “ad hoc designed” specimens reported in Fig.1 [5] have been used, according to Eq. (2a,b), to calculate 48 different values of the overall friction coefficient µm and of the nut factor K: T is given by a torque wrench, whereas FV has been evaluated by means of a strain gauge, located on the external surface of the specimen, which is able to provide the axial compression strain !C. The compression force acting on the specimen is equal, in magnitude, to the preloading force acting on the screw, since the system works like series of mechanical stiffness during the tightening phase. EPJ Web of Conferences the results presented in Ref.[2, 3] using, as loading input parameter for the calculation, the tightening torque (in the place of the preloading force), which is actually controlled during the manufacturing process. the results presented in Ref.[2, 3] using, as loading input parameter for the calculation, the tightening torque (in the place of the preloading force), which is actually controlled during the manufacturing process. 2 Methodology The whole cross section (tubular) in the central part of the specimen (“calibrated length” in Fig.1), has the same strain and, therefore, the same stress. Thus, it is possible to calculate the compression force acting on the specimen, which is the same tensile force (preloading) acting on the screw. The study considers M8x1.25, SAE Standard 8.8 galvanized screws. a( ) µm = T FV " 0.16 # p 0.58 # d2 + 0.5# du b( ) K = T FV # d a( ) µm = T FV " 0.16 # p 0.58 # d2 + 0.5# du b( ) K = T FV # d (2) (2) 15002-p.2 14th International Conference on Experimental Mechanics The Analysis of Variance (ANOVA) statistical approach has been then applied to the results in order to obtain the mathematical equations of friction coefficient µm (Eq.3) and nut factor K (Eq.4) as functions of the investigated variables (or their interactions) which are actually significant in changing the values [5]. Fig.1 The specimen useful for relating the tightening torque T to the preloading force FV Strain gauge Calibrated length Strain gauge Fig.1 The specimen useful for relating the tightening torque T to the preloading force FV (3) (4) (3) (4) As highlighted by equations (3) and (4), friction conditions are strongly affected by surface finishing (C variable), lubrication (A variable) and number of tightening and loosening (D variable); conversely, the forming process (cast or forged aluminium alloy) seems to have no significant influence on friction conditions [5]. Spray-painted specimens (C=0) present the lower values of µm and K (the higher preloading forces FV). Lubrication (A=1) always increases the preloading forces, while the tightening replicas (D=1) progressively decrease the preloading forces, mainly in case of dry surfaces, as demonstrated also in Ref.[10]. As highlighted by equations (3) and (4), friction conditions are strongly affected by surface finishing (C variable), lubrication (A variable) and number of tightening and loosening (D variable); conversely, the forming process (cast or forged aluminium alloy) seems to have no significant influence on friction conditions [5]. Spray-painted specimens (C=0) present the lower values of µm and K (the higher preloading forces FV). Lubrication (A=1) always increases the preloading forces, while the tightening replicas (D=1) progressively decrease the preloading forces, mainly in case of dry surfaces, as demonstrated also in Ref.[10]. Secondly the outcomes of the present analysis have been applied to an unlubricated, spray-painted, cast aluminium wheel clamp, realized by the Paioli Meccanica S.p.A. of Bologna (IT), which produces front motorbike suspensions. Two M6x1, SAE Standard 8.8 galvanized screws (d=6mm, p=1mm, d2=5.35mm and du=8mm), have been tightened up to six times each. Strain gauges have been placed on the critical section of the clamp (loaded by a bending stress, as deeply demonstrated in [2]), where a stress concentration factor Kt occurs, mainly due to the presence of spot-facings and holes, as reported in Fig.2. 14th International Conference on Experimental Mechanics Fig.2 Example of wheel clamp: stress concentration factor and geometrical dimensions Kt Critical section ical ion Fig.2 Example of wheel clamp: stress concentration factor and geometrical dimensions 15002-p.3 15002-p.3 EPJ Web of Conferences Referring to Fig.2, the theoretical bending stress in the critical section can be evaluated by applying Eq.(5) [2], in which FV is the preloading force, a [mm] the lever arm of FV with respect to the critical section, n the number of bolts, b [mm] and h [mm] are the width and the height of the rectangular cross section, respectively. " b _ th = M b Wb = n # FV # a b # h2 6 (5) " b _ th = M b Wb = n # FV # a b # h2 6 (5) " b_ max = " b_th # Kt = n # FV # a b # h2 6 # Kt Kt_M 6 = 2.438 + 0.548 # hs$ f h $1.131# a ds$ f $ 0.393# v ds$ f % & ' ' ( ' ' (6) (5) " b_ max = " b_th # Kt = n # FV # a b # h2 6 # Kt Kt_M 6 = 2.438 + 0.548 # hs$ f h $1.131# a ds$ f $ 0.393# v ds$ f % & ' ' ( ' ' (6) (6) 3 Results The maximum bending stress on the clamp can be calculated by applying Eq.(6) in which the stress concentration factor Kt takes into account the perturbation produced by the spot-facings located close to the coupling zone. The geometrical dimensions of clamps under investigation are reported in Tab.2: the actual Kt value calculated applying Eq.(6) is equal to 1.37 [2, 3]. Table 2. Wheel clamp geometrical dimensions useful for the calculation according to Fig.2 Table 2. Wheel clamp geometrical dimensions useful for the calculation according to Fig.2 Description Symbol Value Wheel pivot diameter D [mm] 20 Distance between the wheel pivot centre and the bolt axis L [mm] 15.5 Lever arm of the preloading force (L-D/2) a [mm] 5.5 Distance between the wheel pivot and the clamp side c [mm] 16 Spot-facing diameter ds-f [mm] 11 Clamp total height htot [mm] 45 Clamp height h [mm] 18.5 Thread height hth [mm] 24.5 Spot-facing height hs-f [mm] 4.3 Clamp width b [mm] 36 Distance between bolt axes v [mm] 17.5 Mill radius r [mm] 2 A tightening torque T=9.5Nm has been applied to the wheel clamp: the preloading forces produced by the tightening have been evaluated by means of Eq.(7), in which the overall friction coefficient µm is equal to 0.108 during the first tightening (A=B=C=D=0, according to Tab.1 and Eq.3) and equal to 0.118 during the sixth tightening (A=B=C=0, D=1, according to Tab.1 and Eq.3). FV = T 0.16" p+ 0.58"µm "d2 + 0.5"µm "du [ ] (7) T (7) V = T 0.16" p+ 0.58"µm "d2 + 0.5"µm "du [ ] (7) By applying the Von Mises equivalent stress criterion, the maximum preloading forces, with respect to the bolt yielding or failure, can be evaluated by means of Eq.(8) (see also the sketch reported in Fig.3), in which SY [MPa] is the yielding point and SU [MPa] is the ultimate point of the screw (SAE Standard 8.8: SY=640MPa, SU=800MPa), while At [mm2] is the screw tensile stress area and dt its diameter (At=20.1mm2, dt=5.06mm, in case of bolt M6x1) [4]. 3 Results By applying the Von Mises equivalent stress criterion, the maximum preloading forces, with respect to the bolt yielding or failure, can be evaluated by means of Eq.(8) (see also the sketch reported in Fig.3), in which SY [MPa] is the yielding point and SU [MPa] is the ultimate point of the screw (SAE Standard 8.8: SY=640MPa, SU=800MPa), while At [mm2] is the screw tensile stress area and dt its diameter (At=20.1mm2, dt=5.06mm, in case of bolt M6x1) [4]. 15002-p.4 14th International Conference on Experimental Mechanics FV _ yielding = SY 1 At " # $ % & ' 2 + 3( 0.16 ( p + 0.58 ( µm ( d2 ) ( dt ( ) 3 16 " # $ $ $ $ % & ' ' ' ' 2 FV _ failure = SU 1 At " # $ % & ' 2 + 3( 0.16 ( p + 0.58 ( µm ( d2 ) ( dt ( ) 3 16 " # $ $ $ $ % & ' ' ' ' 2 Fig.3 Sketch of the load diagrams (axial and torsional) acting on the bolt during the tighten FV _ yielding = SY 1 At " # $ % & ' 2 + 3( 0.16 ( p + 0.58 ( µm ( d2 ) ( dt ( ) 3 16 " # $ $ $ $ % & ' ' ' ' 2 FV _ failure = SU 1 At " # $ % & ' 2 + 3( 0.16 ( p + 0.58 ( µm ( d2 ) ( dt ( ) 3 16 " # $ $ $ $ % & ' ' ' ' 2 (8) Fig.3 Sketch of the load diagrams (axial and torsional) acting on the bolt during the tightening The numerical values of the preloading forces are reported in Tab.3, as a function of the overall friction coefficient µm (Eq.3). The numerical values of the preloading forces are reported in Tab.3, as a function of the overall friction coefficient µm (Eq.3). Table 3. Preloading force values as a function of the overall friction coefficient (bolt: M6x1, 8.8) Clamp parameters (Tab.1) T [Nm] m FV [kN] (Eq.7) FV_yielding [kN] (Eq.8) FV_failure [kN] (Eq.8) A=B=C=D=0 9.5 0.108 10.223 10.641 13.301 A=B=C=0, D=1 9.5 0.118 9.497 10.430 13.038 Table 3. 3 Results Preloading force values as a function of the overall friction coefficient (bolt: M6x1, 8.8) During the first tightening (fresh and unspoiled surfaces) the strain gauge applied on the clamp (Fig.4), gives a strain value that is equivalent to a stress of 79MPa. A numerical (FEM) nonlinear analysis, with contact elements between the shaft and the hub, has been performed on the same joint by imposing the preloading force accurately calculated by the theoretical formulae (FV=10.223kN of Tab.3). The stress evaluated via FEM is equal to 82MPa, as shown in Fig.4, so that the difference between the experimental test and the FEM values is lower than 4%. During the sixth tightening (spoiled surfaces) the test provides a strain value equivalent to a stress of 73MPa: the ratio between the sixth and the first tightening bending stresses (73/79=0.924) is very close to the ratio between the first and the sixth preloading force calculated by the theoretical formulae (9.497/10.223=0.929). 15002-p.5 15002-p.5 EPJ Web of Conferences Fig.4 Experimental tightening test (T=9.5Nm) vs FEM analysis (FV =10.223kN) Fig.4 Experimental tightening test (T=9.5Nm) vs FEM analysis (FV =10.223kN) The engineering design formulae of Ref.[2], reported in Eq.(5) and (6), are able to provide, during the first tightening, a maximum bending stress !b_max equal to 75MPa (theoretical bending stress !b_th equal to 55MPa and stress concentration Kt equal to 1.37): the discrepancy with respect to the experimental test value is equal to 5%. The aforementioned findings are summarized in Tab.4. Another clamp with the same geometry has been studied during the tightening phase: the bolt has been tightened until its failure, in correspondence of the sixth tightening. Since the yielding of the screw has been overcome, the torque-preloading relationship (Eq.7) is no more effective. However, The engineering design formulae of Ref.[2], reported in Eq.(5) and (6), are able to provide, during the first tightening, a maximum bending stress !b_max equal to 75MPa (theoretical bending stress !b_th equal to 55MPa and stress concentration Kt equal to 1.37): the discrepancy with respect to the experimental test value is equal to 5%. The aforementioned findings are summarized in Tab.4. A th l ith th t h b t di d d i th ti ht i h th b lt h The engineering design formulae of Ref.[2], reported in Eq.(5) and (6), are able to provide, during the first tightening, a maximum bending stress !b_max equal to 75MPa (theoretical bending stress !b_th equal to 55MPa and stress concentration Kt equal to 1.37): the discrepancy with respect to the experimental test value is equal to 5%. The aforementioned findings are summarized in Tab.4. Another clamp with the same geometry has been studied during the tightening phase: the bolt has been tightened until its failure, in correspondence of the sixth tightening. Since the yielding of the screw has been overcome, the torque-preloading relationship (Eq.7) is no more effective. However, according to Eq.(8), it is possible to calculate the preloading force in correspondence of the bolt failure, which is equal to 13.038kN (Tab.3). When the bolt failure occurs (Fig.5) the strain gauge applied on the clamp gives a strain value equivalent to a stress of 107MPa. Another clamp with the same geometry has been studied during the tightening phase: the bolt has been tightened until its failure, in correspondence of the sixth tightening. 15002-p.5 Since the yielding of the screw has been overcome, the torque-preloading relationship (Eq.7) is no more effective. However, according to Eq.(8), it is possible to calculate the preloading force in correspondence of the bolt failure, which is equal to 13.038kN (Tab.3). When the bolt failure occurs (Fig.5) the strain gauge applied on the clamp gives a strain value equivalent to a stress of 107MPa. Table 4. Comparison between the values provided by the experimental (strain gauge), the numerical (FEM) and the design formulae Clamp parameters (Tab.1) T [Nm] FV [kN] !strain_gauge [MPa] !FEM[MPa] (e%) !design_formulae [MPa] (e%) A=B=C=D=0 9.5 10.223 79 82 (+4%) 75 (-5%) A=B=C=0, D=1 9.5 9.497 73 76 (+4%) 70 (-4%) Fig.5 Experimental failure of a bolt, during the tightening phase parison between the values provided by the experimental (strain gauge), the numerical (FEM) and the design formulae Fig.5 Experimental failure of a bolt, during the tightening phase Fig.5 Experimental failure of a bolt, during the tightening phase 15002-p.6 14th International Conference on Experimental Mechanics 14th International Conference on Experimental Mechanics By performing again the numerical nonlinear analysis on the joint and imposing the failure preloading force FV_failure=13.038kN a bending stress equal to 104MPa is reached, as shown in Fig.4: the discrepancy with respect to the experimental result is equal to 3%. The engineering design formulae (Eq.6) provide a maximum bending stress !b_max equal to 96MPa: the discrepancy with respect to the experimental result is equal to 10%. Finally, an original Software, Front Suspension Design©, realised by the authors in Visual Basic® programming language [3], has been updated with the presented findings. The Software is a useful tool for the designing and the validation phases of any type of clamps in front motorbike suspensions. It is possible to calculate quickly the maximum bending stress on the clamp and the mean coupling pressure (output window in Fig.7) by simply inserting the total applied torque T, the clamp geometry and the production parameters of the clamp (input window in Fig.6) that are useful to evaluate the actual friction coefficient and, therefore, the actual preloading force. 4 Conclusions Experimental tightening tests on clamped joints used in front motorbike suspensions have been studied in the present work. The maximum bending stress on the clamp, generated during the tightening, has been evaluated by applying some strain gauges on the actual component. The tightening has been performed by means of a calibrated torque wrench. In order to calculate the corresponding preloading force the overall friction coefficient of the joint has been calculated on some specific specimens, designed and realised with the same process and surface finishing of the actual components. The experimental bending stresses obtained by the strain gauges have been compared both with those provided by some numerical nonlinear analyses and by some design formulae proposed and developed by the Authors. The input parameter for the experimental tightening is the tightening torque applied to the screw by a torque wrench, whereas the input parameter for the numerical simulation and for the design formulae application is the preloading force. By leveraging the accurate definition of the tightening torque-preloading force relationship the discrepancies in evaluating the maximum bending stress between experimental tests, numerical analyses and design formulae remains under a threshold of 10%, both in the elastic and in the elastic-plastic field for the bolt. In light of the proposed results, it is possible to estimate precisely the preloading force starting from the tightening torque and without performing any experimental tests. Furthermore it is possible to evaluate accurately the maximum stress generated on the clamp by applying the proposed design formulae: it is, therefore, easy to relate the applied tightening torque directly to maximum clamp stress. An original software, Front Suspension Design©, realised by the authors in Visual Basic® programming language, has been finally updated with the presented results. The Software is a useful tool for the designing and the validation phases of any clamps in front motorbike suspensions. 15002-p.5 , , p g Fig.6 Input windows of Front Suspension Design© Fig.7 Output windows of Front Suspension Design© Clamp Mechanical properties "b_th Kt "b_max Clamp Safety Ratios Fig.6 Input windows of Front Suspension Design© Fig.6 Input windows of Front Suspension Design© Fig.6 Input windows of Front Suspension Design© Fig.7 Output windows of Front Suspension Design© Clamp Mechanical properties "b_th Kt "b_max Clamp Safety Ratios Fig.7 Output windows of Front Suspension Design© 15002-p.7 References 1. N. Eliaz, G. Gheorghiu, H. Sheinkopf, O. Levi, G. Shemesh, A. Ben-Mordechai, H. Artzi. Engineering Failure Analysis 10, 443–451(2003). 2. D. Croccolo, R. Cuppini, N. Vincenzi, Finite Elements in Analysis and Design 45, 406-414 (2009). 3. D. Croccolo, M. De Agostinis, N. Vincenzi, Engineering Failure Analysis DOI: 10.1016/j.engfailanal.2010.02.002, in press (2010). 4. VDI Handbuch Konstruktion 2230, Systematic calculation of high duty bolted jo with one cylindrical bolt (2001). 5. D. Croccolo, M. De Agostinis, N. Vincenzi, Experimental study of friction in aluminium bolted joints, 14th International Conference on Experimental Mechanics, paper n.262, Poitiers, France (2010). 6. D. Croccolo, R. Cuppini, N. Vincenzi, Strain 44, 170-179 (2008) 7. DIN946, Determination of coefficient of friction of bolt/nut assemblies under specified conditions (1991). 8. S.A. Nassar, P.H. Matin, Journal of Pressure Vessel Technology 127, 387-393 (2005). 9. J.H. Bickford, An Introduction to the Design and Analysis of Bolted Joints, 3rd ed., Marcel Dekker, New York (1997). 10. W. Eccles, I.Sherrington, R.D.Arnell, Tribology International 43, 700-707 (2010). 15002-p.8
https://openalex.org/W2806981202	https://ttu-ir.tdl.org/bitstream/2346/95237/1/Main%20article%20with%20TTU%20Libraries%20cover%20page.pdf	English	null	A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair	Scientific reports	2,018	cc-by	11,706	Zachary K. Boswell, Samiur Rahman, Marella D. Canny & Michael P. Latham Zachary K. Boswell, Samiur Rahman, Marella D. Canny & Michael P. Latham The Mre11-Rad50 protein complex is an initial responder to sites of DNA double strand breaks. Many studies have shown that ATP binding to Rad50 causes global changes to the Mre11-Rad50 structure, which are important for DNA repair functions. Here we used methyl-based NMR spectroscopy on a series of mutants to describe a dynamic allosteric pathway within Rad50. Mutations result in changes in the side chain methyl group chemical environment that are correlated with altered nanosecond timescale dynamics. We also observe striking relationships between the magnitude of chemical shift perturbations and Rad50 and Mre11 activities. Together, these data suggest an equilibrium between a ground state and an “active” dimerization competent state of Rad50 that has locally altered structure and dynamics and is poised for ATP-induced dimerization and eventual ATP hydrolysis. Thus, this sparsely populated intermediate is critical for Mre11-Rad50-directed DNA double strand break repair. The conserved and essential Mre11-Rad50 complex in archaea and bacteria and larger Mre11-Rad50-Nbs1 (or Xrs1) complex in higher eukaryotes is responsible for detecting and initiating the repair of DNA double strand breaks (DSBs)1,2. Mre11 is an obligate dimer that has Mn2+-dependent 3′-to-5′ exonuclease and ssDNA endo- nuclease activity3,4. Each Mre11 dimer is bound to two molecules of Rad50 (i.e., M2R2), a member of the ATP binding cassette (ABC) family of proteins5,6 whose structure is shown in Fig. 1. Nbs1 (or Xrs1) is a flexible scaf- folding protein that uses phospho-protein binding domains to recruit downstream effectors to the site of the DNA break7,8. The proper function of this protein complex is absolutely required for the repair of mutagenic or cyto- toxic DNA DSBs1,2. A number of hypomorphic mutations have been observed in Mre11-Rad50-Nbs1 that lead to neuromuscular developmental disorders, immunodeficiency, and a predisposition to cancer9–11, and spontaneous mutations have been observed in a number of cancer types characterized by gross genomic instabilities12,13. yp y g g Rad50 has a unique structural arrangement whereby the N- and C-terminal sub-domains, which are separated by ~200–600 Å coiled-coil and apical Zn-hook domains, fold back onto each other to form a functional ABC ATPase domain (“NBD”s in Fig. 1a)5,6. As with other members of this protein family, the binding of two ATP molecules induces dimerization of the two Rad50 molecules within the M2R2 complex6. A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair The Texas Tech community has made this publication openly available. Please share how this access benefits you. Your story matter to us. Citation Boswell, Z.K., Rahman, S., Canny, M.D., & Latham, M.P.. 2018. A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-19908-8 Citable Link https://hdl.handle.net/2346/95237 Terms of Use © 2018 The Author(s). cc-by Citation Boswell, Z.K., Rahman, S., Canny, M.D., & Latham, M.P.. 2018. A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-19908-8 Citable Link https://hdl.handle.net/2346/95237 Terms of Use © 2018 The Author(s). cc-by Title page template design credit to Harvard DASH. www.nature.com/scientificreports www.nature.com/scientificreports Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79423, USA. Correspondence and equests for materials should be addressed to M.P.L. (email: michael.latham@ttu.edu) Received: 22 November 2017 Accepted: 10 January 2018 Published: xx xx xxxx A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair Received: 22 November 2017 Accepted: 10 January 2018 Published: xx xx xxxx Zachary K. Boswell, Samiur Rahman, Marella D. Canny & Michael P. Latham Several high-resolution X-ray crystal structures have revealed that Rad50 dimerization results in large structural rearrangements of the entire M2R2 complex, dramatically rotating Rad50 from an “open” to a “closed” form when ATP is bound14–18. It is known that the functionality of the M2R2 complex changes depending on the ATP state of Rad50: Mre11 is exonuclease active in the ATP-free, “open” form; M2R2 DNA tethering activities appear to be stimulated in the ATP-bound, “closed” form; and Mre11 ssDNA endonuclease activity may arise from a proposed ADP-bound intermediate state4,19–21. X-ray crystal structures and biochemical data have shown that ATP binding leads to the local rearrangement of salt-bridge and hydrogen bonding interactions within Rad5018,19. This shuffling of ~20 charge-pair interactions is thought to transmit Rad50 ATP binding state information through the coiled-coil domain to Mre11 and out to the apical Zn-hook domain - long-range allostery that would thus span several hundreds of Ångstroms18,22,23. Included in these ionic interactions is a critical contact involving an arginine res- idue in the extended signature helix, the so called basic switch (R805 in Fig. 1b). Disruption of this basic switch via mutation of this residue from arginine to glutamate leads to changes in Rad50 ATP hydrolysis activity and ATP-dependent dimerization and affects the ability of Mre11-Rad50-Nbs1 to repair DNA DSBs, an indication that long-range allostery is being altered19. g g y g Here, we used nuclear magnetic resonance (NMR) spectroscopy to describe a system of residues spanning the Rad50 ABC ATPase domain whose chemical shifts change upon mutation to the hinge region (V156 and Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 1 www.nature.com/scientificreports/ Walker A His-loop D-loop Signature Motif Mre11HLH Hinge Extended Signature Helix Arg805 Q-loop Walker B Coiled-Coil Val156 & 160 1H (ppm) 13C (ppm) 0 1 2 10 15 20 25 -2M 840M 808M 1M 123M 100M 84I 146I 119I 748I 71I 839I 851I 138I 820I 143I 131I 103L 47L 800L 19V 778L 47L 800L 103L 176L 27L 161L 51L 51L 780V 6V 881V 733V 729L 806L 819L 127I 798I 53I 792L 157V 878V 850V 768V Zn Hook NBD N- -C Coiled-Coil Linker a b c P-loop C - - N Rad50 Rad50NBD NBD Figure 1. Domain architecture and NMR spectra of Rad50. (a) Cartoon representation of full length Rad50 (top) and Rad50NBD (bottom). (b) Crystal structure of P. furiosus Mre11HLH-Rad50NBD (3QKS)18. Colors in (a) and (b) illustrate conserved domains important for Rad50 function. Zachary K. Boswell, Samiur Rahman, Marella D. Canny & Michael P. Latham (c) 2D 13C,1H methyl-TROSY HMQC25 spectrum of Mre11HLH-Rad50NBD collected at 14.1 T and 50 °C. The side chain methyl group assignments are given. Walker A His-loop D-loop Signature Motif Mre11HLH Hinge Extended Signature Helix Arg805 Q-loop Walker B Coiled-Coil Val156 & 160 13C (ppm) 10 15 20 25 Zn Hook NBD N- -C Coiled-Coil Linker a b P-loop C - - N Rad50 Rad50NBD NBD a b His-loop Figure 1. Domain architecture and NMR spectra of Rad50. (a) Cartoon representation of full length Rad50 (top) and Rad50NBD (bottom). (b) Crystal structure of P. furiosus Mre11HLH-Rad50NBD (3QKS)18. Colors in (a) and (b) illustrate conserved domains important for Rad50 function. (c) 2D 13C,1H methyl-TROSY HMQC25 spectrum of Mre11HLH-Rad50NBD collected at 14.1 T and 50 °C. The side chain methyl group assignments are given. V160) and basic switch (R805) (see Fig. 1b). Analysis of these chemical shift changes via a covariation method reveals three distinct clusters within Rad50. These clusters form an allosteric network that couples residues in the vicinity of ATP binding to residues at the base of the coiled-coil and Mre11 interacting domains as well as those in the Rad50 dimerization regions. Methyl group relaxation data shows that this pathway is not static in nature, as the amplitudes of methyl group psec – nsec timescale motions correlate with the chemical shift changes. Furthermore, we show that interruption of this network leads to changes in Rad50 that increase both its propen- sity for dimerization and ATP hydrolysis activity. We finally demonstrate that disruption of this allosteric pathway within Rad50 has a profound effect on Mre11 exonuclease activity. When the effects of mutation on the structure and dynamics are compared with biochemical activity, we see significant correlations for methyl groups in two of the three clusters. Together, these data suggest a model whereby many local changes within the Rad50NBD struc- ture are coupled with a general increase in side chain flexibility, ATP-induced dimerization, and ATP hydrolysis – activities required for M2R2-directed DNA DSB repair activities. Results Sid h (b) Structure of Mre11HLH-Rad50NBD emphasizing side chain methyl groups whose CSPs upon mutation cluster according to CHESCA as described in the Methods. Clusters are listed in Supplementary Table S1 and were determined via the dendrogram in Supplementary Fig. S4. Methyl groups not affected by the mutations are not shown. o methyl groups within a ~20 Å radius of the probe28 (Supplementary Fig. S1), confirming which residues are in s vicinity. Mutations in the Rad50NBD hinge region and basic switch reveal an allosteric network cou- pling ATP binding to the base of the coiled-coil domain. The hinge region of Rad50 (also called the signature-coupling helices) is adjacent to the conserved ABC-ATPase signature motif (Fig. 1b) and cou- ples ATP binding, as sensed by the Q-loop and Walker B motif, to the base of the coiled-coil domain, where Mre11HLH binds, through a series of switchable ionic and hydrogen bonding interactions18,19. This region posed a particularly difficult area in the structure for the NOE-based assignment process because it is densely packed with ILVM residues, and an unambiguous network of side chain methyl groups was challenging to identify from methyl-methyl NOE data. Four mutations were therefore made to this region to aid in assignments: V156M, V157M, and V160M in the signature-coupling α2 helix and L163M at the base of the coiled-coil domain. Even though these mutations did indeed provide methyl group assignments via the disappearance of peaks for the mutated residue and changes to peak positions for nearby residues in the methyl-TROSY correlation spectra (Supplementary Fig. S2), other widespread and systematic changes were surprisingly observed in the data for V156M, V157M, and V160M, shown in the spectral overlay of Fig. 2a (L163M only showed the expected local changes). These chemical shift perturbations (CSPs) radiated out from the site of mutation, and in the case of V156M and V160M, included regions where ATP binds, the base of the coiled-coil domain, the P-loop, and β-strands 8, 9, and 10, which are adjacent to the signature motif and would be near the ATP-binding site of the second Rad50NBD upon dimerization (Figs 1b and 2b): a network that spans ~50 Å. Also shown in the overlay of methyl-TROSY correlation spectra in Fig. 2a is the data for the Mre11HLH-Rad50NBD(R805E) basic switch mutant (full spectra presented in Supplementary Fig. S2). Results Sid h Side chain methyl group assignment of P. furiosus Rad50NBD. P. furiosus (Pf) Rad50 and Mre11 have been used as a model M2R2 complex for a number of structural biology and biophysical studies4,5,17–19. Our initial NMR studies have focused on a common truncated form of Pf Rad50 containing the N- and C-terminal ABC-ATPase nucleotide binding sub-domains connected by a six-residue flexible linker (hereafter, Rad50NBD) as depicted in Fig. 1a. Because of the size of the Mre112-Rad50NBD 2 complex (~160 kDa), we utilized uniformly deuterated, side chain methyl Ileδ1-13CH3; Leuδ/Valγ-13CH3/12CD3; Metε-13CH3-labeled (referred to as ILVM- labeled) protein samples coupled with methyl-Transverse Relaxation Optimized SpectroscopY (TROSY)24,25. For ILVM side chain methyl group assignments of Rad50, we made monomeric ILVM-labeled Rad50NBD in complex with protonated Mre11 helix-loop-helix (Mre11 HLH; light blue in Fig. 1b) domain. Mre11HLH is responsible for complex formation between Rad50 and full length Mre1118 and is necessary for well-behaved Rad50NBD (see Supplementary Information). Figure 1c shows the 2D 13C,1H methyl-TROSY correlation spectrum of Mre11HLH - Rad50NBD; 152 out of 168 (~90%) Rad50NBD side chain methyl group assignments have been determined through the comparison of experimental methyl-methyl distance observations (nuclear Overhauser effects – NOEs) with theoretical distances calculated from a Pf Rad50NBD crystal structure (pdb: 1II85). Methyl group assignments were supplemented and validated with conservative mutations of ILVM-residues based on a sequence alignment26 or with the mutation of surface ILVM-residues to cysteine followed by covalent modification with a paramag- netic spin label27. The spin label broadens resonances in the methyl-TROSY correlation spectra corresponding Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 2 www.nature.com/scientificreports/ Figure 2. Mutations reveal an allosteric network in Rad50NBD. (a) 2D 13C,1H methyl-TROSY HMQC spectral overlay of WT and mutant Mre11HLH-Rad50NBD recorded at 14.1 T and 50 °C. Arrows highlight side chain methyl groups experiencing CSPs. (b) Structure of Mre11HLH-Rad50NBD emphasizing side chain methyl groups whose CSPs upon mutation cluster according to CHESCA as described in the Methods. Clusters are listed in Supplementary Table S1 and were determined via the dendrogram in Supplementary Fig. S4. Methyl groups not affected by the mutations are not shown. Figure 2. Mutations reveal an allosteric network in Rad50NBD. (a) 2D 13C,1H methyl-TROSY HMQC spectral overlay of WT and mutant Mre11HLH-Rad50NBD recorded at 14.1 T and 50 °C. Arrows highlight side chain methyl groups experiencing CSPs. Results Sid h relaxation delay time for wildtype and mutant Mre11HLH-ILVM labeled Rad50NBD. These data were fit to equation (3) to determine the η rates36, as described in the Methods, with errors determined from the covariance matrix of the fit48. The coloring of the curves corresponds to the spectra of the mutants in Fig. 2a. (b) Structure of Mre11HLH-Rad50NBD showing side chain methyl groups with altered dynamics upon mutation. Red and orange spheres represent methyl groups that become more flexible upon mutation, whereas green and blue spheres represent methyl groups that become more rigid upon mutation. Red and green coloring denotes “Correlated” methyl groups with significant CSPs upon mutation (i.e., the range in δMethyl > 0.13 ppm) that also have a correlation for η vs δMethyl of \|RP\| > 0.7. Orange and blue coloring denotes “Not Correlated” methyl groups with small CSPs upon mutation (i.e., the range in δMethyl < 0.13 ppm) but have a large difference in η rates between wildtype and the mutants (\|ηWT – the average ηmutants\| > 8 sec−1). Figure 3. Chemical shift perturbations and dynamics changes are correlated. (a) Representative η vs δMethyl scatter plots for Met808Cε (top) and Ile131Cδ1 (bottom). δMethyl values were determined according to equation (1), see Supporting Information. Pearson’s correlation coefficients (RP) are given in the upper right corner. Insets show the build-up curves for the ratio of intensities arising from methyl group 1H triple-quantum “forbidden” experiments36 (Iforbid/Iallow) vs. relaxation delay time for wildtype and mutant Mre11HLH-ILVM labeled Rad50NBD. These data were fit to equation (3) to determine the η rates36, as described in the Methods, with errors determined from the covariance matrix of the fit48. The coloring of the curves corresponds to the spectra of the mutants in Fig. 2a. (b) Structure of Mre11HLH-Rad50NBD showing side chain methyl groups with altered dynamics upon mutation. Red and orange spheres represent methyl groups that become more flexible upon mutation, whereas green and blue spheres represent methyl groups that become more rigid upon mutation. Red and green coloring denotes “Correlated” methyl groups with significant CSPs upon mutation (i.e., the range in δMethyl > 0.13 ppm) that also have a correlation for η vs δMethyl of \|RP\| > 0.7. Results Sid h Again, a number of expected CSPs were observed for residues in the area of the structure adjacent to the R805E mutation, and widespread methyl group CSPs were also observed in the same regions that were affected by V156M and V160M.i gf y We initially used a median-absolute-deviations approach29,30, a common method to find outliers within a distribution, to identify side chain methyl groups that have substantial changes in their resonance positions upon mutation (Supplementary Fig. S3). For the residues that have significant deviations (modified Z-score > 0.25), analysis of the CSPs like those shown in Fig. 2a revealed a linear change in peak position as a function of muta- tion. The linear movement demonstrates that these CSPs are not simply the result of structural changes resulting from the mutations to methionine, as one would expect those effects to not have systematic differences in the methyl-TROSY spectra. Instead, the data of Fig. 2a can most easily be explained as a shift in equilibrium between two states, where each of the mutants has a different population of the two states, that is fast on the NMR chemical shift timescale and results in the observed population weighted average of chemical shifts31. We observed signif- icant CSPs in the network of salt-bridge and hydrogen bond interactions that connect the Q-loop and R805 to the P-loop (Supplementary Fig. S3)18. The disruption of this network has been noted in the crystal structure of a further truncated Pf Rad50NBD(R805E) construct (i.e., it does not contain any of the coiled-coil domain) and is thought to be responsible for the increased ATP-binding affinity observed for the R805E mutation19 (see below). Furthermore, we also observed methyl CSPs at the base of the coiled-coil domain and in methyl groups down the extended signature helix to the R797 basic switch residue, which couples this helix to the ABC signature motif and is important for dimerization18. Perturbations to the extended signature helix also affected methyl groups that Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 3 www.nature.com/scientificreports/ Figure 3. Chemical shift perturbations and dynamics changes are correlated. (a) Representative η vs δMethyl scatter plots for Met808Cε (top) and Ile131Cδ1 (bottom). δMethyl values were determined according to equation (1), see Supporting Information. Pearson’s correlation coefficients (RP) are given in the upper right corner. Insets show the build-up curves for the ratio of intensities arising from methyl group 1H triple-quantum “forbidden” experiments36 (Iforbid/Iallow) vs. Results Sid h Orange and blue coloring denotes “Not Correlated” methyl groups with small CSPs upon mutation (i.e., the range in δMethyl < 0.13 ppm) but have a large difference in η rates between wildtype and the mutants (\|ηWT – the average ηmutants\| > 8 sec−1). line a hydrophobic cavity that has been shown to collapse upon ATP binding19. However, a careful inspection of the data shows that the effect of mutation is not uniform for every residue. For example, the V160M and R805E mutations have the same CSP for L778, whereas the CSP for R805E is greater than V160M for L800 (Fig. 2a).t We therefore turned to chemical shift covariance analysis (CHESCA), which is a method to cluster CSPs in fast exchange like those in Fig. 2a into groups of residues experiencing the same shift in populations upon per- turbation31–34. Using the median-absolute-deviations filtered methyl residues (Supplementary Fig. S3), CHESCA produced three distinct clusters of side chain methyl groups within Rad50NBD (Supplementary Fig. S4 and Supplementary Table S1). Figure 2b highlights those clusters on the structure of Mre11HLH-Rad50NBD. Cluster 1 (blue spheres in Fig. 2b) largely encompasses the extended signature motif, β-strands 8, 9, and 10, P-loop, and Walker A motif; cluster 2 (magenta spheres in Fig. 2b) consists of residues near the sites of the mutations but also includes methyl groups along the salt-bridge and hydrogen bonding network that switch upon ATP binding18; cluster 3 (green spheres in Fig. 3b) contains methyl groups within the hydrophobic core – an area that experiences compaction upon dimerization19. Again, these three clusters connect the nucleotide binding site through the Q-loop, R805 basic switch, and signature-coupling helix α2 to the coiled-coil domain at one end of Rad50NBD and to the base of the signature motif at the other. The Rad50NBD allosteric network is a dynamic system. We next questioned the effect that the above mutations would have on methyl group dynamics. The data in Fig. 2a is indicative of fast conformational exchange on the chemical shift timescale, which can be quantified with relaxation dispersion Carr-Purcell-Meiboom-Gill (CPMG) methods35. However, we did not observe any relaxation dispersions in wildtype or mutant Mre11HLH-Rad50NBD (see Supporting Information) indicating that if exchange is happening it is faster than the Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 4 www.nature.com/scientificreports/ msec timescale. Results Sid h Fast timescale (psec – nsec) motions were quantified from the rate of the build-up for “forbidden” methyl 1H triple quantum coherences36 (η) in Mre11HLH- Rad50NBD complexes (Fig. 3a, insets). The calculated η rates directly report on the amplitude of methyl side chain motion (order parameter - Saxis 2 ) as well as the global tumbling time (τc)36. These experiments were performed on the nucleotide-free state for each mutation, where Mre11HLH-Rad50NBD is monomeric, and therefore relate to changes in methyl group dynamics upon mutation and not changes in global tumbling resulting from dimerization. When the η rates for each Rad50NBD (i.e., WT or V156M, V160M, or R805E) are plotted against the weighted combined methyl chemical shift37 (δMethyl), as shown in Fig. 3a (and Supplementary Table 1), a clear linear relationship is observed for many of the residues that had significant chemical shift deviations upon mutation (i.e., the range in δMethyl > 0.13 ppm corresponding to a mod- ified Z-score > 0.25). Thus, the amplitudes of methyl side chain dynamics also experience a population weighted shift upon mutation. We then analyzed the trend in the η rates versus δMethyl to extract a change in the amplitude of the methyl group dynamics upon mutation. The changes in fast timescale methyl dynamics are highlighted on the structure of Mre11HLH-Rad50NBD in Fig. 3b: red and green spheres represent methyl groups with η rate vs. δMethyl correlations that are experiencing increasing and decreasing dynamics, respectively. We also examined methyl groups with small changes in methyl chemical shift (range in δMethyl < 0.13 ppm). Although the local envi- ronment of these methyl groups was not affected by mutations, their fast timescale dynamics might be. Indeed, the orange and blue spheres in Fig. 3b represent those methyl groups with small changes in δMethyl but with a sub- stantial increase or decrease in side chain mobility, respectively. Together, these dynamics data, shown on the structure in Fig. 3b, illustrate a dynamic allosteric network that is generally becoming more flexible upon muta- tion. Specifically, we see increased flexibility (red and orange spheres) in and around the P-loop, the hinge (signa- ture coupling helix α2) and base of the coiled-coil domain, including β-strands 8, 9, and 10, as well as parts of the extended signature helix near a hydrophobic cavity. Results Sid h On the other hand, isolated regions where the hinge (both signature coupling helices α1 and α2) interacts with the extended signature helix as well as the area around the H-loop are becoming more rigid (green and blue spheres). Changes in the Rad50NBD allosteric network affect Rad50 activity. To determine the functional role of the allosteric network shown in Figs 2b and 3b, we performed biochemical activity assays on Rad50NBD to assess changes in ATP and ADP affinity, ATP hydrolysis, and ATP-induced dimerization upon mutation. We hypothesized that since the mutations near the coiled-coil domain altered the structure and dynamics of methyl groups at the sites of ATP binding and Rad50 dimerization then these biochemical processes would be affected by mutation as well. y ATP binding affinities were determined by monitoring the increase in the fluorescence polarization of a BODIPY-ATP analog as a function of increasing Mre11HLH-Rad50NBD concentration. From these titrations (Supplementary Figs S5 and S6), we determined a KD,ATP of 16.9 ± 3.9 μM for the wildtype complex. As pre- dicted from our hypothesis, mutations to the signature coupling helix α2 (V156M and V160M) and basic switch (R805E) did result in altered ATP binding affinities. The experimentally determined KD,ATPs (Supplementary Table 2) for the mutants show a lower KD,ATP (i.e., a higher affinity) compared to wildtype. We used fluores- cence resonance energy transfer (FRET) to determine the affinity of Rad50NBD for the hydrolysis product ADP. Titration of Mre11HLH-Rad50NBD to a modified MANT-ADP (Supplementary Figs S5 and S6) yielded an increase in FRET as the acceptor MANT-ADP comes into proximity of a Rad50NBD tryptophan residue, likely W78219. From these data, a KD,ADP of 2.3 ± 0.2 μM was calculated for wildtype Mre11HLH-Rad50NBD, and Supplementary Table 2 demonstrates that mutations to the hinge region and basic switch have similar KD,ADP values as wildtype. g g D,ADP yp Next, Rad50NBD ATP hydrolysis Michaelis-Menten kinetics were calculated from the increase in inorganic phosphate product concentration, as detected by the BioMol green reagent. Because of the very low rate of ATP hydrolysis observed for isolated Rad50NBD, we performed this assay with full-length Mre112-Rad50NBD 2 complex, which has a much greater rate of ATP hydrolysis5. Similar to ATP binding, we observed a ~2-fold lower KM for V160M and R805E mutants as compared to WT (Supplementary Table 2 and Supplementary Fig. S5), while the kcat for these mutants increased by nearly 4-fold. Results Sid h V156M has Michaelis-Menten parameters that fall between WT and V160M/R805E. Finally, the influence of the mutations on ATP-induced dimerization was assessed by size exclusion chromatography. As previously observed, the R805E mutation has a dramatic effect on Rad50NBD ATP-induced dimerization, increasing the dimer population to ~79% compared to the ~35% seen for wildtype (Supplementary Table 2 and Supplementary Fig. S5)19. In line with the results above, the hinge mutations also serve to enhance Mre11HLH-Rad50NBD ATP-induced dimerization, shifting the population of dimer upon ATP binding to 64% and 57% for V156M and V160M, respectively.f g p y Figure 4a illustrates the relative effect that each mutation has on Rad50NBD ATP hydrolysis and ATP-induced dimerization activities. For each of these, the bars are ordered based on the general movement of peak positions away from WT resonances in the methyl-TROSY correlation spectra (gray arrows in Fig. 2a and in the upper inset of Fig. 4a). This representation shows the relationship between peak position in Fig. 2a and a higher rate of ATP hydrolysis (Fig. 4a, left) and a greater propensity to form ATP-induced dimer (Fig. 4a, middle). Importantly, this relationship demonstrates that altering the Rad50NBD dynamic allosteric network highlighted in Figs 2b and 3b results in increased activities, which could possibly arise from either lowering the energy barrier for Rad50NBD activity and/or increasing the population of an “active” Rad50NBD conformation in solution (i.e., conformational capture). Mutations that probe Rad50NBD allostery affect Mre11 exonuclease activity. Since a correlation was observed between the equilibrium peak positions of the hinge region and basic switch mutations in 2D 13C,1H methyl-TROSY spectra and Rad50NBD activity, we next sought to determine if these Rad50 mutations had any effect on Mre11 nuclease activity. 3′-to-5′ Mn2+-dependent Mre11 exonuclease activity was monitored as an increase in fluorescence of a 2-aminopurine (2-AP) nucleotide analog, which has low fluorescence when Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 5 www.nature.com/scientificreports/ Figure 4. Basic switch and hinge region mutations affect Rad50 activity. (a) Bar chart showing the effect of mutation, relative to wildtype activity, for ATP hydrolysis (left - kcat/KM), ATP-dependent dimerization (middle - % dimerization), and Mre11 exonuclease activity (right – relative fluorescence). Green, orange, purple, and blue bars represent wildtype, V156M, V160M, and R805E, respectively. The order of the bars follows the general order of peaks that experience significant CSPs upon mutation. Results Sid h For ATP hydrolysis and exonuclease assays, bars represent the average of at least three independent measurements, and the error bars are the standard deviation of the replicate experiments. Dimerization assays were performed once. , * and *** Represent p-values less than 0.05, 0.01, and 0.001, respectively. The inset above shows the CSPs for L47Cδ1. (b) Structure of Mre11HLH-Rad50NBD highlighting the side chain methyl groups that have a \|mean[RP,Hydrolysis, RP,Dimerization, RP,Exonuclease]\| > 0.65. Figure 4. Basic switch and hinge region mutations affect Rad50 activity. (a) Bar chart showing the effect of mutation, relative to wildtype activity, for ATP hydrolysis (left - kcat/KM), ATP-dependent dimerization (middle - % dimerization), and Mre11 exonuclease activity (right – relative fluorescence). Green, orange, purple, and blue bars represent wildtype, V156M, V160M, and R805E, respectively. The order of the bars follows the general order of peaks that experience significant CSPs upon mutation. For ATP hydrolysis and exonuclease assays, bars represent the average of at least three independent measurements, and the error bars are the standard deviation of the replicate experiments. Dimerization assays were performed once. , * and *** Represent p-values less than 0.05, 0.01, and 0.001, respectively. The inset above shows the CSPs for L47Cδ1. (b) Structure of Mre11HLH-Rad50NBD highlighting the side chain methyl groups that have a \|mean[RP,Hydrolysis, RP,Dimerization, RP,Exonuclease]\| > 0.65. incorporated into a DNA duplex but high fluorescence as the free nucleotide. This probe is placed at the second position from the 3′-end of a 29 bp DNA duplex and is released upon exonuclease cleavage of the DNA. As expected, the presence of the non-hydrolyzable ATP analog AMP-PNP resulted in a dramatic reduction in exo- nuclease activity, as the closed form of the M2R2 complex is stabilized and the exonuclease active site of Mre11 is occluded (Supplementary Fig. S7)17,38,39. Although the Rad50 mutations did not have a significant effect on Mre11 exonuclease activity in the absence of ATP, as seen in Supplementary Fig. S7, the addition of nucleotide caused a statistically significant increase in 2-AP signal for all the mutants when compared to the WT complex (p-value < 0.05). Again, when the relative activity is plotted in the order of NMR peak position, as shown in Fig. 4a right, the observed increase in Mre11 exonuclease function correlates strongly with the observed CSPs shown in the 2D 13C,1H methyl-TROSY spectra in Fig. 2a. Results Sid h Thus, the increase in Rad50NBD activity upon mutation is also seen for Mre11 exonuclease activity.t To quantify the relationship between the shift in population of Rad50NBD structure and dynamics (Figs. 2 and 3) and the changes in M2R2 activity (Fig. 4a and Supplementary Table S2), we calculated the Pearson’s correlation coefficients (RP) of δMethyl versus Rad50NBD nucleotide binding, ATP hydrolysis, and dimerization activities as well as M2R2 exonuclease activity (Supplementary Table S1). 15 of 25 residues in cluster 1 and eight of ten residues in cluster 2 have a meaningful correlation (RP > 0.65) between dynamic changes in structure and ATP hydrolysis, dimerization, and/or exonuclease activities. And 20 of these 23 residues show a meaningful correlation to all three of these activities, underscoring the intimate association between Rad50 ATP-induced dimerization and subse- quent ATP hydrolysis and Mre11 exonuclease activity within M2R2. Interestingly, all of the correlations in cluster 1 are positive, while all the correlations in cluster 2 are negative – a result that emphasizes the power of CHESCA to cluster residues into relevant groups. None of the residues in cluster 3 correlate with these M2R2 activities (i.e., ATP-induced dimerization, ATP hydrolysis, or exonuclease). The side chain methyl groups of clusters 1 and 2 that are correlated with activity are highlighted on the structure of Mre11HLH- Rad50NBD in Fig. 4b. This view shows a path around the perimeter of Rad50NBD, from the signature motif to the hinge across the P-loop and down to the Walker A motif, linking the two sites of ATP-binding with the dimerization interface (Supplementary Fig. S8). Discussion V156 is on the same face of the signature-coupling α2 helix as V160, but the side chain points toward the signature-coupling α1 helix. We pos- tulate that the V156M mutation begins the process of splaying these two helices apart, which occurs upon ATP binding18. Although the movement of these two helices will affect the conserved Q-loop leading to changes in basic switch R805 interactions, the effects would be lesser, resulting in the intermediate changes that we observe in methyl group chemical shift.h y g pt The dynamics information supplied by our NMR data greatly enhances the pictures provided by X-ray crys- tallography. The data presented in Figs 2 and 3 are unique in that many of the CSPs observed with mutation are correlated with a change in the amplitude of side chain methyl group motion. Changes in the salt-bridge and hydrogen bonding network noted by Deshpande et al.19, and seen in our CSPs (Fig. 2b), have been implicated in the altered activities of the R805E mutation. Here, we show that a general loosening of side chain motions (~65% of residues in the network are experiencing an increase in dynamics – Supplemental Table S1), probed by the build-up of “forbidden” methyl 1H triple quantum coherences, also accompanies these previously observed changes in structure. Among the residues with the largest change in η rates upon mutation, signifying a change in Saxis 2 , are L802 and L806 which are in the extended signature helix and line a hydrophobic cavity. The R805E muta- tion causes a decrease in their η rates that corresponds to ∆Saxis 2 , or change in methyl axis order parameters, of ~−0.3–−0.4 (when using an estimated τc of 30 nsec; see Supporting Information). This is a considerable decrease for a parameter that ranges between 0 and 1 for a completely flexible or rigid methyl group, respectively. Previous biochemical studies have shown that filling this cavity, via a L802W mutation, resulted in decreased ATP binding and hydrolysis and ATP-induced dimerization19. The data in Fig. 3 now also suggest a dynamic role for this hydro- phobic cavity in addition to a simple structural role. Discussion Even though X-ray crystallography has revealed the large structural changes that occur to the M2R2 complex upon Rad50 ATP binding14–17, there are still unanswered questions about how this global change and underlying local motions affect DNA DSB recognition, repair initiation, and downstream signaling. High-resolution solution-state studies should be able to provide key information about structures and dynamics not accessible by static crystal- lography or low-resolution small angle X-ray scattering (SAXS) data. Here, we use methyl-based solution state NMR to further the existing structural biology studies on the essential M2R2 complex. We coupled our NMR data 6 Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 www.nature.com/scientificreports/ on Pf Rad50NBD with biochemical data to learn more about a dynamic allosteric network that transmits informa- tion throughout Rad50 to promote function.hii g p The basic switch residue R805 was first identified through the comparison of monomer and ATP-induced dimer Mre11HLH -Rad50NBD crystal structures18. Subsequent X-ray crystallography, biochemical, and in vivo data has shown that the R805E mutant has altered ATP hydrolysis activity and a higher propensity to form dimers in the presence of ATP, which in turn alters M2R2 DNA end tethering, Mre11 endonuclease activity, and down- stream signaling for DNA DSB repair in yeast19. The methyl-TROSY 13C,1H correlation spectrum of Mre11HLH -Rad50NBD (R805E) shows many large CSPs, including regions perturbed in the crystal structure of Pf Rad50NBD (R805E) (i.e., the P-loop) and others that are not (i.e., the base of extended signature helix and β strands 8, 9, and 10). The X-ray crystal structures of WT Rad50NBD (pdb: 3QKS18) and Rad50NBD(R805E) (pdb: 4NCI19) are similar with a Cα and all atom r.m.s.d. of ~1.7 Å and ~2.0 Å, respectively. Yet, our data demonstrates that this basic switch mutation affects every part of Rad50NBD involved in ATP binding and hydrolysis and dimerization. We have also identified two additional mutations, V160M and V156M in the signature-coupling α2 helix, that affect the same residues as R805E in the Mre11 HLH-Rad50NBD methyl correlation spectra. V160 packs against R805, so we hypothesize that the longer side chain of the V160M mutation acts to sterically hinder R805 from its main chain interaction with the carbonyl group of N134. It is therefore not surprising that V160M has similar effects as R805E in the methyl correlation spectra and in the biochemical assays. Discussion y While these data provide a description for altered structure and dynamics (i.e., breaking non-covalent inter- actions and increasing motions) of R805E, V160M, and V156M, these data more importantly provide a different view into the changes in conformation and mobility within Rad50NBD that have to be overcome when ATP binds in order to make a dimer and consequently productive ABC ATPase. Each of these mutations causes many small changes across the structure of Rad50NBD spread across ~50 Å, in contrast to the large global rearrangements that occur upon dimerization, which prepares Rad50NBD for ATP binding and subsequent dimerization. Moreover, these mutations appear to increase conformational entropy, probed through a general increase in methyl group dynamics40,41, unlocking dynamics that need to be accessed for ATP binding. These effects are what we observe in the structural and dynamics data presented in Figs 2 and 3. As illustrated in the model in Fig. 5, these structural and dynamics changes appear to lead to a more “active,” dimerization competent Rad50NBD conformation, either through destabilizing the ATP-free ground state and/or increasing the population of this dimerization competent state. The model for the ATP binding competent state in Fig. 5 is simplified as the data in Fig. 2 suggests the pres- ence of more than one excited state. Yet, these states are very likely in a dynamic equilibrium interconverting on a fast timescale, with one (or more) responsible for on-pathway ATP-dependent dimerization. Given the fast exchange on the chemical shift timescale of Fig. 2a and the lack of observable relaxation dispersion CPMG, a lower limit on the observed rate of exchange (kex) can be placed at ~2000 sec−1. Because of the populated weighted average of the fast timescale methyl amplitudes shown in Fig. 3a, it is tempting to place the conformational exchange on the nanosecond timescale; however, these correlations do not arise because exchange is fast relative to τc or the timescale of Saxis 2 but rather fast relative to the η rates, which is actually slower than the CPMG win- dow. Thus, we hypothesize an upper limit on the observed kex of ~107 sec−1 – a range of conformational exchange rates that have yet to be accessed for methyl groups by NMR spectroscopy. The increased probability of an active Rad50NBD then manifests itself in the observed increases in ATP hydrolysis, Rad50 dimerization, and Mre11 exo- nuclease activity (Fig. 4). Materials and Methods P i i d Protein expression and purification. Codon optimized Pf Rad50 nucleotide binding domain (Rad50NBD: aa1-195; GGAGGAGG linker; aa709-882) was synthesized by Life Technologies and subsequently cloned into a modified pET-29 vector (Novagen). Point mutations were made using the Quikchange (Stratagene) approach. E. coli BL21(DE3) C41 cells (Sigma) were transformed and grown in deuterated M9 minimal media with 1 g/L 15NH4Cl and 3 g/L 2H,12C-glucose. U-[2H,15N], Ileδ1-[13CH3], Leuδ/Valγ-[13CH3,12CD3], Metε-[13CH3] (ILVM) labeled samples of 6xHis-Rad50NBD were produced following the protocol of Tugarinov et al. with precursors added to the bacterial culture 45 min before induction42. Protein expression was induced with 1 mM IPTG for ~16 h at 25 °C. Cells were lysed in 25 mM HEPES, pH 7.0, 300 mM NaCl, 25 mM imidazole via homogeniza- tion, and then heated for 30 min at 65 °C. Clarified cell lysate was loaded onto a 5 mL HisTrap HP column (GE Healthcare) and eluted with 300 mM imidazole. The 6xHis tag was removed with TEV protease and Rad50NBD was repurified on the HisTrap column. The sample was subsequently diluted to decrease the NaCl concentration to ~80 mM, then loaded onto a HiTrap SP HP column (GE Healthcare), and eluted in a linear gradient into 25 mM HEPES, pH 7.0, 1 M sodium acetate, 0.1 mM EDTA. The SP elution peak was concentrated using ultrafiltration (Vivaspin, Sartorius) before loading onto a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) equilibrated in 25 mM HEPES, pH 7.0, 200 mM sodium acetate, 0.1 mM EDTA. Pf Mre11HLH (aa344-426) was sub-cloned from codon optimized full–length Pf Mre11 (synthesized by Life Technologies) into a pETM-60 vector containing an N-terminal NusA-6xHis tag. The plasmid was transformed into E. coli BL21(DE3) C41 cells, and protein expression was induced with 1 mM IPTG for 4 h at 37 °C. Cells were lysed in 50 mM HEPES, pH 7.5, 30% (v/v) glycerol via homogenization. Clarified cell lysate was loaded onto a 5 mL HisTrap HP column and eluted with 300 mM imidazole. The NusA-6xHis tag was removed with TEV protease, followed by dialysis into 50 mM HEPES, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol. Mre11HLH was repurified on the HisTrap column. The sample was then brought up to 1.5 M ammonium sulfate and loaded onto a HiTrap Phenyl HP column (GE Healthcare). Mre11HLH was eluted in a step gradient of decreasing ammonium sulfate. Discussion An allosteric pathway between Mre11 and Rad50 has been implicated in the past with a likely conduit being the Rad50 coiled-coil domain and Mre11HLH interaction18,19. However, we did not see any changes in methyl group chemical shift or fast timescale dynamics in this area (Figs 2 and 3) with our mutations. We do observe increased mobility at the base of the coiled-coil domain, which could lead to rigid body motions that would be transmitted to Mre11 via the helix-loop-helix motif18, but the NMR data presented here would not be sensitive to such rigid body motions. Furthermore, this route would require transmission of the Rad50 ATP state via a long flexible linker between Mre11HLH and its capping domain. On the other hand, we observe changes in structure and dynamics at the base of the extended signature helix, the signature motif, and β strands 8, 9, and 10. This area Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 7 www.nature.com/scientificreports/ Hinge Extended Signature Helix Signature Motif D-loop Walker A P-loop Q-loop Wildtype Mutants Rad50 Dimer Rad50 Monomer ATP Binding ATP Hydrolysis Dimerization Competent State Conformational Equilibrium ATP Hydroly TP Hydro rium ATP + 1 2 2 ADP + 1 Figure 5. Model for Rad50NBD “activation” to a binding competent state. Left, illustrates the “ground” state Rad50NBD with stable interactions between the hinge, extended signature helix, basic switch, and Q-loop. This state is in equilibrium (green reaction arrow) with an ensemble of “Dimerization Competent States,” middle, in which the stable interactions are starting to break and increased side chain dynamics are present. The effect of V156M, V160M, and R805E is to increase the population of this state, as denoted by the orange, purple, and blue reaction arrows. ATP binds to this state, and dimerization can subsequently occur, right. The process is reset by ATP hydrolysis and dimer dissociation. Hinge Extended Signature Helix Signature Motif D-loop Walker A P-loop Q-loop Wildtype Mutants Rad50 Dimer Rad50 Monomer ATP Binding ATP Hydrolysis Dimerization Competent State Conformational Equilibrium ATP Hydroly TP Hydro rium ATP + 1 2 2 ADP + 1 Dimerization Competent State Rad50 Monomer Figure 5. Model for Rad50NBD “activation” to a binding competent state. Left, illustrates the “ground” state Rad50NBD with stable interactions between the hinge, extended signature helix, basic switch, and Q-loop. Discussion This state is in equilibrium (green reaction arrow) with an ensemble of “Dimerization Competent States,” middle, in which the stable interactions are starting to break and increased side chain dynamics are present. The effect of V156M, V160M, and R805E is to increase the population of this state, as denoted by the orange, purple, and blue reaction arrows. ATP binds to this state, and dimerization can subsequently occur, right. The process is reset by ATP hydrolysis and dimer dissociation. of Rad50NBD makes contact with the Mre11 capping domain, a region thought to be important for exonuclease substrate DNA unwinding and translocation4. Rad50NBD ATP-induced dimerization and subsequent hydrolysis could power the conformational changes in this domain, which are necessary for efficient M2R2 nuclease activity. Thus, this second interaction could be an alternative or additional pathway for Mre11-Rad50 allostery. The muta- tions that we have produced and characterized here are perfect tools for upcoming methyl-based NMR studies on ILVM-labeled Mre11 to pin down the role of the Rad50 ABC ATPase in M2R2 DNA DSB activity. In conclusion, we have shown that methyl-based NMR methods are sensitive to the subtle structural and dynamics changes that accompany mutation in Rad50NBD and provide a view into the processes that underlie Rad50NBD functions required for DNA DSB repair. Materials and Methods P i i d To determine which methyl groups experience a deviation due to mutation, we used a median-absolute-deviation (MAD) approach29,30, which is routinely applied to distributions to find outliers. Here we assume that in the absence of an effect, there will be small, random fluctuations in the peak position due to noise which will be reflected in a small standard deviation. If one or two mutations have an effect then a slightly larger standard devi- ation will be calculated. Finally, if the mutations have a systematic effect on the peak position, a large standard deviation will be obtained. Thus, for each methyl group, the standard deviation of the four δMethyl values (i.e., WT, R805E, V160M, and V156M) was determined. We then calculated the median of the δMethyl standard deviations, and for each methyl group calculated the absolute value of the difference between the median and the methyl group standard deviation. This value along with its median was then converted into a Z-score (see Supplementary Fig. S3 for flow chart of analysis). Methyl groups with Z-scores greater than 0.25, 1.5, and 2.5 were used to color atoms in Supplementary Fig. S3 light blue, blue, and dark blue respectively.t pp y g g p y Chemical shift covariation analysis (CHESCA)32 was performed with in-house written python scripts that make use of the scientific python (scipy) library. Using methyl groups that had a significant deviation according to the MAD approach (Z-score > 0.25), unit-less inter-residue correlation distances45,46 (dij) were calculated between each side chain methyl group using the δMethyl values for each mutation according to δ δ δ δ δ δ δ δ = − − ⋅ − − − ( ) ( ) ( ) ( ) d 1 (2) ij methyl i methyl i methyl j methyl j methyl i methyl i methyl j methyl j , , , , , , 2 , , 2 (2) where δmethyl i, (δmethyl j, ) and δmethyl i, (δmethyl j, ) are a vector and mean of the δMethyl values for the i-th (j-th) residues, respectively, the · is the dot product, and \|\| \|\|2 is the norm. The correlation distance is similar to the Pearson’s cor- relation coefficient; however, the correlation distance is zero only if the random vectors (i.e., δmethyl i, and δmethyl j, ) are truly independent45,46. dij values were not filtered prior to clustering. Materials and Methods P i i d A final HisTrap column purification was performed on the 95% B elution peak containing Mre11HLH and Mre11HLH was collected in the flow through. HLH NBD NBD HLH l g Mre11HLH-Rad50NBD complexes were made by mixing Rad50NBD with a slight excess of Mre11HLH and heating at 60 °C for 10 min. Once cooled, the Mre11HLH-Rad50NBD complex was purified on the HiTrap SP HP column. 8 Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 www.nature.com/scientificreports/ For NMR samples, Mre11HLH-Rad50NBD complexes were buffer exchanged into deuterated NMR buffer (25 mM HEPES, pH 7.0 [uncorrected], 150 mM NaCl) and concentrated. NMR assignments. Assignments were made using 3D 13C,13C,1H HMQC-NOESY-HMQC data (250 msec mixing time) recorded on a 900 MHz (21.1 T) Varian VNMRS NMR spectrometer equipped with a cryogeni- cally cooled z-axis gradient probe (Rocky Mountain Regional 900 MHz Facility, University of Colorado, Denver). NOE-based assignments were validated and extended by recording 2D 13C,1H methyl-TROSY HMQC spectra on cysteine point mutations (I190C, V768C, V866C, L103C, L176C, and L51C) in the absence and presence of MTSL nitroxide spin label (Toronto Research Chemicals). Additionally, Rad50 hinge region mutations (V156M, V157M, V160M, and L163M) were produced to help resolve a crowded region in the Rad50NBD structure. The choice of the mutation to methionine was derived from a sequence alignment of Rad50, which showed the need for a hydro- phobic residue at these positions, and the desire to put the newly labeled methyl group in an uncrowded region of the methyl-TROSY spectra. HMQC spectra on these mutations were recorded on a 600 MHz (14.1 T) Agilent DD2 NMR spectrometer equipped with a room temperature z-axis gradient probe. All NMR data were collected at 50 °C, and spectra were processed with NMRPipe43 and analyzed with CCPN analysis44. Methyl chemical shift perturbation analysis. To help analyze the effect that mutation has on each of the side chain methyl groups, a combined weighted methyl chemical shift37 value (δMethyl) was calculated from the 13C (δC) and 1H (δH) chemical shifts according to δ δ δ = + w w / / Methyl C C H H δ δ δ = + w w / / (1) Methyl C C H H (1) where wc = (1.65, 1.6, 1.4, and 1.54) and wH = (0.29, 0.28, 0.27, and 0.41) are the standard deviations for δC and δH from the Biological Magnetic Resonance Data Bank for Ileδ1, Leuδ, Valγ, and Metε methyl groups, respectively. Materials and Methods P i i d Parallel and perpendicular fluorescence were measured using a Synergy Neo2 multimode reader equipped with a FL 485/530 filter (Biotek); polarizations were calculated via BioTek software. Binding affinities were calculated by fitting polarization vs. unlabeled nucleotide concentra- tion with an in-house script to the previously described inhibition function competitive binding function50. The calculated KI,ATP and KI,ADP are in agreement with the data presented in Supplementary Table S2, showing the same trends as observed for the KD’s determined with the fluorescently labeled nucleotide analogs. Dimerization. Mre11HLH-Rad50NBD dimerization was analyzed on a Superdex 200 Increase 10/300 GL col- umn (GE Healthcare) as described by Desphande et al.19. Each sample contained 75 μM Mre11HLH-Rad50NBD and 5 mM MgCl2 in 50 μL with and without 5 mM ATP. Samples were incubated at 60 °C for 10 min, cooled briefly on ice, and centrifuged for 10 min at 14,000 rpm. The samples were then loaded onto the column equilibrated in 25 mM HEPES, pH 7.0, 150 mM NaCl, and 0.1 mM EDTA at 4 °C. The fraction of dimeric Mre11HLH-Rad50NBD was determined by measuring the area under the curve, using a box sum, for monomer and dimer peaks and dividing the area of the dimer by the total area of the two peaks. ATP hydrolysis assay. ATP hydrolysis kinetics were measured in 50 mM Tris, pH 7.75, 100 mM NaCl, and 10 mM MgCl2. 60 μL reactions containing either 0.5 μM (for V160M and R805E) or 2 μM Mre112-Rad50NBD 2 (For wildtype and V156M) with 1.2-fold excess Mre11 and 0–150 μM ATP were incubated for 60 min at 65 °C, then cooled on the bench for 2 min before centrifuging at max speed for 1 min. Reactions without protein at each ATP concentration (i.e., an ATP only titration) were included for each experiment as a control for ATP degradation/ PO4 contamination. 50 µL of each reaction was transferred into clear, flat-bottom 96-well plates, then 100 µL of cold BIOMOL Green reagent (Enzo Life Sciences) was added to each well. The plates were centrifuged at 500 × g for 1 min and the BIOMOL reaction was allowed to proceed at room temperature for 30 min. The plate was read for BIOMOL Green signal at A640 in the Synergy Neo2 multi-mode reader using the path length correction function to correct for any differences in volumes between the wells. Materials and Methods P i i d protein concentration ([P]) with an in-house script to the quadratic binding function, = + − + + − + + − ∗ F F F F K ATP P K ATP P ATP P ATP ( )( [ ] [ ]) ( [ ] [ ]) 4[ ][ ] 2 [ ] Max D D 0 0 2 (4) where F0 and FMax are initial and final polarization values. Reported errors are the standard deviation of n ≥ 3 experiments. ADP binding affinity. Mre11HLH-Rad50NBD binding affinity to ADP was measured using FRET between the intrinsic tryptophan residues in Rad50 and MANT-ADP (2′-(or-3′)-O-(N-Methylanthraniloyl) Adenosine 5′-Diphosphate, Thermo Scientific). Reactions were performed in 25 mM HEPES, pH 7.0, 150 mM NaCl, 0.1 mM EDTA. MANT-ADP was titrated (0–50 µM) into a black, flat-bottom 384-well plate, where each well contained 5 µM Mre11HLH-Rad50NBD and 5 mM MgCl2. The plate was then centrifuged at 500 × g for 2 min and incubated at room temperature for 10 min. Tryptophan residues were excited at 290 nm and MANT emission from FRET was detected at 450 nm on the Synergy Neo2 plate reader. Spectral overlap was corrected using the tryptophan emission from an unlabeled ADP titration with the appropriate protein concentration and the emission from a titration of MANT-ADP alone, where the signal from FRET has the background contribution from donor and acceptor subtracted from each point49. Binding affinities were calculated by fitting FRET intensity vs. protein concentration to the quadratic binding function equation (4). Reported errors are the standard deviation of n ≥ 3 experiments. Nucleotide competition studies. Binding affinities for Mre11HLH-Rad50NBD to ATP and ADP were also measured via a competition assay, as shown for the data in Supplementary Fig. S6. BODIPY FL ATP was pre-bound to Mre11HLH-Rad50NBD (wildtype or R805E) to achieve ~80% fraction bound, and a decrease in fluo- rescence polarization was monitored as a function of increasing unlabeled ATP or ADP (Supplementary Fig. S6). Reactions were performed in 25 mM HEPES, pH 7.0, 150 mM NaCl, and 0.1 mM EDTA. ATP or ADP (0–1 mM) was titrated into a black, flat bottom 384-well plate where each well contained 5 nM BODIPY FL ATP, 5 mM MgCl2, and 25 μM Mre11HLH-Rad50NBD (wildtype or R805E). The plate was then centrifuged at 500 × g for 2 min and then incubated at room temperature for 10 min. Materials and Methods P i i d Connections were determined using the complete linkage algorithm, which has been shown to minimize false positives compared to the original single linkage method47. A unit-less distance cut off of 1.5, which was empirically determined based on the dendrogram in Supplemental Fig. S4, was used to derive the three clusters. The python script to perform CHESCA on methyl residues is available upon request. Methyl group relaxation and analysis. The build-up and decay of methyl 1H ‘forbidden’ triple-quantum coherences were measured at 50 °C as described by Sun et al. using an Agilent 600 MHz (14.1 T) DD2 NMR spec- trometer36. Relaxation delays (T) of 2, 4, 7, 12, 17, 22, 27, 32, 37, and 42 msec were recorded in an interleaved manner. Datasets were processed and analyzed using NMRPipe and associated programs43. Intra-methyl 1H - 1H dipolar cross-correlated relaxation rates (η) were calculated by fitting ratios of peak intensities as a function of relaxation time, η η δ η δ δ η δ = + + − + I I C T T ( tanh( )) tanh( ) (3) forbid allow 2 2 2 2 2 2 (3) where Iforbid and Iallow are the intensity of the 1H triple- and single-quantum coherences, respectively, C = 0.75, and δ accounts for relaxation from external protons36. The reported errors were calculated from the covariance matrix of the fit48. where Iforbid and Iallow are the intensity of the 1H triple- and single-quantum coherences, respectively, C = 0.75, and δ accounts for relaxation from external protons36. The reported errors were calculated from the covariance matrix of the fit48. ATP binding affinity. ATP binding affinity of Mre11HLH-Rad50NBD was measured as described previously by Majka et al. by observing the fluorescence polarization of BODIPY FL ATP (Adenosine 5′Triphosphate, Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 9 www.nature.com/scientificreports/ BODIPY FL 2′-(or-3′)-O-(N-(2-Aminoethyl)-Urethane), Life Technologies) in a Synergy Neo2 multi-mode reader (BioTek)17. Reactions were performed in 25 mM HEPES, pH 7.0, 150 mM NaCl, 0.1 mM EDTA. Mre11HLH-Rad50NBD (0–70 μM) was titrated into a black, flat bottom 384-well plate where each well contained 5 nM BODIPY FL ATP and 5 mM MgCl2. The plate was centrifuged at 500 × g for 2 min and then incubated at room temperature for 10 min. Parallel and perpendicular fluorescence were measured using a FP 485/530 filter; polarizations were calculated via BioTek software. Binding affinities were calculated by fitting polarization (F) vs. References References 1. Rupnik, A., Lowndes, N. F. & Grenon, M. MRN and the race to the break. Chromosoma 119, 115–35 (2010). 1. Rupnik, A., Lowndes, N. F. & Grenon, M. MRN and the race to the break. Chromosoma 119, 115–35 (2010). 2. Williams, R. S., Williams, J. S. & Tainer, J. A. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, d strand break signaling, and the chromatin template. Biochem. Cell Biol. 85, 509–20 (2007). 3. Paull, T. T. & Gellert, M. The 3′ to 5′ Exonuclease Activity of Mre11 Facilitates Repair of DNA Double-Strand Breaks. Mol. Cell 1, 969–979 (1998). 4. Williams, R. S. et al. 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The 2-aminopurine (2-AP) exonuclease assays were performed in 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% PEG-6000, and 2.5% glycerol as described by Williams et al.4. Two DNA strands Exo2-5 (5′-GGCGTGCCTTGGGCGCGCTGCGGGCGG(2-AP)G-3′) and Exo2-3 (5′-CTCCGCCCGCAGCGCGCCCAAGGCACGCC-3′) were resuspended in 10 mM Tris, pH 7.5 at a concen- tration of 100 μM. Equal volumes of strands were combined, heated for 5 min at 95 °C, then slowly cooled to room temperature to allow the strands to anneal. 30 μL exonuclease reactions contained 0.5 μM Mre112-Rad50NBD 2 complex (with 1.2-fold excess Rad50NBD) and 1 μM 2-AP DNA duplex plus 1 mM MnCl2 or 1 mM MnCl2/5 mM MgCl2/1 mM ATP or 1 mM MnCl2/5 mM MgCl2/1 mM AMP-PNP. Reactions were incubated at 60 °C for 45 min in a heat block (and in the dark to prevent quenching of the fluorophore), then cooled on the bench for 2 min before centrifuging at max speed for 1 min. 25 μL of each reaction was transferred to black, flat-bottom 384-well plates. 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An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule FEBS Lett. 360, 111–114 (1995). Acknowledgements g We thank all members of the Latham laboratory for assistance and discussion. We are grateful to Dr. Lewis E. Kay (University of Toronto) for critically reading this manuscript. This work was funded by a Welch Foundation Grant D-1876-20150314 (M.P.L.). The Rocky Mountain Regional 900 MHz NMR facility is supported by the University of Colorado Cancer Center Support Grant (P30 CA046934). We thank all members of the Latham laboratory for assistance and discussion. We are grateful to Dr. Lewis E. Kay (University of Toronto) for critically reading this manuscript. This work was funded by a Welch Foundation Grant D-1876-20150314 (M.P.L.). The Rocky Mountain Regional 900 MHz NMR facility is supported by the University of Colorado Cancer Center Support Grant (P30 CA046934) Author Contributions Z.K.B., M.D.C., and M.P.L. conceived and designed the research; Z.K.B., S.R., M.D.C., and M.P.L. performed the research and analyzed the data; Z.K.B. and M.P.L. contributed analysis tools; Z.K.B., M.D.C., and M.P.L. wrote the paper; and M.P.L. supervised the project. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1. Boulton, S. & Melacini, G. Advances in NMR Methods to Map Allosteric Sites: From Models to Translation. Chem. Rev. 116 6267–6304 (2016). 2. Selvaratnam, R., Chowdhury, S., Vanschouwen, B. & Melacini, G. Mapping allostery through the covariance analysis of NMR chemical shifts. Proc. Natl. Acad. Sci. USA 108, 6133–6138 (2011).h t ( ) 33. Selvaratnam, R., Mazhab-Jafari, M. T., Das, R. & Melacini, G. The Auto-Inhibitory Role of the EPAC Hinge Helix as Mapped by NMR. PLoS One 7, 1–9 (2012). 3. 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Detecting outliers: Do not use standard deviation around the mean, use absolute deviation around the median. J. Exp. Soc. Psychol. 49, 764–766 (2013). Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 11 Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19908-8. Supplementary information accompanies this paper at https://doi.org/10.1038/s4 Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. 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 an institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific ReportS \| (2018) 8:1639 \| DOI:10.1038/s41598-018-19908-8 12
W2954760479.txt	https://www.mdpi.com/1996-1944/12/13/2131/pdf?version=1562065810	en		The Effects of Temperature on the Hydrothermal Synthesis of Hydroxyapatite-Zeolite Using Blast Furnace Slag	Materials	2,019	cc-by	6,446	materials Article The Effects of Temperature on the Hydrothermal Synthesis of Hydroxyapatite-Zeolite Using Blast Furnace Slag G.U. Ryu 1 , G.M. Kim 2 , Hammad R. Khalid 3 1 2 3 * and H.K. Lee 1, * Department of Civil and Environmental Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea Center for Carbon Mineralization, Climate Change Mitigation and Sustainability Division, Korea Institute of Geoscience and Mineral Resources, 124 Gwahak-ro, Yuseong-gu, Daejeon 34132, Korea Civil & Environmental Engineering Department, King Fahd University of Petroleum & Minerals, Dhahran 31261, Saudi Arabia Correspondence: haengki@kaist.ac.kr; Tel.: +82-42-350-3623; Fax: +82-42-350-3610 Received: 4 June 2019; Accepted: 1 July 2019; Published: 2 July 2019 Abstract: Blast furnace slag, an industrial by-product, is emerging as a potential raw material to synthesize hydroxyapatite and zeolite. In this study, the effects of temperature on the hydrothermal synthesis of hydroxyapatite-zeolite from blast furnace slag were investigated. Specimens were synthesized at different temperatures (room temperature, 50, 90, 120, or 150 ◦ C). The synthesized specimens were analyzed qualitatively and quantitatively via X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), BET/BJH, and scanning electron microscopy/energy dispersive using X-ray analysis (SEM/EDX). It was found that the hydroxyapatite phase was synthesized at all the reaction temperatures, while faujasite type zeolite appeared in the specimens synthesized at 90 and 120 ◦ C. Moreover, faujasite was replaced by hydroxysodalite in the specimens synthesized at 150 ◦ C. Additionally, the crystals of the hydroxyapatite tended to become larger and total crystallinity increased as the reaction temperature increased. Keywords: hydroxyapatite; zeolite; adsorbent; hydrothermal method; blast furnace slag 1. Introduction Hydroxyapatite (Ca10 (PO4 )6 (OH)2 ) and zeolites (alumino-silicate materials represented by Mm [Alm Sin O2(m+n) ] · H2 O where M represents alkali cations electrostatically bonded to the extra skeleton) have been used for various applications, i.e., adsorption and catalysis [1,2]. Specifically, they have been studied as adsorbents due to their high specific surface area and the ionic forces induced by the presence of exchangeable ions in their structures [3–5]. The adsorption performance of zeolites for cations is much higher than that for anions since the exchangeable ions in zeolites are usually alkali cations [6,7]. On the other hand, hydroxyapatite phases have high adsorption performance for both the cations and anions [4,6,8,9]. Cations are exchanged with calcium ions present in hydroxyapatite [6,10], while anions are exchanged with hydroxyl ions [4]. Hydroxyapatite and zeolites are normally synthesized by hydrothermal methods [11–13]. The reaction temperature for hydrothermal treatment is an important variable for the characteristics of the resulting products [14,15]. If the reaction temperature is not high enough, hydroxyapatite and zeolites cannot be synthesized, and it can result in a low specific surface area [14,16]. The formation rate of different crystals and the aspect ratio of hydroxyapatite crystals also get affected by the reaction temperature [17]. Similarly, an increase in the reaction temperature was favorable to synthesize zeolite crystals through a hydrothermal process [16]. Materials 2019, 12, 2131; doi:10.3390/ma12132131 www.mdpi.com/journal/materials Materials 2019, 12, 2131 2 of 12 In this study, blast furnace slag, which is a by-product of the iron industry, was used to synthesize hydroxyapatite and zeolite as main phases. Both of these materials were targeted, considering the fact that zeolites are efficient adsorbents for cations, and they can support hydroxyapatite to adsorb cations and anions simultaneously. Because of its low price and mineral composition, it has been used in various fields, especially the construction and environmental fields [1,18,19]. However, about 35% of the slag is still being wasted in Europe alone [20]. Owing to the chemical composition of slag, it can be used to synthesize hydroxyapatite and zeolites [12,21,22]. Recently, Khalid et al. [12] proposed a robust one-step hydrothermal treatment method to synthesize geopolymer-supported zeolite adsorbents using blast furnace slag and fly ash. Guo et al. [23] synthesized NaA-zeolite from blast furnace slag and studied their performance for removal of ammonium. Kuwahara et al. [1] and Li et al. [24] studied the effects of reaction time on the synthesis of zeolite and sodalite using Ti-bearing electric arc furnace slag. Despite the high potential of slag utilization for the synthesis of these adsorbents, only two studies have been reported on the synthesis of hydroxyapatite using blast furnace slag [1,11]. Furthermore, it is known that the reaction temperature during hydrothermal treatment can significantly affect the resulting crystalline phases and total crystallinity [14,15,25]. Therefore, it is crucial to investigate the effects of the reaction temperature on the synthesis of hydroxyapatite using blast furnace slag, which has not been reported in the literature to the best of our knowledge. Hence, this study specifically focused on the investigation of effects of reaction temperature on the crystallinity, content, and dimension of synthesized hydroxyapatite and zeolite via the hydrothermal synthesis method. The specimens were synthesized at different reaction temperatures (room temperature, 50 ◦ C, 90 ◦ C, 120 ◦ C, or 150 ◦ C). The characteristics of the specimens were investigated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy/energy dispersive using X-ray analysis (SEM/EDX), while microstructural characteristics were investigated via BET/BJH method. 2. Materials and Methods 2.1. Materials The chemical composition of the blast furnace slag used in the present study was investigated by XRF; results are listed in Table 1. The slag was mainly composed of CaO, Al2 O3 , and SiO2 components, which are needed to synthesize hydroxyapatite and zeolites. Table 1. Chemical components of blast furnace slag. Compound Proportion (wt.%) Al2 O3 SiO2 SO3 K2 O CaO TiO2 MnO Fe2 O3 NiO CuO SrO ZrO2 7.50 18.20 3.10 0.76 67.60 0.95 0.44 1.0 0.04 0.046 0.019 0.082 Materials 2019, 12, 2131 3 of 12 2.2. Test Methods The synthesis process for hydroxyapatite-zeolite consisted of three steps. In the first step, 25 g of slag and 100 mL of 1.77 M phosphoric acid (H3 PO4 , Samchun Pure Chemical, Pyeongtaek, Korea) solution were stirred at 350 rpm using a magnetic stirrer at ambient temperature for 2 h. In this step, the molar ratio of Ca/P was fixed at 1.67 considering the theoretical Ca/P ratio of hydroxyapatite is 1.67. In the second step, 350 mL of 2 M sodium hydroxide (NaOH, Daejung Chemical & Materials Co., LTD, Goryeong, Korea) solution was added to the prepared suspension. Mixtures were stirred at 450 rpm for 4 h at their respective temperatures (such as room temperature, 50, 90, 120, or 150 ◦ C) on the bottom of the beakers and specimens were denoted by HZ-R, HZ-50, HZ-90, HZ-120, and HZ-150, respectively. The mixture containers were fully covered with an aluminum foil to prevent the mixtures from water evaporation during stirring. In the last step, the prepared suspensions were transferred to sealed Teflon bottles and heated for 24 h in an oven again at their respective temperatures (i.e., room temperature, 50, 90, 120, or 150 ◦ C). After the last step, white powders were obtained. The obtained powders were washed several times with distilled water, followed by filtration and drying. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) measurement, scanning electron microscopy/energy dispersive using X-ray analysis (SEM/EDX), and BET/BJH tests were conducted to characterize the crystallinity of the prepared specimens. The XRD analysis was conducted in a 5◦ –65◦ 2θ scan range at 0.2◦ /min scanning rate using PANalytical X’Pert PRO-MPD (DA107 at KBSI Daegu Center). Then, to investigate the quantities of hydroxyapatite and zeolites produced under the different synthesis conditions, the content of the crystalline phases was estimated from XRD diffraction patterns. It should be noted that the reference intensity ratio method, which is suitable method for zeolitic samples synthesized from blast furnace slag, was used to approximate the amount of crystalline phase of the samples [12,26]. SEM images were obtained to investigate the morphology of the crystalline phases in the synthesized specimens. FT-IR measurements (6300FV and IRT5000 device, JASCO, Tokyo, Japan) were conducted in the range of 4000–450 cm−1 under a vacuum state using the KBR tablets in the transmission mode. The specific surface areas of the specimens were measured by BET test, and BJH pore volume tests were conducted to investigate the pore size distribution (PSD). EDX analysis was carried out to investigate the elemental composition of the reaction products. Before the EDX analysis, the samples were coated with Pt to enhance the conductivity of the samples, thereby increasing the visibility of the morphologies. It should be noted that the EDX spectra of the samples presented in this study excluded the intensity originated from the presence of Pt for a better comparison with other spectra. 3. Results and Discussion 3.1. Crystalline Characteristics The XRD test results of the specimens synthesized at different reaction temperatures are shown in Figure 1. The XRD pattern of the blast furnace slag indicated that the slag included amorphous phases and crystalline peaks of anhydrite (PDF #01-072-0916) and melilite (solid solution of gehlenite, PDF #00-035-0755 and åkermanite, PDF #01-074-0990) [1,12]. The XRD patterns of the specimens synthesized at room temperature and 50 ◦ C showed broader peaks corresponding to hydroxyapatite (PDF #98-003-4457), possibly due to low crystallinity. Alternatively, the peaks of the specimens synthesized at 90 ◦ C, 120 ◦ C, and 150 ◦ C were quite sharp, showing well-developed crystals in these specimens as verified with SEM images. Materials 2019, 12, 2131 4 of 12 The XRD patterns of the specimens synthesized at room temperature and 50 ◦ C showed only hydroxyapatite peaks (PDF #98-003-4457), while those of the specimens synthesized at 90 ◦ C and 120 ◦ C showed faujasite (FAU) type zeolite (PDF #98-008-5622) peaks as well. This indicated that zeolite phases can also be synthesized when the reaction temperature was 90 ◦ C or more. However, the XRD patterns of the specimens synthesized at 150 ◦ C showed the inclusion of hydroxysodalite (PDF #98-016-3788) instead of faujasite because hydroxysodalite can be synthesized at a higher temperature than that to synthesize zeolite [27]. Hydroxysodalite is zeolite-like material, which also possess an affinity to adsorb cations such as heavy metals [12,28]. The crystallinity and quantitative content of the specimens synthesized at different reaction temperatures are listed in Table 2. It was observed that the crystallinity of the specimens fabricated at room temperature and 50◦ C was similar, meaning that reactivity was the same when the reaction temperature increased to 50◦ C. The crystallinity of the specimens got doubled when the temperature increased to 90 ◦ C, compared to that of specimens synthesized at lower temperatures. Moreover, the crystallinity of specimens tended to increase with reaction temperature. Compared to the content in specimens synthesized at reaction temperatures lower than 50 ◦ C, the content of the hydroxyapatite phase increased when the reaction temperature was more than 90 ◦ C. The content of the hydroxyapatite phase did not change significantly at reaction temperatures more than 90◦ C. Meanwhile, the content of FAU type zeolite or hydroxysodalite increased as the reaction temperature increased. This means that zeolite or zeolite-like phases formed well when the reaction temperature increased beyond 90 ◦ C. On the other hand, the reaction temperature did not significantly influence the quantity of formation of hydroxyapatite, although crystallinity does increase with reaction temperature. The intensity of the peaks at 603 and 566 cm−1 , representing PO4 3− groups in hydroxyapatite, increased with the increase in reaction temperature from room temperature to 90 ◦ C; this peak intensity slightly decreased with further increase in the reaction temperature [1]. This indicates the formation of zeolite crystals in the specimens synthesized above 90 ◦ C. On the other hand, the peak intensities of the SiO4 4− and PO4 3− groups decreased with the increase of the reaction temperature to 90 ◦ C. Moreover, the intensities of the CO3 peaks (1465–1468 and 1417 cm−1 ) and the dicalcium phosphate dehydrate peak (875 cm−1 ) were highest in the specimen synthesized at room temperature. This seems to be because dicalcium phosphate dehydrate was transformed into hydroxyapatite as the reaction temperature increased. The intensity of two water peaks at 3449–3438 and 1640 cm−1 decreased with increasing reaction temperature due to the evaporation of water. It should be noted that the samples were oven dried at the identical temperature used for their synthesis, which resulted in the decrease in the water content in the samples synthesized at high temperatures. Table 2. Crystallinity and quantitative content of the materials synthesized at the different reaction temperature. Reaction Temperature Amorphous Phase (%) Crystallinity (%) Phases Quantitative Content (%) Room temperature 50 ◦ C 64 66 36 34 90 ◦ C 37 63 120 ◦ C 43 57 150 ◦ C 30 71 Hydroxyapatite Hydroxyapatite Hydroxyapatite FAU type zeolite Hydroxyapatite FAU type zeolite Hydroxyapatite Hydroxysodalite 36 34 50 13 50 7 46 25 Materials 2019, 12, 2131 5 of 12 Materials 2019, 12, x FOR PEER REVIEW 5 of 12 H: Hydroxyapatite F: FAU type zeolite S: Hydroxysodalite A: Anhydrite M: Melilite S H S S H H S H H H H S H H H H S H H S S H H H H S S S 150 ℃ F H F F Intensity (a.u.) F F F F H H H F F FH H F H H H H H 120 ℃ F H H F F H F H F F F H H H H H H 90 ℃ H H H H H H H H 50 ℃ H H M A H H Room temperature M A Raw slag A 0 10 20 30 40 50 60 70 2θ(◦ ) Figure The pattern XRD pattern rawand slag the specimens synthesized at the different Figure 1. The1.XRD of rawofslag theand specimens synthesized at the different reaction reaction temperature. temperature. FT-IR spectra of the specimens synthesized at different reaction temperatures are presented in FT‐IRspectra spectraof ofthe the synthesized specimens synthesized at showed different clear reaction temperatures areatpresented in 963, Figure 2. The specimens absorption peaks 1093, 1037, Figure 2. The spectra of the synthesized specimens showed clear absorption peaks at 1093, 1037, 963, 875, 603, and 566 cm−1 , which are generally assigned to the vibration of the PO 3− groups, although −1, which are generally assigned to the vibration of the PO43− 4groups, although 875, 603, and 566 cm 3− some parts of the PO4 absorption peaks are difficult to discern due to the superposition of the some parts of the PO43− absorption peaks are difficult to discern due to the superposition of the absorption bands with those of the silica shouldbebenoted noted that silica matrix absorption bands with those of the silicagroup group [1,29]. [1,29]. ItItshould that thethe silica matrix and and 4− the hydroxyapatite share many similar vibration Thisisisdue duetotothe the similarity of the 44− 4 the hydroxyapatite share many similar vibrationmodes modes [1]. [1]. This similarity of the SiOSiO 3− −1 −1 (Si–O–Si and PO tetrahedral units [1,29]. modesare are460 460cm cm (Si–O–Si stretch/P–O bending), and units [1,29].The Thevibration vibration modes stretch/P–O bending), 43− tetrahedral 4 PO −1 −1 −1 −1 963 cm (Si–O/P–O symmetric stretch), 1037 cm (Si–OH deformation vibration/vibration mode of of 963 cm (Si–O/P–O symmetric stretch), 1037 cm (Si–OH deformation vibration/vibration mode 3− −1 3− −1 group), 1093 (Si–O/P–O stretch) stretch) [1,29]. thethe four peaks at 1465–1468, 1417,1417, 4 PO4 PO group), andand 1093 cmcm (Si–O/P–O [1,29].However, However, four peaks at 1465–1468, −1 are distinctly indicative of hydroxyapatite [1]. The peaks at 1465–1468 and 1417 −1 cm 603,565 andcm 565 603, and are distinctly indicative of hydroxyapatite [1]. The peaks at 1465–1468 and 1417 cm−1 3− 32− groups replacing the PO cm−1 are assigned to the carbonate thehydroxyapatite. hydroxyapatite. The are assigned to the carbonate CO3 2− CO groups replacing the PO4 3−4 ininthe Thepeaks peaks of of the carbonate groups usually appear in all the hydroxyapatite synthesized in the air [30]. The last the carbonate groups usually appear in all the hydroxyapatite synthesized in the air [30]. The last two two peaks at 603 and −1 566 cm−1 can represent hydroxyapatite formation [1]. The small peak at 875 cm−1 −1 peaks at 603 and 566 cm can represent hydroxyapatite formation [1]. The small peak at 875 cm is is due to HPO42− groups from the reactant, which is dicalcium phosphate dehydrate (CaHPO4·2H2O), 2− ·2H2 O), due to HPO4 groups from the reactant, which dicalciumacid phosphate dehydrate (CaHPO synthesized from blast furnace slag mixed withis phosphoric without NaOH solution [1,31].4On synthesized from blast furnace slag mixed with phosphoric acid without NaOH solution [1,31]. On the −1 the other hand, the left two peaks at 3449–3438 and 1640 cm represent water [32]. −1 other hand, the left two peaks at 3449–3438 and 1640 cm represent water [32]. Materials 2019, 12, 2131 Materials 2019, 12, x FOR PEER REVIEW 6 of 12 6 of 12 Figure 2. The FT-IR spectra of the materials synthesized at the different reaction temperature. Figure 2. The FT‐IR spectra of the materials synthesized at the different reaction temperature. 3.2. Microstructural Characteristics 3.2. Microstructural Characteristics BET/BJH test was conducted to investigate the specific surface area and microstructure of the specimens synthesized this study.toThe specific surface area and pore characteristics of specimens BET/BJH test wasinconducted investigate the specific surface area and microstructure of are the presented in Table 3. The test results show that the specific surface area of the specimens synthesized at specimens synthesized in this study. The specific surface area and pore characteristics of specimens ◦ 50 decreased in compared the specimens synthesized room temperature. the specimens are Cpresented Table 3.toThe test results show thatatthe specific surfaceHowever, area of the specimens ◦ C had the largest specific surface area because FAU type zeolite formed, while synthesized at 90 synthesized at 50 °C decreased compared to the specimens synthesized at room temperature. ◦ C decreased continuously. the specificthe surface area ofsynthesized the specimens at 120 ◦specific C and 150 However, specimens at 90synthesized °C had the largest surface area because FAU type This tendency is similar for the cumulative volume of pores from BJH adsorption. zeolite formed, while the specific surface area of the specimens synthesized at 120The °C decrease and 150 in °C the specific surface area of the synthesized specimens was possibly attributable to changes content decreased continuously. This tendency is similar for the cumulative volume of pores in from BJH among hydroxyapatite, FAU typespecific zeolite,surface and hydroxysodalite (see Table specimens 2). The reason the adsorption. The decrease in the area of the synthesized was that possibly ◦ C was lowest seems to be the formation of specific surface area of the specimens synthesized at 150 attributable to changes in content among hydroxyapatite, FAU type zeolite, and hydroxysodalite (see hydroxysodalite, which has lower specific surface than that of the FAU type zeolite [12]. The BJH Table 2). The reason that the specific surface areaarea of the specimens synthesized at 150 °C was lowest ◦ adsorption pore diameter of the specimens synthesized 120 Csurface was thearea largest of seems to beaverage the formation of hydroxysodalite, which has lower at specific thanand thatthat of the ◦ C was the smallest. These results seem to be due to differences in pore the specimens synthesized at 50 FAU type zeolite [12]. The BJH adsorption average pore diameter of the specimens synthesized at distribution of specimens at differentsynthesized reaction temperatures. the specific 120 °C was the largest andsynthesized that of the specimens at 50 °C wasOverall the smallest. Thesesurface results area decreases as the temperature increases within the temperature range for producing the same seem to be due to differences in pore distribution of specimens synthesized at different reaction crystal phase [33,34]. Moreover, the higher specific surface area can help to improve the adsorption temperatures. Overall the specific surface area decreases as the temperature increases within the capacity [35,36]. temperature range for producing the same crystal phase [33,34]. Moreover, the higher specific surface area can help to improve the adsorption capacity [35,36]. Table 3. BET/BJH results of the materials synthesized at the different reaction temperature. Table 3. BET/BJH results of the materials synthesized at the different reaction temperature. Room Reaction Temperature 50 ◦ C 90 ◦ C 120 ◦ C 150 ◦ C Temperature Reaction Temperature Specific surface area (m2 /g) 84.84 BJH Adsorption Specific surfacecumulative area (m2/g) 0.28 volume of cumulative pores (cm3 /g)volume of BJH Adsorption 3/g) pore BJH Adsorption average pores (cm 11.30 diameter (nm) BJH Adsorption average pore diameter (nm) Room 50 90 69.35 98.74 °C 78.57 Temperature °C 84.84 69.35 98.74 0.19 0.30 0.28 9.44 11.30 0.19 0.19 9.96 0.30 13.37 9.44 9.96 120 78.57 150 °C 51.34 0.19 0.18 51.34 °C 0.18 13.18 13.37 13.18 The pore size distributions (PSD) of the specimens are plotted in Figure 3. For all the specimens, the pores were mostly distributed in the mesoporous range (10 nm and 30 nm). The specimens synthesized at room temperature and 90 °C have a relatively high specific surface area and, compared Materials 2019, 12, 2131 7 of 12 The pore size distributions (PSD) of the specimens are plotted in Figure 3. For all the specimens, Materials 2019, 12, x FOR PEER REVIEW 7 of 12 the pores were mostly distributed in the mesoporous range (10 nm and 30 nm). The specimens ◦ C have a relatively high specific surface area and, compared synthesized at room temperature and 90 to the other specimens, were found to contain a large number of pores with sizes between 10 and 30 to thenm. other specimens, found to contain abecause large number of pores with sizes 10 andfor 30 nm. This pore sizewere range is encouraging mesoporous materials are between advantageous This pore size range is encouraging because mesoporous materials are advantageous for adsorbing adsorbing contaminants [12,21,37]. However, it should be noted that the contaminant adsorption contaminants [12,21,37]. However, it should be noted thatthan the contaminant adsorption capacity capacity of the hydroxyapatite‐zeolite could be higher that of pure hydroxyapatite becauseof the zeolite has the higher specific surface area andofadsorption capacity than those of hydroxyapatite, hydroxyapatite-zeolite could be higher than that pure hydroxyapatite because zeolite has the higher implying zeolite, which can only adsorb cations, can complement other specific surfacethat area and adsorption capacity than thoseand of hydroxyapatite hydroxyapatite, implying thateach zeolite, which [1,11]. can only adsorb cations, and hydroxyapatite can complement each other [1,11]. Figure 3. The pore distribution of the materials synthesized at the different reaction temperature. Figure 3. The pore distribution of the materials synthesized at the different reaction temperature. 3.3. SEM/EDX Test 3.3. SEM/EDX Test SEMSEM images of crystals areare shown Thehydroxyapatite hydroxyapatite crystals in the specimens images of crystals shownininFigure Figure 4. 4. The crystals in the specimens ◦ C look like irregular clusters of needles [38] (Figure 4a). The size of the hydroxyapatite synthesized at 120 synthesized at 120 °C look like irregular clusters of needles [38] (Figure 4a). The size of the crystals was about 300 µm. The withμm. a size approximately 120ofµm and hierarchical poreand system hydroxyapatite crystals wascrystals about 300 Theofcrystals with a size approximately 120 μm hierarchical pore which systemwas are FAU type zeolite, which was observed[39] in the identical [39] are FAU type zeolite, observed in the identical specimens (Figure 4b).specimens Hydroxysodalite, 4b). Hydroxysodalite, had spherical that aggregate form agglomerates with which(Figure had spherical crystals thatwhich aggregate to form crystals agglomerates with a to size of approximately 40 µm, ◦ a size of approximately 40 μm, was observed in the specimens synthesized at 150 was observed in the specimens synthesized at 150 C, as shown in Figure 4c [28]. °C, as shown in Figure 4c [28]. The EDX test results for each crystal are presented in Figure 5. The (Ca + Mg + Na)/P molar ratio The EDX test results for each crystal are presented in Figure 5. The (Ca + Mg + Na)/P molar ratio of the hydroxyapatite crystals, shown in Figure 5a, corresponding to a stoichiometric composition of the hydroxyapatite crystals, shown in Figure 5a, corresponding to a stoichiometric composition of of 1.68, which means that Ca is partially substituted with Mg and Na components [1]. From an 1.68, which means that Ca is partially substituted with Mg and Na components [1]. From an elemental elemental analysis, the FAU typeappears zeolitetoappears to beX‐zeolite a Na-type X-zeolite molar ratio of analysis, the FAU type zeolite be a Na‐type having a molarhaving ratio of aSiO 2/Al2O3 = SiO2 /Al 2.47. The ratio of the hydroxyapatite crystals wasofhigher than that of 2 OThe 3 = SiO 2 O3hydroxyapatite 2/Al 2O3 SiO 2.47. ratio2 /Al of the crystals (4.24) was higher(4.24) than that the FAU type the FAU typecrystals. zeolite This crystals. This the indicates theAlSi3+4+ions andbound Al3+ ions bound to the hydroxyapatite lattice zeolite indicates Si4+ and to the hydroxyapatite lattice during hydroxyapatite formation [1]. during hydroxyapatite formation [1]. Materials 2019, 12,12, 2131 Materials 2019, x FOR PEER REVIEW of 12 8 of 812 Materials 2019, 12, x FOR PEER REVIEW (a) 8 of 12 (b) (c) (a) (b) (c) Figure4.4.SEM SEMimages images of of (a) (a) hydroxyapatite, hydroxyapatite, (b) hydroxysodalite. Figure (b)FAU FAUtype typezeolite, zeolite,and and(c)(c) hydroxysodalite. Figure 4. SEM images of (a) hydroxyapatite, (b) FAU type zeolite, and (c) hydroxysodalite. (a) (a) (b) (b) (c) (c) Figure 5. EDX spectraofof(a) (a) hydroxyapatite, hydroxyapatite, (b) type zeolite, andand (c) hydroxysodalite. Figure 5. EDX spectra (b)FAU FAU type zeolite, (c) hydroxysodalite. Figure 5. EDX spectra of (a) hydroxyapatite, (b) FAU type zeolite, and (c) hydroxysodalite. Lastly, the effects of reaction temperature on hydroxyapatite are shown in Figure 6. As the reaction temperature increased, the crystallinity and the size of the hydroxyapatite crystals tended to increase. The hydroxyapatite crystals synthesized at room temperature were about 500 nm in size, and the Materials 2019, 12, x FOR PEER REVIEW 9 of 12 Materials 2019, 12, Lastly, the2131 effects 12 of reaction temperature on hydroxyapatite are shown in Figure 6. As9 ofthe reaction temperature increased, the crystallinity and the size of the hydroxyapatite crystals tended to increase. The hydroxyapatite crystals synthesized at room temperature were about 500 nm in size, crystallinity seemed to be low. The hydroxyapatite crystals synthesized at 50 ◦ C showed good growth and the crystallinity seemed to be low. The hydroxyapatite crystals synthesized at 50 °C showed good compared to those synthesized at room temperature; needle-like shapes of specimens can be confirmed growth compared to those synthesized at room temperature;◦needle‐like shapes of specimens can be in the SEM images [40,41]. In the specimens synthesized at 90 C, hydroxyapatite crystals had irregular confirmed in the SEM images [40,41]. In the specimens synthesized at 90 °C, hydroxyapatite crystals cluster needle shapes, which is similar to the shape of the specimen synthesized at 120 ◦ C, but smaller. had irregular cluster needle shapes, which is similar to the shape of the specimen synthesized at 120 The growth of the hydroxyapatite crystal size, as the reaction temperature increase, is consistent with °C, but smaller. The growth of the hydroxyapatite crystal size, as the reaction temperature increase, other studies [33,42,43]. This is the reason why the specific surface area decreases as the temperature is consistent with other studies [33,42,43]. This is the reason why the specific surface area decreases increases with the same crystal phase (see Table 3) [33,42]. as the temperature increases with the same crystal phase (see Table 3) [33,42]. Figure 6. Hydroxyapatite crystalline synthesized at different reaction temperature. Figure 6. Hydroxyapatite crystalline synthesized at different reaction temperature. 4. Concluding Remarks 4. Concluding Remarks In this study, the effects of reaction temperature on the synthesis of hydroxyapatite-zeolite from In this study, effects of reaction temperature the synthesisSpecimens of hydroxyapatite‐zeolite from blast furnace slag the by the hydrothermal method wereon investigated. were synthesized at blast furnace slag by the hydrothermal method were investigated. Specimens were synthesized at five different reaction temperatures; followed by characterization through XRD, FT-IR, BET/BJH, and five different reaction temperatures; followed bystudy characterization through XRD, FT‐IR, and SEM/EDX analyses. The results presented in this will contribute to broadening the BET/BJH, knowledge of SEM/EDX analyses. The results presented in this study willmaterials contribute to broadening the knowledge physicochemical characteristics of hydroxyapatite-zeolite synthesized from blast furnace of physicochemical hydroxyapatite‐zeolite from blast slag, especially withcharacteristics regard to the of reaction temperature. Thematerials findingssynthesized from the present studyfurnace can be slag, especially with regard to the reaction temperature. The findings from the present study can be summarized as follows. summarized as follows. ◦ C reaction temperature, only hydroxyapatite was synthesized, while FAU type zeolite Up to to 50 50 °C 1. 1. Up reaction temperature, only hydroxyapatite was synthesized, while FAU type zeolite ◦ was formed 120 ◦120 C in addition to hydroxyapatite. With further increase in temperature was formedatat9090C and °C and °C in addition to hydroxyapatite. With further increase in ◦ to 150 C, hydroxysodalite was synthesized instead of FAU type zeolite along with hydroxyapatite. temperature to 150 °C, hydroxysodalite was synthesized instead of FAU type zeolite along with 2. hydroxyapatite. As the reaction temperature increased, the crystallinity of the specimens tended to increase, andthe thereaction contenttemperature of crystallineincreased, phases also The content of hydroxyapatite 2. As thechanged. crystallinity of the specimens tended toincreased increase,with and ◦ temperature up to 90 C, while no change was observed with further increment in the temperature. the content of crystalline phases also changed. The content of hydroxyapatite increased with Materials 2019, 12, 2131 3. 4. 5. 10 of 12 The specimens synthesized at 90 ◦ C had the highest specific surface area of 98.7 m2 /g, and the specimen synthesized at 150 ◦ C had the lowest specific surface area (51.3) potentially due to the formation of the hydroxysodalite, which has a relatively small specific surface area. The pore size distributions of the specimens prepared at room temperature and 90 ◦ C were largely in the mesoporous range, which is advantageous for adsorbing contaminants. SEM test results showed that hydroxyapatite and FAU type zeolite phases could be clearly observed at the reaction temperature of 120 ◦ C, while hydroxysodalite phase was observed at the reaction temperature of 150 ◦ C instead of FAU type zeolite. Hence, it can be concluded that 90 ◦ C was the most optimized reaction temperature to synthesize the hydroxyapatite-zeolite using blast furnace slag by the hydrothermal treatment. The results of this study indicate the potential of the synthesized materials to be used as absorbents to remove contaminants [6,7,10,35,38]. However, further study will be needed to investigate the contaminant adsorption capacity of hydroxyapatite-zeolite synthesized by the hydrothermal method. 5. Patents This section is not mandatory, but may be added if there are patents resulting from the work reported in this manuscript. Author Contributions: Conceptualization, G.M.K.; methodology, G.M.K. and G.U.R.; validation, G.U.R.; formal analysis G.U.R.; investigation, G.U.R.; writing—original draft preparation, G.U.R.; writing—review and editing, G.U.R., G.M.K., H.R.K., H.K.L.; supervision, H.K.L.; project administration, H.K.L.; funding acquisition, H.K.L. Funding: This research was supported by a grant (18CTAP-C143331-01) from Technology Advancement Research Program (TARP) Program funded by Ministry of Land, Infrastructure and Transport of Korean government. 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https://openalex.org/W4232541687	https://periodicos.ufpa.br/index.php/nra/article/download/10021/6973	Portuguese	null	Ver ou baixar revista completa	Nova Revista Amazônica	2,021	cc-by-sa	113,481	REVISTA DO PROGRAMA DE PÓS-GRADUAÇÃO EM LINGUAGENS E SABERES NA AMAZÔNIA UNIVERSIDADE FEDERAL DO PARÁ – CAMPUS BRAGANÇA VOLUME IX – NÚMERO 01 – MARÇO 2021 – ISSN – 2318-1346 QUALIS B3 Os artigos publicados na Nova Revista Amazônica são indexados por: Periódicos – CAPES; Diadorim; LivRe – Revistas de Livre Acesso; latindex – Sistema Regional de Información en Línea para Revistas Científicas de América Latina, el Caribe, España y Portugal; ROAD – Directory of Open Access Scholarly Resources; CiteFactor – Academic Scientific Journals Os artigos publicados na Nova Revista Amazônica são indexados por: Periódicos – CAPES; Diadorim; LivRe – Revistas de Livre Acesso; latindex – Sistema Regional de Información en Línea para Revistas Científicas de América Latina, el Caribe, España y Portugal; ROAD – Directory of Open Access Scholarly Resources; CiteFactor – Academic Scientific Journals REVISTA DO PROGRAMA DE PÓS - GRADUAÇÃO EM LINGUAGEN S E SABERES NA AMAZÔNIA UN IVER SID ADE FED ERA L DO PARÁ – CAMPUS BRAGANÇA VOLUME IX – NÚMERO 01 – M REVISTA DO PROGRAMA DE PÓS GRADUAÇÃO EM LINGUAGEN S E Karley dos Reis Ribeiro Raquel Amorim dos Santos Camila de Cássia Brito ____ Karley dos Reis Ribeiro Raquel Amorim dos Santos Camila de Cássia Brito ____ _63 DOSSIÊ II - PRÁTICAS SOCIOAMBIENTAIS COMUNITÁRIAS APRESENTAÇÃO Cristina Maria Coimbra Vieira Norma Cristina Vieira ________ APRESENTAÇÃO Cristina Maria Coimbra Vieira Norma Cristina Vieira _________ _80 NARRATIVAS DE CRIANÇAS SOBRE O SABER/FAZER EM FESTAS AMAZÔNICAS: O CASO DA MARUJADA DE SÃO BENEDITO E SÃO SEBASTIÃO EM TRACUATEUA/PA Dilma Oliveira da Silva Dilma Oliveira da Silva Nazaré Cristina Carvalho _ _83 Rita Cassia de Quadros Castro Gisela Macambira Villacorta __ _143 SEÇÃO LIVRE _163 _205 REVISTA DO PROGRAMA DE PÓS - GRADUA ÇÃO EM LIN GUAGENS E rotinas sociais, enquanto o segundo, nas fases da juventude e posteriores, podemos ser desafiados a questionar criticamente estes processos de socialização, muitas vezes construídos de maneira acrítica e pouco consciente. Por isso mesmo é que, a educação para as primeiras idades da vida deve ser predominantemente formativa, enquanto a destinada às idades do segundo período deve assumir uma dimensão transformativa. Para que seja possível garantir o direito de todas as pessoas à educação, propiciando atividades educativas transformativas, condizentes com os avanços do conhecimento neste domínio, é preciso defender políticas públicas responsáveis e credíveis, integradas por profissionais com formação adequada e envolvida por uma pesquisa preocupada e atuante. Estas serão as condições para a educação e o desenvolvimento (social, cultural, político e econômico) se desafiarem mutuamente, tendo a transformação contínua da interação das pessoas e dos seus espaços de vida como preocupação dominante. No ano em que celebramos o centenário do nascimento de Paulo Freire, um dos nomes maiores e mais reconhecidos, a nível mundial, neste domínio, a Nova Revista Amazônica, publica um dossiê testemunhando os esforços de pesquisadores, na tarefa de contribuir para dignificar a educação de jovens e adultos, com textos que se orientam, em particular, a reflexão sobre as práticas e a análise dos contextos. Assim, os artigos integrantes do dossiê “Educação de Jovens e Adultos em diferentes espaços da vida” promoveram um diálogo entre países, Brasil e Portugal, estados brasileiros e se evidenciou a diversidade étnica, com ênfase aos povos quilombolas. A Amazônia se destacou em grande parte das investigações apresentadas e Paulo Freire foi figura presente em todas as pesquisas. Temas atuais como a Pandemia da Covid-19 e as Metodologias ativas também estão presentes na coletânea. No artigo Formação profissional e desenvolvimento local: relações e oportunidades a partir da elaboração do projeto educativo de um município Português promoveu-se a discussão das políticas de formação profissional alinhadas às inovações tecnológicas por meio de um projeto estratégico para a educação em contexto municipal, projeto desenvolvido entre a Universidade de Coimbra e o município de Sintra, em Portugal. No artigo A EJA em tempos de Pandemia de Covid -19: Reflexões sobre os direitos e políticas educacionais na Amazônia bragantina analisou-se as políticas educacionais em tempos de pandemia na Amazônia bragantina, com olhar voltado para a Educação de Jovens e Adultos. Evidenciou-se as políticas compensatórias de diminuições de tempos e conteúdos, fechamento de turmas, redução do número de matrículas, além do fechamento dos programas específicos para a EJA, agravando a situação pandêmica. No artigo Aprendizagem Significativa como alicerce para Metodologias Ativas no ensino de Ciências: uma interlocução em prol da Educação de Jovens e Adultos, as reflexões sobre a EJA na busca de uma participação ativa do aluno, frisou a importância da formação docente em diálogo com os autores Freire, Ausubel, Moran e Chassot, os quais promoveram uma nova compreensão de estratégias eficazes à Aprendizagem Significativa em Ciências. Paulo Freire é o ponto central do artigo A questão do tema gerador em Paulo Freire: práticas e experimentações em Tracuateua-PA, suas bases teóricas e práticas foram tomadas como referência nas escolas do município de Tracuateua-PA e suas ideias possibilitaram a resistência às pressões no cenário político atual brasileiro, mantendo o trabalho pedagógico alicerçado na escuta e dialogicidade. Por fim, o artigo Retratos da EJA na Amazônia Marajoara: desafios do ensino da Língua Inglesa na educação escolar Quilombola, discutiu o ensino da Língua Inglesa para alunos da Educação de Jovens e Adultos na perspectiva da Educação Escolar Quilombola, do município de Salvaterra. A pesquisa realçou as questões étnico-raciais nas aulas como sendo um campo desafiador no alcance da qualidade do ensino. A necessidade de formação continuada dos professores de Língua Inglesa pautada nas relações étnico-raciais é uma necessidade emergencial, pois os dados indicaram desafios associados especialmente às suas práticas pedagógicas. Desejamos uma boa leitura deste rico e denso material, que ora vos apresentamos. Dr. Luís Alcoforado Doutor em Ciências da Educação pela Universidade de Coimbra, Professor na Faculdade de Psicologia e de Ciências da Educação da Universidade de Coimbra e Investigador do Grupo de Políticas Educativas e Dinâmicas Educacionais (GRUPOEDE) do Centro de Estudos Interdisciplinares do Século XX (CEISXX). Foi Coordenador do Mestrado em Educação e Formação de Adultos e Intervenção Comunitária (2012 - 2018), é Coordenador do Mestrado em Ciências da Educação e Membro da Comissão de Coordenação do Doutoramento em Ciências da Educação, da Faculdade de Psicologia e de Ciências da Educação da Universidade de Coimbra. E-mail: lalcoforado@fpce.uc.pt. Dra. Nívia Maria Vieira Costa Doutora em Educação pela Universidade Federal do Ceará- UFC. Pós-doutora em Educação de Adultos pela Faculdade de Psicologia e de Ciências da Educação da Universidade de Coimbra. Professora do Instituto Federal de Educação, Ciência e Tecnologia do Pará- IFPA/Campus Bragança. Pesquisadora da Cátedra Paulo Freire da Amazônia e Coordenou o Projeto de Pesquisa Interinstitucional "Paulo Freire: Diversidade e Educação", entre IFPA e UFPA. Avaliadora do INEP/MEC de Instituições e Cursos Superiores; Membro do grupo de pesquisa ETTHOS, do IFPA/ Campus Bragança e NUPEEFT, do IFG/ Campus Goiânia. E- mail: nivia.costa@ifpa.edu.br. VOLUME DOSSIÊ AMAZÔNIA 9 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 10 By combining professional training policies with technological innovations, changes in production and work organization seek to define strategies that result in the individual’s effective participation in their production environment. With an integrative intention and by taking into account the processes and contexts’ changes of the economy sectors, professional training entities should, by adopting a broader view of action, focus on the development of training initiatives adjusted to society’s real needs and, specially, adjusted to the local community in which the citizens act. As a result, within a municipal context’s education strategic project – Local Educational Project – developed between the University of Coimbra and a Lisbon’s Metropolitan Area County (Sintra), still in a diagnostic phase and by using the Focus Group technique, some companies and training institutions representatives of the territory counties were heard about the business characterization and its needs in terms of human resources and training areas. Assuming that local development calls for the participation of all agents and all people, based on a holistic preparation, professional education, as a vital contribution to economic, social and cultural development, becomes a multidimensional phenomenon essentially focused on promoting action capacities and strengthening the conditions for individual and group participation, in the processes of integrated and sustained development, based on the realities of people and their communities. eywords: Local educational project. Professional education. Partnerships and local developmen Data da submissão 15.10.2020 Data da aprovação 07.12.2020 – – VOLUME NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 11 científica e técnica ou tecnológica, variando o peso de cada componente de acordo com a tipologia do curso”, situação que se mantém até 2004 (fase de nova reforma do ensino secundário), em cursos predominantemente orientados para o prosseguimento de estudos – os cursos gerais - e cursos predominantemente orientados para o ingresso na vida ativa – os cursos tecnológicos (CERQUEIRA & MARTINS, 2011). Para além das iniciativas levadas a cabo pelo Ministério da Educação, no âmbito dos ensinos básico e secundário, há que salientar a experiência acumulada nesta área, mormente através dos cursos de formação em alternância, pelo Instituto do Emprego e Formação Profissional (IEFP), sob a tutela do Ministério da Segurança Social e do Trabalho. g ç As Escolas Profissionais surgem, portanto, na sequência das tendências verificadas a partir da década de oitenta, nas políticas educativas portuguesas, numa situação favorável de existência de recursos económicos (provenientes dos fundos estruturais da, então, Comunidade Económica Europeia para a formação profissional), sendo encaradas como uma via alternativa de ensino secundário, “de iniciativa eminentemente local” para os jovens que tivessem concluído o 9º ano de escolaridade. Tal como se enunciava nas decisões políticas, com as Escolas Profissionais (Decreto-Lei nº 26/89, de 21, de janeiro, Artº 3º) pretendia-se, […] contribuir para a realização pessoal dos jovens, proporcionando, designadamente, a preparação adequada para a vida activa; fortalecer, em modalidades alternativas às do sistema formal de ensino, os mecanismos de aproximação entre a escola e o mundo do trabalho; facultar aos jovens contactos com o mundo do trabalho e experiência profissional; prestar serviços directos à comunidade, numa base de valorização recíproca; dotar o País dos recursos humanos de que necessita, numa perspectiva de desenvolvimento nacional, regional e local; preparar o jovem com vista à sua integração na vida activa ou ao prosseguimento de estudos numa modalidade de qualificação profissional; proporcionar o desenvolvimento integral do jovem, favorecendo a informação e orientação profissional, bem como a transição para a vida activa, numa modalidade de iniciação profissional, a nível do 3.º ciclo do ensino básico e até à efectivação da escolaridade obrigatória de nove anos.” (PORTUGAL, 1989). 1 FORMAÇÃO PROFISSIONAL EM PORTUGAL- BREVE CONTEXTUALIZAÇÃO As políticas educativas portuguesas ganham especial enfoque em 1986, ano que ficou marcado por dois acontecimentos charneira, “com repercussões que podemos considerar muito importantes para a evolução futura do sistema de ensino, particularmente, para a componente técnica e profissional” (PARDAL et al., 2003, p. 105). Se, por um lado, assistimos à aprovação em 14 de outubro, da Lei de Bases do Sistema Educativo (LBSE), que definiu o enquadramento legal para a educação, nomeadamente, estabilizando o lugar do ensino técnico e profissional de nível secundário, por outro lado, verifica-se a integração de Portugal na então Comunidade Económica Europeia, facto que se assumiria como de extrema relevância, designadamente, para o financiamento da formação profissional para jovens e adultos (ALCOFORADO et al., 2013) com o objectivo, explicitamente enunciado, de incrementar a produtividade do trabalho e a competitividade da economia. A formação profissional surgiu, deste modo, com a pretensão de se adequar “às necessidades conjunturais nacionais e regionais de emprego”, sendo “dever das escolas básicas e secundárias do país assegurar o funcionamento destes cursos” destinados a jovens que frequentavam o ensino básico e secundário, através do estabelecimento de protocolos de colaboração com o tecido empresarial e as autarquias locais, fomentando uma ligação entre a formação e o emprego (RODRIGUES, 2011, p. 46). A LBSE, na sequência deste processo de reconstrução da oferta de formação profissional inicial, possibilitou, igualmente, o aparecimento de Escolas Profissionais de iniciativa privada, apoiadas pelo Estado Português e pela União Europeia (UE), criadas pelo Decreto-Lei nº 26/89, de 21 de janeiro (PORTUGAL, 1989). A criação destas escolas constituiu-se, desta forma, como um instrumento essencial para a afirmação do ensino profissionalmente qualificante, como modalidade alternativa ao ensino secundário regular (DIAS, 2012). No âmbito da Revisão Curricular dos Ensinos Básico e Secundário, consagrada no Decreto-Lei nº 286/89, de 29 de agosto, prevê-se que o ensino secundário se organize numa estrutura curricular constituída por três componentes: “geral ou sociocultural, específica ou NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 12 Estamos, portanto, na actualidade (PELIZ, 2014), perante uma via de ensino secundário que se alarga a dimensões profissionais, desportivas e artísticas, procurando contribuir decisivamente para o cumprimento da escolaridade obrigatória (entretanto, aumentada para doze anos), sendo capaz, em simultâneo, de proporcionar oportunidades a diferentes níveis, nomeadamente o prosseguimento de estudos e a perspetiva de desenvolvimento pessoal e social, mas sempre com a finalidade central de preparar para o incremento da empregabilidade, formando trabalhadores capazes de aumentar a produtividade do trabalho e das empresas. No que concerne à formação profissional, para pessoas adultas, a adesão de Portugal à Comunidade Económica Europeia, constituiu, igualmente, um momento fundacional de novas opções e de concentração de recursos para enfrentar novas prioridades (CRUZ et al., 2013). Na verdade, se da aposta na formação de jovens poderíamos esperar, na lógica da dimensão ideológica predominante, um futuro com maior produtividade do trabalho e uma economia mais competitiva, a dimensão dos défices que Portugal apresentava, em relação aos seus parceiros do novo bloco económico, nos indicadores que eram privilegiados pelas políticas europeias, eram tão significativos que se tornava urgente empreender mudanças imediatas. E essas, naturalmente, só poderiam advir do investimento na formação das pessoas em idade ativa. Foram, então, disponibilizados meios muito significativos, primeiro, para uma formação profissional muito próxima das, eventuais, necessidades das empresas e depois, já no final do século passado e início deste século, orientadas para uma aposta muito forte na elevação dos níveis de qualificação da população portuguesa, considerando ser esse o melhor preditor para, em simultâneo, atender às necessidades de aumentar a empregabilidade, promover a mobilidade e contribuir para uma economia competitiva, mais suportada no conhecimento e em contributos mais intelectuais. Já no presente século, torna-se imperativo atribuir particular destaque à Resolução do Conselho de Ministros n.º 173/2007 (PORTUGAL, 2007), que aprovou uma nova reforma da formação profissional. A Iniciativa Novas Oportunidades surgiu (ALCOFORADO et al., 2018), nesse contexto, como uma estratégia direcionada para a concretização de um amplo conjunto de reestruturações que, na verdade, vinham colocar novas ambições e disponibilizar novos recursos para os objectivos já enunciados para o sistema de formação profissional, suportando-se em dois pilares fundamentais. Em primeiro lugar, visava fazer do ensino profissionalizante, de nível secundário, uma verdadeira e real opção, dando oportunidades novas aos jovens. DOSSIÊ AMAZÔNIA O surgimento do ensino técnico e profissional era, então, apresentado com o objetivo primordial de fornecer ao mercado de trabalho mão-de-obra especializada, através de profissionais de nível intermédio, com uma boa formação integral, permitindo contribuir para o desenvolvimento socioeconómico do país, para a minimização do desemprego jovem e para a redução do insucesso e abandono escolares. Anteriormente, o ensino técnico e profissional sempre esteve associado a um meio de seleção escolar precoce, que reproduzia a origem social (já que os jovens pertencentes às classes mais baixas eram “orientados” para esta tipologia de ensino, tendo em vista a necessidade de um emprego a curto prazo) e promovia uma formação sem grandes possibilidades de continuidade, não dando, de forma primordial, acesso direto ao ensino superior contribuindo, assim, para a ideia de um ensino desprestigiado. Estas iniciativas procuravam fazer estes percursos valer pela sua qualidade intrínseca, devendo constituir-se como uma via com igual dignidade, sendo, por isso mesmo, uma opção entre outras. Se é certo que entre o legislado e o conseguido existiu sempre uma distância muito considerável, ao longo dos últimos anos, na sequência de compromissos internacionais e do esforço para garantir o ensino secundário como nível mínimo de formação para todos os portugueses, o ensino profissionalizante, mesmo que nunca tenha sido alcandorado ao mesmo prestígio e dignidade de outras vias, passou a envolver mais de metade dos jovens portugueses do ensino secundário, tendo-se procedido a uma reformulação dos objetivos e conteúdos curriculares, procurando reforçar, progressivamente, o reconhecimento social do ensino técnico e profissional, como uma alternativa credível, proporcionando, inclusivamente, possibilidades de continuação para o ensino superior. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 13 bem como das respectivas prioridades nos processos negociais dos contratos-programa plurianuais com a União Europeia. Ainda que no caso dos jovens se consiga identificar uma certa persistência do essencial das opções políticas, no caso das pessoas adultas foram sendo registradas demasiadas oscilações para que hoje possamos assinalar a existência de uma política estável e previsível. Em qualquer dos casos, contudo, nos anos mais recentes, mantendo-se o essencial das dimensões ideológicas, também se criaram condições para um evidente reforço da centralidade discursiva e de práticas com um novo apelo à participação do poder e sociedade civil locais. Têm sido, exactamente, estas novas responsabilidades e competências atribuídas e assumidas pelos municípios, também no domínio da formação profissional, que nos tem ocupado ao longo dos anos mais recentes, em diferentes trabalhos colaborativos de pesquisa e elaboração de políticas de base local. Fomos assumindo a necessidade de reequacionar os pressupostos de elaboração dos princípios orientadores de novas opções, partindo de um entendimento alargado sobre a relação da educação com o desenvolvimento local, acreditando que as políticas públicas municipais devem resultar de uma articulação entre diferentes parceiros, constituindo-se como um instrumento facilitador das dinâmicas das pessoas e das diferentes comunidades que elas integram. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 O segundo pilar estava voltado para elevar a formação de base dos ativos (ALCOFORADO, 2008; ALCOFORADO et al., 2018; ALCOFORADO et al., 2020). Para estas duas dimensões são estabelecidas metas muito ambiciosas, enunciando uma absoluta profissão de fé na necessidade de elevar definitivamente os níveis de qualificação da população e no conhecimento como base para a transformação desejada dos processos produtivos. Em síntese, era declarada a intenção de que toda a formação pudesse assegurar uma progressão escolar e profissional, aumentando a sua relevância para a modernização empresarial, através de uma agenda reformista centrada em cinco linhas fundamentais: estruturar uma oferta formativa, relevante e certificada; reformar as instituições e a regulação da formação; definir prioridades e modelos de financiamento adequados; promover a qualidade da formação e facilitar o acesso e promover a procura da formação (PORTUGAL, 2007). Como tem sido demonstrado (ALCOFORADO et al., 2018), as políticas de Educação e Formação de Adultos, em Portugal, se têm vindo a revelar alguma persistência em termos de recursos disponíveis e finalidades enunciadas, do ponto de vista das iniciativas nacionais e da organização, têm reflectido, de forma recorrente, as sensibilidades dos diferentes governos, NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 14 vizinhança…), ajudando a dar sentido às aspirações e projetos dos seus membros, humanizando-os e humanizando as suas diferentes ações. vizinhança…), ajudando a dar sentido às aspirações e projetos dos seus membros, humanizando-os e humanizando as suas diferentes ações. Olhando de um outro viés, a sociedade atual vem-se caracterizando por céleres mudanças tecnológicas, sociais e económicas, sendo hoje atribuído aos sistemas educativos, pelas diferentes instâncias de poder, o mandato de acompanhar ou antecipar essas transformações. Assiste-se, assim, nos últimos anos, a uma mudança das relações educação- economia e educação-mercado de trabalho, na qual vão sendo definidos, continuamente, novos perfis escolares e profissionais. Por um lado, as idiossincrasias da globalização económica e as ambições competitivas das economias apelam ao reforço das competências relacionadas com a produtividade do trabalho, enquanto, ao mesmo tempo, o conhecimento das Ciências da Formação procuram, ainda que timidamente, influenciar políticas públicas promotoras de dimensões mais qualitativas e de contracorrente, alargando o conceito de formação profissional para lá de um alinhamento natural com as transformações produtivas, as inovações tecnológicas e com a organização do trabalho, mas, principalmente, possibilitando experiências educativas mais holísticas, capazes de preparar os/as trabalhadores/as para uma acção responsável e autónoma na transformação dos seus diferentes contextos de vida. ç Desde há várias décadas, como oportunamente lembrou Lesourne (1988), que os desafios económicos estão a exigir, tanto a nível individual como coletivo, um aumento considerável das competências da população ativa, jovem ou adulta. Este facto remete para uma “melhoria na formação geral dos jovens, no sentido de desenvolver as atitudes e aptidões desejáveis, uma formação profissional facilitadora do acesso ao primeiro emprego, tendo em conta que esta será tanto mais frágil quanto mais especializada, e, por último, uma formação contínua que acompanhe o indivíduo ao longo do seu desenvolvimento profissional e pessoal” (MADEIRA, 2006, p. 124). p É, então, crucial e determinante proporcionar aos jovens uma formação inicial que assegure uma base cultural e técnico-científica alargada que possibilite a adaptabilidade e a mobilidade exigidas numa sociedade em constante mudança. Para além da preparação para o exercício de uma atividade profissional, é essencial atender a dimensões como a inovação, a iniciativa e a aprendizagem ao longo da vida. 1.1 FORMAÇÃO PROFISSIONAL E DESENVOLVIMENTO LOCAL O binómio educação e trabalho sempre deixou transparecer uma relação controversa, complexa e dialética, desafiando a que sejam considerados os princípios educativos e formativos, independentes e resultantes da interceção dos dois campos, os vínculos entre vida produtiva, ciência, cultura e humanismo e levando à necessidade de assumir, em primeiro lugar, o entendimento da formação profissional, como um fator determinante do processo de construção de conhecimentos totalizadores e libertadores do sujeito integrado numa determinada comunidade. Associada a esta relação entre educação e trabalho encontra-se a necessidade do desenvolvimento socioeconómico, político e cultural da sociedade, o qual necessita de um tempo e de um espaço para acontecer, relacionados diretamente com a procura de formas integradas e sustentadas de desenvolvimento local (PIVETEAU, 2010; ALCOFORADO et al., 2014), devendo a formação profissional impulsionar o aparecimento de indivíduos críticos e conscientes das relações que se estabelecem no mundo do trabalho (SOUZA, 2011) e nos contextos territoriais, onde todas as dimensões da vida acontecem. A compreensão do desenvolvimento local, a partir da educação, pressupõe a aceitação de que o pensamento recebe influência do contexto, sendo esta ideia reforçada por Freire (1983, p.17), ao afirmar que “como não há homem sem mundo, nem mundo sem homem, não pode haver reflexão e ação fora da relação homem-realidade”. Assim, para o mesmo autor, faz parte do leque de responsabilidades da escola potenciar o despertar crítico do indivíduo e trazer à realidade este “homem” no seu mundo, com o compromisso de organizar os seus “saberes históricos e escolares”, estruturando currículos de maneira a considerarem saberes significativos para este indivíduo e sua comunidade, de forma que se internalizem as transformações sociais e culturais da sociedade envolvida, numa conceção de interação com o seu meio. Através da formação e da educação, o indivíduo constrói-se e reconstrói-se, adapta-se e transforma as condições dadas pelo seu contexto social, enfrenta desafios pessoais e coletivos, devendo assumir-se como agente de desenvolvimento, em termos pessoais e sociais. Dito de outra forma, acredita-se que a apropriação do saber pelo indivíduo provoca direta e efetivamente o progresso de uma comunidade (profissional, cultural, de 2 METODOLOGIA Na sequência do desafio lançado aos municípios portugueses para assumirem responsabilidades e competências, no domínio da educação, foi sendo criada a necessidade de ser desenvolvido, por cada executivo municipal, um planeamento estratégico, primeiro, no domínio da rede escolar e, depois, de todas as ações que se deseja que possam vir a contribuir para o início da configuração de uma real política local de educação e formação profissional. Dando sentido a estes reptos e obrigações e corporizando a vontade de pensar estrategicamente a evolução da educação, a nível municipal, foi desenvolvido um trabalho conjunto entre uma equipa de investigação interdisciplinar da Universidade de Coimbra e o Departamento de Educação do Município de Sintra e respectivos responsáveis políticos e diferentes agentes com ação e interesse nesta temática, nesse espaço territorial, que resultou em diagnósticos circunstanciados da realidade existente e se propôs elaborar um projeto com um sentido e recursos para o prosseguir, capaz de se traduzir como denominador comum de aspirações e vontades no âmbito educativo, para o curto e o médio prazo – Projeto Educativo Local (PEL). Assumindo este planeamento estratégico no âmbito da educação, para além das suas funções tradicionais, o Município acumula a responsabilidade de desenvolvimento permanente de uma função educadora, orientada para a formação, promoção e desenvolvimento de todos os cidadãos e das respectivas comunidades, tornando-se essencial desenvolver práticas ecoeducativas capazes de permitir que todos aprendam em conjunto, nos seus espaços e em todas as idades da vida, num desafio indutor de desenvolvimento integrado e sustentável com o meio em que vivem e que partilham. Neste processo, a articulação entre as organizações educativas e outros parceiros locais com responsabilidades no sucesso do cidadão municipal revela-se determinante, na medida em que, esta visão estratégica da educação e da formação, procura potenciar todos os recursos educativos do território para construir o melhor caminho que responda à necessidade de uma formação humanista integral, que incremente a criação de riqueza, com emprego digno, multiplique as oportunidades de acesso e produção de bens culturais e vise um bem-estar individual e coletivo, ancorado na ambição da construção continuada de um Município inteligente, inovador, inclusivo e participado. O planeamento do Projeto Educativo Local (PEL), suportado por todas estas características e convicções, assentes nas necessidades dos cidadãos e, concludentemente, num quadro desejável com todos e para todos, suportou-se numa metodologia totalmente participada, ao longo das diferentes etapas do processo. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 A este nível ressalta a importância das competências transversais, como são exemplo a criatividade, a capacidade de comunicação e de resolução de problemas, a capacidade de trabalhar em equipa e de avaliar as contínuas situações de imprevisibilidade com que, inevitavelmente, se irão deparar (AZEVEDO, 1991). Um tal entendimento de formação obriga a uma relação mutuamente desafiante com um projecto de desenvolvimento local ideologicamente consonante e, por maioria de razão, impele à construção de comunidades locais de educação e formação, integradas por todos os parceiros, capazes de garantir a diversidade e continuidade de experiências que estas opções holísticas, integradas e transformadoras exigem. Por isso mesmo, pensar e planear políticas locais exige o compromisso de escutar todos os parceiros e criar hábitos de diálogo e trabalho conjunto para encontrar novas respostas, baseadas em processos mais democráticos, específicos, participados e inovadores. Conscientes desta necessidade e obrigação, e no âmbito dos trabalhos para a elaboração de um plano estratégico educativo municipal, foram implementadas metodologias participativas de envolvimento de todos os agentes, dando nota, neste artigo, das perceções iniciais de um grupo de representantes das empresas e instituições de formação do Município de Sintra, ao nível das necessidades e da disponibilidade para o envolvimento sincero numa outra forma de planear e desenvolver actividades de formação de jovens e adultos. 15 DOSSIÊ AMAZÔNIA NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 16 elegendo uma abordagem moderadamente estruturada, cujo guião de debate terá assumido a forma de um conjunto de linhas orientadoras para a discussão pretendida. elegendo uma abordagem moderadamente estruturada, cujo guião de debate terá assumido a forma de um conjunto de linhas orientadoras para a discussão pretendida. Assim, foram lançadas várias questões para reflexão: O que podemos dizer do tecido empresarial do concelho? E sobre a sua evolução a curto e médio prazo? Como podemos perspectivar a sua evolução em termos de qualificações? De que trabalhadores necessitam as empresas do concelho em termos de formação geral e formação técnica específica? Como podem ser formados esses trabalhadores? Que contributos podem dar as empresas na formação inicial de jovens, na formação contínua e em programas de informação profissional para os adolescentes das escolas do município? 3 RESULTADOS Tendo como referência algumas das linhas orientadoras de reflexão e debate lançadas aos representantes das empresas e entidades de formação do concelho, antecipadamente, torna-se possível estruturar a informação recolhida por blocos temáticos, os quais permitirão evidenciar algumas das características do tecido empresarial do município, bem como principais lacunas e necessidades sentidas no âmbito da formação dos seus recursos humanos. Assim, de forma a facilitar a análise dos dados recolhidos optou-se pela construção de um quadro que sintetiza as áreas temáticas abordadas, unidades de registro e exemplos de transcrições das declarações dos participantes (Tabela 1). ç ç p p Uma análise global das diversas intervenções das entidades presentes tornou visível a opinião generalizada no que concerne à pouca atratividade e falta de mão-de-obra no setor da indústria, resultando em entraves ao nível dos processos de “recrutamento de recursos humanos qualificados” (nomeadamente, jovens) em áreas técnicas (e.g. serralharia, mecânica, canalização, soldadura, química, manutenção industrial, carpintaria, eletricidade, mecatrónica, entre outros), principalmente, quando surgem picos de produção. A mobilidade dos trabalhadores constituiu mais uma das temáticas evidenciadas por vários empresários, tendo estes referido que a ausência de transportes públicos, em determinadas zonas do concelho, torna a viatura própria um requisito obrigatório para a admissão de recursos humanos. Tal facto traduz-se numa acentuada dificuldade no que diz respeito aos processos de recrutamento. No âmbito dos cursos profissionais foi referido o facto de alguns deles terem sido extintos, com particular ênfase na área da química, colocando ao dispor das empresas apenas licenciados e mestres em áreas afins, o que não corresponde às suas necessidades. Importante será também mencionar a disponibilidade manifestada pelas empresas para apoiar as Escolas Profissionais na criação de cursos/ações de formação voltadas para as áreas em análise. Tabela 1. Organização da informação recolhida – Temas, unidades de registo e principais indicadores Temas Unidades de Registo Exemplos Setor industrial 8 “(...) pouca atratividade e falta de mão de obra. Temos dificuldade em recrutar jovens para áreas técnicas, principalmente quando surgem picos de produção”. Mobilidade 3 “Não existe transporte público em algumas zonas, o que dificulta bastante a vida de quem procura trabalho (...) ”. Cursos profissionais extintos 4 “Alguns cursos profissionais simplesmente deixaram de existir, como por exemplo, a química (...) acabamos por apenas ter ao dispor licenciados e mestres em áreas afins”. Falta de reconhecimento 6 “ (...) hoje em dia ainda é evidente o estigma associado ao ensino ela 1. 2 METODOLOGIA Não obstante o alargado conjunto de procedimentos metodológicos elencados na construção do PEL, atendendo às diferentes áreas temáticas e respetivos parceiros locais, neste trabalho, em concreto, centramo-nos, exclusivamente numa das dimensões do eixo estratégico do trabalho, qualificação e inovação. Neste sentido, considerando a importância que o tecido empresarial encerra no desenvolvimento concelhio, ainda numa fase de diagnóstico do PEL, através de um Focus Group, foi proposto a diferentes entidades locais de âmbito empresarial e com intervenção ao nível da qualificação da população jovem e adulta, um momento de reflexão conjunta em torno das características do tecido empresarial do município e respectivas necessidades de formação. Para esta etapa do diagnóstico e, atendendo ao vasto número de atores implicados neste processo (empresários), com a colaboração do Gabinete de Apoio Empresarial do Município, foi estabilizada a participação de vinte e quatro elementos, entre representantes do tecido empresarial e entidades promotoras de formação do município. Tendo em consideração o elevado número de elementos, optou-se por formar dois grupos de doze participantes, 3 RESULTADOS Organização da informação recolhida – Temas, unidades de registo e principais indicadore DOSSIÊ AMAZÔNIA 17 Fonte: Elaborada pelos autores do Ensino Profissional profissional e áreas técnicas (...) é preciso atuar, trabalhar para o reconhecimento social do ensino profissional e de algumas profissões”. Formação prática no Ensino Profissional 5 “Falta prática nestes cursos, é um facto (...) é essencial aumentar as atividades dentro das empresas”. Estágios-Emprego (IEFP) 5 “O processo para financiamento de um estágio profissional é extremamente lento (...) é urgente criar estratégias que permitam agilizar todo este processo”. Competências transversais 3 “Sentimos grandes lacunas nas competências pessoais e sociais dos jovens (...) ”. Colaboração Empresas – Escolas Profissionais 6 “A nossa empresa está completamente disponível para colaborar com as escolas profissionais no sentido da criação de ações de formação voltadas para estas áreas” “ (...) e portanto, reconhecemos a necessidade de ajustar a formação às necessidades das empresas. Temos trabalhado neste sentido com a ajuda das empresas ao nível da adaptação dos planos curriculares (...) e têm surgido novas áreas de formação”. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 18 próprias entidades de formação a mencionar que têm procedido à adaptação dos planos curriculares dos cursos em conjunto com as empresas, no sentido de aproximar os percursos formativos, o mais possível, às necessidades do mercado de trabalho. Ainda de salientar, por parte das entidades formadoras, o reconhecimento de que os cursos profissionais não são consentâneos com as reais necessidades das empresas, destacando-se o ensino tendencialmente teórico e, por outro lado, o esforço de algumas entidades no que se refere à implementação de novas áreas de formação, ajustadas às necessidades das empresas (e.g. mecatrónica automóvel, turismo, etc). Outra das temáticas a merecer destaque neste trabalho prendeu-se com a intervenção de um representante dos serviços públicos de emprego e formação, a qual incidiu sobre os Jovens NEET (Not in Employment Education or Training) - jovens até aos 29 anos que não estão integrados em qualquer modalidade de ensino, formação ou mercado de trabalho -, procurando divulgar a iniciativa Garantia Jovem. Esta iniciativa, da responsabilidade do IEFP, vem reforçar a importância de uma metodologia de intervenção assente num trabalho de (re)construção de redes a nível local, integrando as entidades e os atores melhor posicionados e capacitados, para sinalizar e captar os jovens que se encontrem em situação de maior fragilidade. Neste contexto, a lógica deste trabalho em rede direciona-se para a conciliação de respostas ao nível da qualificação, da inserção e das transições. De notar que, de acordo com dados de 2017, dos serviços públicos de emprego, o ensino básico é o nível mais representativo na estrutura de habilitações da população desempregada em Sintra (52,7%), enquanto dados do ano anterior (2016) indicam que a taxa de desemprego dos jovens (inscritos nos serviços de emprego) atingia 1001 jovens (7.5%) com idade inferior a 25 anos e 2382 jovens (17.8%) na faixa etária dos 25-34 anos. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 O trabalho de diagnóstico por nós realizado no âmbito do projecto educativo local revelou claras preocupações, associadas a uma grande percentagem (18,2%) de população residente com idades entre os 18 e os 24 anos que completou o 3º Ciclo do Ensino Básico (9º ano de escolaridade), mas que não se encontra a frequentar o sistema de ensino, assim como a uma taxa de retenção e desistência do ensino básico superior à média da região de Lisboa (AML) e do país e a uma taxa de transição/conclusão no ensino secundário inferior às unidades desejáveis de referência. Por tudo isto se justifica a especial atenção que devem merecer os jovens afastados do sistema formal de educação, formação e emprego, Jovens Nem-Nem, principalmente, aqueles que estão ocultos nas estatísticas oficiais, dificultando a sua sinalização e aumentando o risco de marginalização e exclusão social. Todos estes indicadores reforçam as novas exigências colocadas ao município e aos diferentes parceiros que nele atuam, no que se refere ao aumento das qualificações de todos os jovens e ativos empregados e desempregados, tendo sempre em linha de conta o equilíbrio entre as necessidades das pessoas/famílias e as das dinâmicas socioeconómicas, numa lógica de trabalho digno. Fonte: Elaborada pelos autores Fonte: Elaborada pelos autores De acordo com alguns participantes, nos dias que correm, ainda se assiste a uma considerável falta de reconhecimento do Ensino Profissional e áreas técnicas, pelo que urge criar um conjunto de estratégias que potenciem a mudança de mentalidades e, consequentemente, o reconhecimento social do Ensino Profissional e de algumas profissões que têm sido desvalorizadas. Esta atuação é, assim, percecionada como profícua para uma completa resposta às necessidades do tecido empresarial. Outro dos tópicos mencionados pelos empresários diz respeito à falta de trabalho prático associada aos cursos, não só profissionais, mas também universitários, implicando (apesar da formação on job que as empresas realizam) um retorno tardio da atividade dos trabalhadores. Esta situação acaba por desencadear um desajustamento entre as competências dos trabalhadores e as necessidades das empresas daí que, no âmbito dos percursos formativos, tenha sido reforçada a importância de um equilíbrio entre a formação teórica e prática, fomentando um aumento das atividades em contexto empresarial. Os estágios-emprego financiados pelos serviços públicos de emprego e formação profissional constituíram, igualmente, um dos temas abordados pelos representantes das empresas, tendo as intervenções incidido na morosidade dos processos. De facto, foi manifestada grande abertura à integração de estagiários do ensino profissional (curriculares e/ou profissionais), porém fica também patente a necessidade de criar uma estratégia que agilize este processo (e.g. após um estágio curricular – 3 meses, criar uma rápida resposta para a integração dos jovens num estágio-emprego – 9 meses). p g ç j g p g Relativamente ao domínio das competências, à semelhança do que tem vindo a ser comprovado através de vários estudos, assiste-se a uma valorização das competências transversais (autonomia, responsabilidade, iniciativa, trabalho em equipa, línguas estrangeiras) em detrimento das competências técnicas, evidenciando-se mais uma vez um desfasamento entre as competências pessoais dos jovens e a realidade do mundo empresarial. A oportunidade de reunir no mesmo espaço representantes quer de entidades locais de âmbito empresarial, quer de entidades com intervenção ao nível da qualificação da população, desencadeou uma importante dimensão: a cooperação entre as mesmas. Se por um lado assistimos à total disponibilidade das empresas para colaborar com as Escolas Profissionais no que concerne à criação de cursos voltados para as suas necessidades, por outro lado, surge a constatação de que esta complementaridade tem vindo a ocorrer, uma vez que são as económicas predominantes. Indubitavelmente, a formação profissional de jovens, associada ao mundo do trabalho, tem de adaptar-se à evolução do mesmo, designadamente no que respeita aos objetivos e conteúdos. Tal como ficou demonstrado, as contribuições dos representantes das empresas do município em análise, apontam para a vontade de verem o ensino profissional encetar o desejável percurso, no sentido da adaptação dos percursos formativos às suas reais necessidades, não só ao nível das áreas de formação, mas também ao nível das competências técnicas e das competências pessoais e sociais (transversais). Sendo bem conhecido o que as empresas pretendem traduzir com este discurso, esta constatação aponta para a vontade de verem emergir processos formativos enquadrados com necessidades de recursos humanos adaptáveis às culturas empresariais e capazes de evoluírem na lógica das suas estratégias futuras. Todavia, embora seja importante levar em consideração as exigências formativas das empresas, é imprescindível que os sistemas de formação potenciem um leque de competências e atitudes que possibilitem aos jovens integrar-se na vida ativa e favoreçam, no futuro, uma navegação profissional sustentada em projetos de vida informados, desafiantes e realistas, capazes de lidar com resiliência e criatividade com as constantes mudanças tecnológicas, sociais e culturais, conjugando dinamicamente, de acordo com a realidade e as possibilidades de mudança, saberes técnicos relacionais e emancipatórios. Numa nova conjuntura, em que as mudanças aceleradas se constituem como regra, é essencial criar condições para ancorar a preparação dos trabalhadores no futuro e criar estratégias diferenciadas, encarando o profissional como um agente no processo de tomada de decisão e de construção de novas realidades, tendo sempre como premissa a necessidade de um contínuo envolvimento em práticas de educação e formação que se devem prologar e alargar longo e a todos os espaços da vida. Partindo das declarações dos participantes no grupo focal, foi notória a preocupação quanto à falta de reconhecimento do ensino profissional e de profissões associadas a áreas mais técnicas. Evidentemente, estamos perante uma dimensão sobre a qual deverão ser tomadas medidas e ser definidas sólidas estratégias que promovam uma nova visão, visando a valorização e o prestígio da formação profissional. A elevada burocracia subjacente aos processos dos serviços públicos, ao representar para as empresas locais um dos entraves à dinâmica do recrutamento e seleção de estagiários, facilitando os seus processos de socialização profissional, deverá ser também objeto de reflexão, passando pela criação estratégias que melhorem a comunicação e agilizem todos os procedimentos. 4 CONSIDERAÇÕES FINAIS Apesar do significativo esforço de qualificação da população, em todos os níveis, feito por Portugal, ao longo das últimas décadas, no sentido de recuperar o atraso que nos distancia de países mais desenvolvidos, a verdade é que a realidade ainda se distancia da situação de grande parte dos países da União Europeia, persistindo baixos níveis de qualificação. Tal facto, associado a elevadas taxas de abandono e saída precoce do sistema de ensino, acaba por dificultar substancialmente o acesso à formação e à aquisição e aplicação de novos conhecimentos, impedindo a criação de uma base sólida de competências e, consequentemente, desencadeando grandes dificuldades ao nível do envolvimento da população ativa em contextos de mobilidade profissional e reorientação das orientações DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 19 económicas predominantes. I d bi l f fi i l d j i d d d b lh económicas predominantes. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 20 melhorar a informação, as práticas de orientação, os recursos necessários ao incremento da qualidade da formação e dos estágios e aumentar as possibilidades e oportunidades de emprego, de acordo com as expectativas e os projetos das pessoas. Revelou-se, igualmente, perentória a aposta num aumento significativo das atividades educativas e de formação para adultos, nomeadamente as unidades de formação de curta duração, aproximando-as a curto prazo do objetivo desejável de envolver, todos os anos, uma maior percentagem de pessoas adultas, com particular atenção aquelas que apresentam menores níveis de escolaridade e de qualificação e se situam em grupos etários mais elevados. Afigurou-se, ainda, determinante potenciar o envolvimento de mais trabalhadores em atividades de Educação e/ou Formação de Adultos, visando a elevação dos níveis de escolaridade e de qualificação da população empregada. p g Entendeu-se, assim, que a capacidade de resposta aos desafios futuros deverá passar por uma adequação da oferta formativa e por uma atuação prospetiva, empenhada e responsável dos atores envolvidos na estrutura das ofertas formativas, promovendo, ao mesmo tempo, novas abordagens junto dos destinatários, incentivando-os a procurar as melhores opções de formação e informando-os, claramente, sobre as condições de empregabilidade. p g De igual modo, fez parte das linhas de ação do projecto educativo local a promoção de uma maior aproximação entre as instituições de ensino e o tecido empresarial do território, solicitando junto destas a colaboração para a definição dos perfis e conteúdos formativos, bem como para a elaboração de programas e metodologias de formação. Procurou-se caminhar no sentido da criação de uma rede colaborativa potenciadora da construção de um território verdadeiramente comprometido com a qualidade de vida dos cidadãos e, consequentemente, com o sucesso dos seus percursos. Para isso, como também se percebeu, é incontornável que as políticas locais potenciem experiências educativas diferenciadas e espaços de interacção e debate, destinados a promover reflexões e acções problematizadoras, a partir e sobre o trabalho. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Face ao indubitável aumento da precariedade do emprego jovem, o projecto educativo local procurou, também, centrar a sua missão na criação de oportunidades para que os jovens que não estão a trabalhar, nem inseridos no sistema educativo/formativo e que abandonaram precocemente o sistema de ensino, concluam um percurso qualificante, por via do ensino profissional ou superior, implementando estratégias que promovam transições bem-sucedidas entre o sistema educativo/formativo e o mercado de trabalho. A construção do Projeto Educativo Local de Sintra revelou-se uma notável oportunidade de criação de políticas locais de educação e formação, associando-as a determinadas opções de desenvolvimento, e consequentemente, aos recursos e dinâmicas existentes ou passíveis de ser mobilizadas e desenvolvidas no município. Neste contexto, o planeamento concertado – agentes educativos, empresários, outros parceiros – de uma oferta diversificada e adaptada às necessidades do município em termos de ensino profissional e tecnológico, a implementação de um sistema de orientação vocacional que preveja e evite situações de insucesso e de abandono do percurso escolar e formativo por parte da população jovem, afigurou-se determinante, face à nova visão para a educação do território. económicas predominantes. Tendo em consideração todas as dimensões abordadas e analisadas pelas empresas e entidades de formação, não ficámos indiferentes à disponibilidade demonstrada para uma efetiva cooperação entre elas, traduzida pela complementaridade e adaptação dos percursos formativos, nas variadas vertentes, às reais necessidades das dinâmicas do trabalho naquele território. Neste sentido, importa reforçar a ideia e a necessidade de trazer novas lógicas de partilha de responsabilidades na articulação e otimização de meios e recursos, essenciais à concretização de práticas concertadas, alcançando o grande desígnio de um trabalho em rede local para a qualificação capaz de promover mudanças ao nível dos processos produtivos e do incremento contínuo de emprego de qualidade. Paralelamente a outras estratégias de envolvimento e auscultação dos parceiros locais, todos os contributos resultantes deste momento reflexivo permitiram consolidar alguns princípios orientadores para a definição de um eixo estratégico com objetivos e ações consentâneas com as transformações ambicionadas no âmbito da qualificação da população. 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Universidade Católica Dom Bosco, Campo Grande, 2011. DOSSIÊ AMAZÔNIA 23 DOSSIÊ AMAZÔNIA REVISTA DO PROGRAMA DE PÓS NÚMERO 01 – M ARÇO GRADUAÇÃO EM LINGUAGENS E UNIVERSIDADE FEDERAL DO PARÁ – INTRODUÇÃO O presente trabalho teve como foco de estudo o silenciamento e negação dos direitos à Educação de Jovens e Adultos – EJA, em especial em tempos da Pandemia do Covid-19. O interesse por esta temática é resultado de inquietações que permeiam enquanto pesquisadoras que têm como campo de estudo a modalidade da EJA. Historicamente o movimento em defesa ao direito de educação aos jovens e adultos que foram excluídos da educação escolarizada, tem se contrapondo às políticas públicas e é com muita frequência ainda visto como educação compensatória, de forma pontual e reproduções de modelos escolares para as crianças. A educação no contexto da pandemia global do novo coronavírus (COVID-19) marcado por incertezas, teve um grande impacto e provocou muitas reflexões, mudanças rápidas e exigiu repensar novas formas de ensinar e aprender a partir da suspensão das aulas presenciais e do isolamento social. Tal cenário, segundo os dados da Organização das Nações Unidas para a Educação, a Ciência e a Cultura - UNESCO no mês de abril de 2020, foram suspensas às aulas em 194 países, atingindo cerca de 1,6 bilhão de crianças e jovens no mundo inteiro (UNESCO, 2020). O reflexo da educação no mundo, agravou ainda mais a realidade da educação brasileira que além de desmascarar a situação precária ficou mais sucateado o ensino das escolas públicas, principalmente, em determinadas regiões e populações como a Amazônica. No contexto amazônico e diante da situação atual pandêmica, a EJA enfrenta grandes desafios por se tratar de sujeitos com processos de exclusões históricas e com diversidades muito específicas. Os diálogos sobre os movimentos em defesa da necessidade de criar e de dar continuidade às políticas públicas para a EJA que tenham por princípios, o respeito às diversidades, às realidades e às especificidades dos alunos, assegurando não apenas o acesso, mas também, a permanência dos mesmos no âmbito escolar, assim como a compreensão da EJA como princípio fundamental do ser humano, trouxeram também novas reflexões entre o que diz a legislação e o que de fato conseguem implementar e materializar, o que nos levou a questionar: que estratégias ou políticas públicas são construídas para a garantia da EJA como direito humano em tempos de pandemia? Essas estratégias conseguem materializar um projeto de EJA que potencializa a cidadania dos jovens e adultos que a acessam? CAMPUS BRAGANÇA IX – NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 24 Data de submissão: 30.09.2020 Data de aprovação: 06.12.2020 Data de submissão: 30.09.2020 Data de aprovação: 06.12.2020 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 25 documentos nacionais (Constituição de 88, LDB 96, Diretrizes Curriculares da EJA, Documentos orientadores dos Fóruns de EJA); c) Os documentos locais (Plano Municipal de Educação, Diretrizes Operacionais da EJA, Conselho Municipal de Educação); as estatísticas da EJA no município e nas plataformas de informação IBGE, DEEPASK, INEP. A escolha do Município de Bragança, na Amazônia bragantina, configura-se em virtude do município ter 407 anos de existência, com uma rica diversidade ambiental e sociocultural, contexto de diferentes formas de produção de existências: agricultores, pescadores, coletores, ribeirinhos e quilombolas. Entretanto, 49,8% da população bragantina possui uma renda média de até meio salário mínimo e ocupa a posição 1.938 na escala de renda entre 5.570 municípios brasileiros. Aliada ainda, ao fato de apresentar um quadro de 11.690 pessoas acima de 15 anos analfabetas, ou seja, 15,33% do total da população do município, necessitam que seus direitos à escolarização sejam garantidos pelo poder público municipal (DEEPASK, 2018). Os dados coletados foram organizados num movimento circulatório onde se cruzaram as informações das diferentes fontes documentais, visando qualificar o corpus de análise (SAMPIERI, CALLADO E LUCIO, 2013). Os resultados desse movimento de cruzar informações e interpretar os sentidos e significados, encontram-se organizados nos três tópicos desse trabalho: o primeiro discorre sobre as políticas educacionais brasileiras e a constituição da Educação de Jovens e Adultos como modalidade para assegurar o direito dessa população a educação; o segundo apresentamos a EJA na Amazônia bragantina, suas políticas, concepções e as implicações no campo do direito à educação em tempos de pandemia do Covid-19; e o terceiro traz reflexões sobre os possíveis caminhos e estratégias de inclusão da EJA ao universo educacional em meio à pandemia ainda em curso. INTRODUÇÃO Tais questionamentos nos impulsionaram à organização da pesquisa; assim traçamos como objetivo geral: analisar os direitos e as políticas para Educação de Jovens e Adultos em um município da Amazônia bragantina, frente ao contexto da pandemia de Covid-19; E para tanto se delineou: a) Estabelecer a relação teórica entre as políticas da educação de jovens e adultos como campo de disputas e lutas de garantia do direito; b) Analisar as formas de organização educacional da EJA em um município da Amazônia bragantina; c) Analisar os possíveis impactos no processo de escolarização de Jovens e Adultos na Amazônia bragantina em tempos de pandemia de Covid-19; d) Propor estratégias que visam a garantia desse direito em contexto de pandemia de Covid-19; Com o objetivo de interpretar o fenômeno em questão, ou seja, a garantia do direito à Educação de Jovens e Adultos em tempos de pandemia, apoiamos nossos estudos na abordagem qualitativa (LÜDKE E ANDRÉ, 2013). Para tal, usamos a pesquisa documental. A pesquisa documental nos permitiu investigar de forma indireta, por meio do estudo dos documentos produzidos a respeito da EJA, dentre os quais destacamos: a) Os documentos internacionais (Declaração dos direitos humanos e as Cartas das CONFINTEAS); b) Os NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 26 mais variadas: alfabetização de adultos, pós-alfabetização, educação rural, educação familiar, educação da mulher, educação em saúde e nutrição, educação cooperativa, educação vocacional, educação técnica, ou ainda, temáticas correlacionada ao desenvolvimento ecológico sustentável, da democracia, da justiça, da igualdade entre os sexos, do desenvolvimento socioeconômico e científico (NEVES, SILVA JÚNIOR E GONÇALVES, 2019). No Brasil em diferentes momentos históricos, projetos de educação jovens e adultos pautados: a) nos setores populares, incentivam a organização e autonomia da classe trabalhadora, engajando-os num projeto de transformação social; b) na lógica da educação escolar supletiva; ou ainda c) na lógica mercadológica, fomentaram/fomentam tensões e disputas que delineiam as políticas públicas escolar. A Constituição Federal de 1988, inaugura no cenário educacional Brasileiro o debate sobre a garantia obrigatória e gratuita da oferta de educação para todos que não tiveram acesso em idade própria, conforme artigo 208, inciso I, em que afirma, O dever do estado com a educação será efetivado mediante a garantia de: I – educação básica obrigatória e gratuita dos quatro aos dezessete anos de idade, assegurada inclusive sua oferta gratuita para todos os que a ela não tiveram acesso na idade própria (BRASIL, 1988). Ressalta-se que o reconhecimento desse direito permite, por sua vez, que a escolarização de jovens e adultos deixe de ser pensada como ensino suplência, voltada para instrução e, provoca mudanças que vão como descreve Soares (2002), muito além do que atualização vocabular que levam a reafirmação da EJA como uma modalidade de educação básica. Contraditoriamente, autores como Neves, Silva Júnior e Gonçalves (2019) destacam que apesar da Lei de Diretrizes e Bases da Educação – LDB, n° 9.394/96 trazer a concepção EJA como modalidade da educação básica, isso não significou a superação por completo da EJA como supletivo. Entretanto, como analisam Di Pierro e Haddad (2015), a mobilização da sociedade civil no Brasil foi propulsora da conquista de direitos, como o reconhecimento dos jovens e adultos dentre os sujeitos do direito humano à educação, antes restrito às crianças e adolescentes. 1 EDUCAÇÃO DE JOVENS E ADULTOS COMO CAMPO DE DISPUTAS POR DIREITOS A Educação de Jovens e Adultos, como modalidade de ensino no Brasil, tem se configurado em um campo de disputas acerca do direito à educação de jovens e adultos que historicamente sofreram e sofrem diferentes formas de exclusão social. A garantia do direito à educação, não se trata apenas do direito à escola, trata-se do direito à própria vida, na medida em que o acesso a ele potencializa afirmação da dignidade humana, [...] o direito à educação enquanto direito humano fundamental assume papel relevante para a afirmação da dignidade humana, pois sem a possibilidade de crescimento intelectual, de desenvolvimento de suas aptidões cognitivas, o ser humano não poderá desfrutar de outros direitos, uma vez que será excluído da sociedade letrada, tratado à margem desse contexto, não sendo visto como igual em direitos e nem tratado com dignidade. Daí o caráter de Direito Humano Fundamental atribuído à educação, tão precioso e necessário quanto a própria vida, pois tal direito é a alavanca para a realização de tantos outros. Sendo assim, a educação deve ser compreendida como um bem fundamental da humanidade. (CARVALHO, BARBOSA, RODRIGUES, TEIXEIRA, 2010, p.3). Neste sentido, versar sobre a Educação de Jovens e Adultos como um direito humano fundamental, implica compreender as políticas construídas em prol desse princípio de que estão marcadas por conflitos e contradições em torno das concepções da educação. Ao longo de todo o século passado em nível internacional a Conferência Internacional de Educação de Adultos-CONFINTEAS, apresenta esse movimento de construções que destacam inicialmente a necessidade da especificidade, da alfabetização para cidadania; da educação para superação da carência cultural, a defesa da educação ao longo da vida, o reconhecimento do direito de aprender, como também, apresentam articulações à temáticas DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 27 Ensino Médio que se desenvolvem em instituições próprias integrantes dos Sistemas de Ensino Federal, Estaduais, Municipais e do Distrito Federal (BRASIL, 2010). Ensino Médio que se desenvolvem em instituições próprias integrantes dos Sistemas de Ensino Federal, Estaduais, Municipais e do Distrito Federal (BRASIL, 2010). Neste contexto, os fóruns de EJA defendem políticas para esta modalidade de ensino que objetivem uma escolarização básica de qualidade para que todos os sujeitos da EJA tenham uma boa qualidade de vida, o que para Machado (2009, p. 34) implica em “processo de convencimento das pessoas não escolarizadas de que faz sentido a luta pelo acesso ao conhecimento”. Entretanto, ressalta-se que nas contradições existentes em projetos educacionais que se encontram mergulhados no neoliberalismo e na busca em torno do direito à educação, os esforços por colocar a EJA na agenda dos governos não resultam em avanços significativos. Como analisa de Di Pierro (2010, p. 952), No plano Nacional da Educação o tópico dedicado à EJA no Eixo VI, da CONAE, intitulado "Justiça Social, Educação e Trabalho: Inclusão, Diversidade e Igualdade", foi ampliado em mais de dez páginas, nas quais se distribui - sem qualquer hierarquia - um extenso rol de proposições de diferentes ordens, relativas às concepções pedagógicas, ao planejamento e às estratégias de políticas públicas, à ampliação da oferta e do financiamento, melhoria da infraestrutura, da formação inicial e continuada dos educadores e das condições de trabalho docente, consideração da diversidade dos educandos, fiscalização do setor privado, produção de estatísticas e pesquisas, supervisão e normas, organização escolar, currículos, materiais e meios de ensino. A Conferência também incluiu todo um tópico sobre a educação básica e profissional das pessoas privadas de liberdade. Fica a impressão de que, para contrapor-se à redação enxuta e compensar o último lugar atribuído à EJA do Documento de Referência, a plenária do Eixo VI optou por acolher toda sorte de propostas, sem estabelecer entre elas uma escala de prioridades, o que dificulta sua tradução em um número limitado de diretrizes e metas no novo PNE. A própria Base Nacional Curricular Comum – BNCC aprovada em fevereiro de 2019, aliada a modelos de ranking educacionais por meio de avaliações externas, silencia a respeito do direito à especificidade da modalidade da EJA e, em sua organização, não considera as singularidades dos seus sujeitos. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 O que terminou por potencializar inúmeros marcos legais dentre os quais: o Parecer CNE/CEB n°11/2000 que institui as Diretrizes Curriculares Nacionais para a Educação de Jovens e Adultos e a Resolução nº 3/ 2010 que Institui Diretrizes Operacionais para a Educação de Jovens e Adultos. O Parecer CNE/CEB n°11/2000, que define a Educação de Jovens e Adultos como, Uma dívida social não reparada para com os que não tiveram acesso e nem domínio da escrita e leitura como bens sociais, na escola ou fora dela, e tenham sido a força de trabalho empregada na constituição de riquezas e na elevação de obras públicas. Ser privado deste acesso é, de fato, a perda de um instrumento imprescindível para uma presença significativa na convivência social contemporânea (BRASIL, 2000, p. 5). Neste documento a EJA reassume as funções reparadoras, equalizadoras e qualificadoras e, como destaca o próprio documento, “representa uma promessa de efetivar um caminho de desenvolvimento de todas as pessoas, de todas as idades” (BRASIL, 2000, p.10). Já a Resolução nº 3 /2010 Institui Diretrizes Operacionais para a Educação de Jovens e Adultos nos aspectos relativos à duração dos cursos e idade mínima para ingresso nos cursos de EJA; idade mínima e certificação nos exames de EJA; e Educação de Jovens e Adultos desenvolvida por meio da Educação a Distância, a serem obrigatoriamente observadas pelos sistemas de ensino, na oferta e na estrutura dos cursos e exames de Ensino Fundamental e 2 A EDUCAÇÃO DE JOVENS E ADULTOS EM TEMPOS DE PANDEMIA DE COVID-19 NA AMAZÔNIA BRAGANTINA De modo espantoso e evidenciando a fragilidade humana para os chamados “seres invisíveis”, o mundo inteiro foi tomado por um vírus, SARS-CoV-2 (do inglês Severe Acute Respiratory Syndrome Coronavirus 2) ou síndrome respiratória aguda grave do coronavírus 2, que iniciou em Dezembro de 2019 na China, na cidade de Wuhan, causando uma suposta pneumonia viral e rapidamente ganhou proporções gigantescas alcançando à todos e impondo novos modos de vida. A Organização Mundial da Saúde (OMS) passou a chamar oficialmente a doença causada pelo novo coronavírus de Covid-19. COVID significa COrona VIrus Disease (Doença do Coronavírus), enquanto “19” se refere ao ano de 2019 (FIOCRUZ, 2020). O planeta parou para que o vírus parasse, mas milhares de pessoas no mundo não puderam parar e ficar em casa, porque milhares não tem casa e outros milhares não tiveram escolha: ou saiam de casa para trabalhar ou perdiam seus empregos evidenciando a desigualdade social existente no mundo e no Brasil, de modo geral. No Brasil o vírus continua se alastrando e alcançou, a título de exemplo, no dia 24 de setembro de 2020, o número de 4.657.702 milhões de casos confirmados e 139.808 mil óbitos, com 831 óbitos nesse dia (BRASIL, 2020a). Quando esse artigo for lido por você certamente os números serão maiores. Os impactos da pandemia são enormes, tanto a nível social, como econômico, político, cultural, histórico e educacional sem precedentes na história recente das epidemias. Na educação, a crise vivenciada pela Covid-19 resultou no encerramento das aulas em escolas e em universidades, afetando mais de 90% dos estudantes do mundo (UNESCO, 2020). Segundo a Unesco, se não forem criadas políticas públicas que invistam em melhorias na infraestrutura, tecnologias, formação, metodologias e salários, além do reforço da merenda, melhor aproveitamento do tempo, tutoria fora do horário usual das aulas e material adicional, os impactos serão sentidos por mais de uma década (UNESCO, 2020). O Ministério da Educação (MEC) publicou a portaria nº 343 de 17 de março de 2020, que orienta as Instituições de Ensino a substituírem as aulas presenciais por aulas em meios digitais pelo prazo de 30 dias ou pelo tempo que durar a pandemia (BRASIL, 2020b). DOSSIÊ AMAZÔNIA Pautada em discursos que desqualificam o serviço público, a BNCC fortalece iniciativas privadas que apresentam como solução para qualidade do ensino, pacotes pedagógicos prontos em detrimento de práticas curriculares da EJA que deveriam considerar “conhecimentos e capacidades que os fortaleçam como coletivo, que os tornem menos vulneráveis, nas relações de poder” (ARROYO, 2007, p.10). Assim, embora a Educação de Jovens e Adultos seja defendida como uma estratégia para escolarizar, como nos diz Arroyo (2007, p.7), “(…) sujeitos concretos, em contextos concretos, com histórias concretas, com configurações concretas”, contraditoriamente vivenciamos nos últimos anos o aprofundamento das desigualdades sociais, cenário em que pessoas que ficaram à margem da sociedade, que não puderam ou foram impedidos de concluir seus estudos, nos fazendo inferir que políticas generalistas não atendem às necessidades educacionais e a diversidade dos sujeitos da EJA. Na esteira das disputas em que a educação de jovens e adultos é defendida como estratégia para escolarizar e ao mesmo tempo em que vivemos o fortalecimento de uma política neoliberal que tem agravado as desigualdades sociais pelas lógica generalista, o mundo é surpreendido pela pandemia Covid-19, as contradições entre o desafio de promoção do direito à educação, a defesa de emprego, da saúde e adoção de alternativas que reproduzem e ampliam as desigualdades sociais demonstrando as impossibilidades dessa saídas imediatista para educação de jovens e adultos na e para Amazônia bragantina. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 28 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 29 Corroboramos com Di Pierro e Ximenes (2011) ao afirmarem que não faz sentido as turmas estarem em redução no Brasil se os números de brasileiros com baixa escolaridade se mantém elevado, conforme segue, Como compreender que a oferta escolar esteja em declínio, se existe uma demanda potencial de mais de 60 milhões de brasileiros com idade igual ou superior a 14 anos que são analfabetos absolutos, pessoas com escolaridade reduzida ou que não concluíram o ensino fundamental? É possível imaginar que não exista demanda social por EJA se o mercado de trabalho requer níveis cada vez mais altos de escolaridade e qualificações profissionais? Por que os jovens e adultos não buscariam oportunidades de estudos se a participação na vida social, cultural e política é cada vez mais mediada por informação, conhecimento e tecnologia? (DI PIERRO E XIMENES, 2011, p.6). A explicação para essa questão da redução na oferta de turma da EJA se pauta na escassez de recursos para essa modalidade de ensino e no pouco estímulo para continuidade da escolaridade em meio à rotina do jovem e adulto trabalhador que se sente exaurido pelo mundo do trabalho, sem ter condições de priorizar sua escolarização. Soma-se a isso o fato de que pela ausência de políticas públicas para incentivá-lo a estudar, por meio de auxílios ou bolsas de estudo, o aluno da EJA fica sem escolha ao fazer a opção entre trabalho e estudo. Essa redução de oferta de turmas de EJA na Amazônia bragantina ocasionou, desde o ano de 2018, em uma nova forma de oferta: a nucleação, ou seja, só seriam ofertadas turmas em “escolas núcleo” da cidade, não mais em todas as escolas como antes. O processo de nucleação se configura como uma oferta de turmas da EJA, cuja distribuição é realizada a partir de processos avaliativos como forma de controle de resultados, de modo que, pouca ou nenhuma evasão passa a ser o critério para manutenção da oferta ou fechamento de turmas em escolas do campo ou cidade, transformando algumas escolas como referência para a oferta da EJA, em bairros da área urbana ou em comunidades para os territórios do campo. Para Messina (1993), há tempos as pesquisas demonstram que a demanda por EJA é modelada pela natureza e características da oferta educativa. 2 A EDUCAÇÃO DE JOVENS E ADULTOS EM TEMPOS DE PANDEMIA DE COVID-19 NA AMAZÔNIA BRAGANTINA O parecer do Conselho Nacional de Educação - CNE do Ministério da Educação do Brasil reconheceu os problemas causados pela pandemia e procurou reorganizar as atividades acadêmicas com a permissão para aulas aos sábados – em horários de contraturno e durante as férias –, para que os alunos da Educação Básica não percam o ano letivo e apontou outras medidas semelhantes àquelas já defendidas pela Unesco (BRASIL, 2020c) Todavia, e diante de toda essa situação mundial pandêmica, os alunos da EJA ficaram esquecidos. São alunos trabalhadores que precisam conciliar trabalho, estudo, família, filhos, e tantas outras responsabilidades e em meio a toda essa pandemia lutam por suas vidas e pela manutenção de seus empregos e sua condição de alunos que lhes é negada. Assim como nas demais regiões do Brasil, na região Norte e na Amazônia bragantina também houve redução das turmas de EJA. No ano de 2005, em Bragança-PA, haviam 1.507 jovens e adultos matriculados, aumentando em 2012 para 2.642 matriculados e diminuindo, em 2017, para 1.388, ou seja, menos 1.254 matrículas, o que significa que em apenas cinco anos houve uma redução de 47,4% de alunos na EJA (NEVES, SILVA JÚNIOR E GONÇALVES, 2019). De acordo com os dados do Instituto Nacional de Pesquisas Educacionais Anísio Teixeira (INEP) houve uma queda de 7,7% no número de alunos da EJA no Brasil, tanto no ensino fundamental quanto no ensino médio (INEP, 2019), isso representa 1.051.919 de matrículas a menos que no ano de 2010, por exemplo. 3 REPENSANDO AS ESTRATÉGIAS DA EJA EM TEMPOS DE PANDEMIA: DESAFIOS E POSSIBILIDADES Diante desse contexto geral apontamos algumas provocações para repensar os caminhos da EJA no território da Amazônia bragantina em tempos da pandemia de Covid-19, no campo das estratégias e das possibilidades, no sentido de materializar uma política de EJA que potencialize a cidadania dos jovens e adultos neste território. Todavia, é de fundamental importância que tenhamos conhecimento de um documento elaborado, em julho de 2020, pelos Fóruns de EJA do Brasil que tratam de ações necessárias para a EJA em tempos da pandemia de Covid-19. O documento aponta três pautas de luta, a saber: 1. O primeiro ponto da pauta de luta diz respeito a disponibilizar banda larga, como direito social, na forma de serviço público gratuito; plataforma pública, com expansão da Rede Nacional de Pesquisa (RNP); integração com TVs públicas, rádios públicas e redes sociais; e infraestrutura de tecnologia virtual, em sala de aula, como instrumentos de inserção no chamado ciberespaço com produção de transvídeos e construção de tipos textuais no celular, demonstrando as funcionalidades e aplicações na vida de cada educando(a) (FÓRUNS EJA BRASIL, 2020, p. 2). 2. O segundo ponto da pauta de luta é que reafirmamos que a oferta da EJA precisa tornar parte da constituição de políticas públicas de Estado (em âmbito federal, estadual, municipal e distrital) e não por meio de programas. Defendemos o direito à educação de qualidade e nos posicionamos contrários à oferta de atendimento compulsório da EJA na modalidade EaD. Exigimos que, para esse atendimento, o poder público e suas instituições (nos âmbitos federal, estadual, municipal e distrital) realizem o recenseamento, a mobilização da sociedade por meio de chamada pública e divulgação de vagas da demanda e garanta a abertura e manutenção de turmas/escolas de EJA na educação básica (Fundamental e Médio) na forma integrada à Educação Profissional nos termos do artigo 5o da LDB 9394/96 e Lei 13.005/2014 do Plano Nacional de Educação (Metas 9 e 10) (FÓRUNS EJA BRASIL, 2020, p. 3). 3.O terceiro ponto da pauta de luta é a defesa de que o ano letivo não precisa coincidir com o ano civil, principalmente, em situações excepcionais como a que nos encontramos. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Se no Brasil os dados apontam que apenas 39% dos domicílios brasileiros possuem computador (CETIC.BR, 2019), imaginem ter computador com acesso à internet na Amazônia bragantina quando as desigualdades sociais são extremas e cerca de 50% das pessoas vivem apenas com uma renda média mensal de até meio salário mínimo (DEEPASK, 2018). Desse modo, a Educação a distância - EaD não pode ser a única solução, uma vez que esta metodologia tende a exacerbar as desigualdades já existentes, que são parcialmente niveladas nos ambientes escolares, simplesmente, porque nem todos possuem o equipamento necessário (SOUZA, FRANCO E COSTA, 2016). Registra-se ainda que há um grande movimento de luta nacional contra a oferta de aulas no modelo EaD para as turmas de EJA, aderir à essa concepção significa negar o direito à educação para todos com as mesmas condições de igualdade de acesso e permanência na escola. ç g p A seguir apresentaremos algumas estratégias que podem se apresentar como alternativas para lidar com a situação da EJA em tempos de Pandemia de Covid-19 na Amazônia bragantina. DOSSIÊ AMAZÔNIA Em São Paulo, por exemplo, desde 2011 já haviam estudos que apontavam a existência da política de nucleação na EJA, conforme afirmam Di Pierro e Ximenes (2011, p.10), “de fato, os dados e documentos reunidos evidenciam que, assumindo ou não as autoridades centrais, há uma política de nucleação e desestímulo em franca aplicação na rede estadual de ensino”. A lógica que permeia essa política é cruel e intencional, deseja o fim da educação pública e a não garantia de direitos aos jovens e adultos brasileiros, uma vez que, “diante do decréscimo de estudantes, reduz-se a oferta, o que, por sua vez, implica em crescente redução da demanda (DI PIERRO E XIMENES, 2011, p. 8). Freire (1997) já destacava que a prática educativa para a EJA deveria ser pautada na realidade, na diversidade do aluno e nos conhecimentos construídos historicamente, para que a construção do saber fosse consolidada de maneira crítica, ou seja, assumir a realidade concreta para transformá-la. Desse modo, em meio à toda essa precariedade na EJA na Amazônia bragantina as aulas são suspensas por conta da pandemia. A primeira proposta, e parece ser a única, que se considera em meio a essa situação vivida é a oferta de atividades de ensino por meio tecnológico, todavia a integração ao mundo tecnológico, midiático e informacional impõe-se como uma exigência quase universal, embora venha se realizando de forma desigual e até mesmo marginal, conforme as diferenças sociais, econômicas, políticas e culturais entre as regiões do planeta e do país, entre os grupos sociais e entre os indivíduos (SOARES E SANTOS, 2012). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 30 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 3 REPENSANDO AS ESTRATÉGIAS DA EJA EM TEMPOS DE PANDEMIA: DESAFIOS E POSSIBILIDADES É possível organizar os dias letivos e horas, de modo a garantir o ensino presencial para todos(as), assegurando- se que: não haja discriminação devido às condições de vida dos(as) trabalhadores(as) estudantes e à estrutura das instituições educacionais; a autonomia aos sistemas (municipais, estaduais, federais e distrital) para definir suas formas de recuperação, com GESTÃO DEMOCRÁTICA, assegurando a participação das comunidades escolares, para propor e deliberar sobre as alternativas; a reorganização dos calendários escolares possibilitarão a reposição das aulas e atividades de modo presencial, assim que seja superada a pandemia da COVID-19, e as condições sanitárias permitam a viabilidade do retorno às escolas, DOSSIÊ AMAZÔNIA 31 garantindo a igualdade de condições .para todos(as), ainda que para isso se reconheça a necessidade de que as atividades do ano letivo de 2020, sejam estendidas até 2021 (FÓRUNS EJA BRASIL, 2020, p. 4). Quanto ao primeiro ponto da pauta de luta, caso haja necessidade de opção pelo ensino remoto nas escolas, será vital que se garanta igualdade de condições para acesso e permanência às aulas aos alunos da EJA. A renda baixa dos alunos trabalhadores do munícipio de Bragança, na Amazônia bragantina, os impossibilita de fornecer a si próprios recursos tecnológicos e acesso à internet banda larga, por isso será necessário que a secretaria municipal de Educação, em parceria a outros órgãos governamentais, lhes dê as condições para tal. Em meio à pandemia sabemos que grande número de trabalhadores perderam seus empregos e, apesar do auxílio emergencial do governo federal que milhares de brasileiros receberam, ele se destina basicamente ao suprimento das necessidades básicas como alimentação e compras de remédios, além de que foi temporário e somente aos que não tinham nenhum vínculo empregatício. Todavia, registra-se que essa oferta por meio do ensino remoto deve ser uma exceção pela situação vivida e que a Amazônia bragantina também deve se posicionar contrariamente à oferta compulsória da Educação a Distância às turmas de EJA, uma vez que se luta nacionalmente por uma educação de qualidade socialmente referenciada para todos. No segundo ponto da pauta de luta exige-se políticas de EJA efetivas e não mais via programas governamentais, para garantir as condições necessárias de acesso, permanência e êxito à escolarização dos jovens, adultos e idosos, não somente neste contexto de pandemia, mas permanentemente. 3 REPENSANDO AS ESTRATÉGIAS DA EJA EM TEMPOS DE PANDEMIA: DESAFIOS E POSSIBILIDADES Nesse sentido, a escola precisa se tornar cada vez mais um lugar de acolhimento das necessidades e interesses do aluno(a) da EJA, contribuindo diretamente na construção das suas histórias de NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 32 vida. Na realidade da Amazônia Bragantina, como as aulas foram suspensas, houve como desdobramento o isolamento do contexto escolar que pode ser superado se a escola mantiver um vínculo comunicativo com os alunos, mesmo sem aulas. vida. Na realidade da Amazônia Bragantina, como as aulas foram suspensas, houve como desdobramento o isolamento do contexto escolar que pode ser superado se a escola mantiver um vínculo comunicativo com os alunos, mesmo sem aulas. O cenário atual que estamos vivendo é uma situação que vai mudar a sociedade, vai exigir da educação um novo design, currículos humanizados e mais interessantes, diálogos mais potentes, olhares mais afetuosos e sensíveis, discussão sobre a cultura escolar, que ainda impera, de expulsar o jovem da escola, e, fomentar a elaboração de outra cultura, do acolher e não excluir. A secretaria de educação do município de Bragança-PA precisa fortalecer os esforços de qualificação dos gestores, professores, coordenadores pedagógicos e técnicos administrativos, para além de motivar, acolher, aprender como desenvolver competências socioemocionais no contexto da escola, realizar escutas mais ativas e fazer uso de novas ferramentas. Para além dos pontos circunstanciados sobre a EJA em tempos de pandemia nos territórios da Amazônia bragantina, os impactos e traumas causados pela pandemia terão de ser considerados e explorados de um novo ponto de vista pedagógico. É um momento que as escolas possuem para repensar a sua função social na vida dos jovens e adultos, com expectativas, projetos e sonhos, como retrata muito bem no documentário “Nunca me Sonharam”, 2017. 3 REPENSANDO AS ESTRATÉGIAS DA EJA EM TEMPOS DE PANDEMIA: DESAFIOS E POSSIBILIDADES A EJA integrada à Educação Profissional e Tecnológica -EPT precisa se tornar política pública para garantir uma melhor qualificação aos alunos da educação básica, bem como a manutenção e a ampliação da oferta presencial nos três turnos como uma opção para os diferentes sujeitos que à acessam. No terceiro ponto de pauta assegura-se a flexibilização do calendário do ano letivo vigente e que este não precisa coincidir com o ano civil, priorizando o aprendizado do aluno, seu processo formativo em detrimento de avaliações, aligeiramento, redução do calendário, bem como por questões meramente burocráticas da educação. Esses três pontos pautam a compreensão da importância do diálogo coletivo para criar alternativas que tenham como foco os sujeitos da EJA e suas lutas pelo direito à educação e alternativas emancipatórias. Nesta perspectiva será necessário que se repense não apenas o conteúdo de ensino, mas o modo de ensinar e de aprender em seus tempos, sempre em diálogo com a diversidade dos alunos da EJA, aqui em especial destaca-se a Amazônia Bragantina. Além das questões ponderadas e proposições acerca do contexto atual pandêmico para a EJA já descritas, provocamos outras reflexões sobre pontos que merecem visibilidade neste momento, tais como: a escola deve oportunizar aos jovens e adultos o protagonismo nos diferentes contextos de vida, assim como o trabalho deveria investir e incentivar no mesmo sentido, mas percebemos que há entraves nesta relação. Todavia, se o trabalho faz o jovem abandonar a escola, ele posteriormente o motiva à sua volta, porque compreende que a elevação da escolaridade melhora a qualificação profissional. Faz-se necessário que a política de escolarização dialogue com os diferentes modos de trabalho e com os tipos de oferta para jovens e adultos no mundo do trabalho. A escola precisa estabelecer e fortalecer ainda mais os vínculos com os jovens, adultos e idosos e não deve travar as portas quando estes não voltarem ou perderem a motivação pelo estudo no decorrer deste período pandêmico e para além dele. Por conseguinte, faz-se necessário observar os alunos que estão vivenciando o luto, que tiveram perda de referências e afetividades em suas vidas, que tem sofrido com queixas de ansiedade, insônia, depressão e ainda precisam trabalhar para fazer a manutenção da vida. 4 CONSIDERAÇÕES FINAIS Este artigo se propôs analisar os direitos e as políticas para Educação de Jovens e Adultos em um município da Amazônia bragantina, frente ao contexto da pandemia de Covid- 19. Ao longo do texto tratamos questões referentes aos direitos, políticas educacionais, a EJA na Amazônia bragantina em tempos de pandemia e apresentamos proposições para a educação da EJA. Todavia, compreende-se que os desafios das políticas educacionais sobre a permanência e êxito das pessoas jovens, adultas e idosas nos contextos escolares em tempos de pandemia precisam ser ainda mais efetivos e cuidadosos no sentido de garantir esse direito que ao longo da história foi negado e compensado com muitas fragilidades. Tal contexto, ainda pode ser mais excludente entre estes jovens se não considerar a relação com trabalho, pobreza, gravidez precoce, violência, problemas emocionais e o sentido da escola para eles. A suspensão das atividades de ensino presenciais de forma regular, embora considerando parte dos estudantes tiveram acesso às atividades remotas, ocasionou uma ruptura na rotina de estudo de todos os estudantes brasileiros, entre eles, crianças, adolescentes, jovens, adultos e idosos de forma substancial, o que não representa e alcança a realidade educacional de onde estamos falando – Amazônia bragantina, que ainda não retornou as atividades de ensino presencial, assim como não conseguiram realizar atividades de ensino remotas a distância devido a dificuldade de acesso à internet e falta de recursos tecnológicos. Outro fator mais desafiante é alcançar os sujeitos da EJA em seus diferentes contextos de vidas, tais como: campo, ribeirinhos, agricultura familiar, territórios pesqueiros, coletores, dentre outros contextos sociais. Assim sendo, em tempos de pandemia não deveria ser um momento de ataque aos direitos humanos conquistados e garantidos, de mais cortes na Educação e de eliminação de direitos trabalhistas, assim como a negação da valorização da modalidade de ensino de jovens e adultos e de outros conceitos e ideias que encontram-se alinhavadas à falta de equidade, qualidade e à amortização desta, mas de repensar as políticas de escolarização que dialoguem com os modos de trabalhos, de escolas que realmente tenham sentidos nos projetos de vidas das pessoas jovens e adultas. 33 DOSSIÊ AMAZÔNIA REFERÊNCIAS ARROYO, Miguel. Balanço da EJA: o que mudou nos modos de vida dos jovens-adultos populares?. REVEJ@ - Revista de Educação de Jovens e adultos, v. 1, n. 0, p. 1-10, ago. 2007. BRASIL. 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NEVES, Joana d’Arc de Vasconcelos; SILVA JUNIOR, Sebastião Rodrigues da; GONÇALVES, Gabriela Oliveira. A Educação de Jovens e Adultos como direito fundamental: um olhar sobre o município de Bragança-PA. 3112 - Trabalho Completo - 2ª DOSSIÊ AMAZÔNIA 35 Reunião Científica Regional Norte da ANPED (2018) GT 18 - Educação de Pessoas Jovens e Adultos. Rio Branco: Acre, 2019. ISSN:2595-7945 SAMPIERI, Roberto Hernández.; COLLADO, Carlos Fernández; LUCIO, Pilar Baptista. Metodologia de pesquisa. (Série Métodos de Pesquisa) 5. ed. Porto Alegre: AMGH, 2013 SOARES, Leôncio José Gomes. Educação de jovens e adultos. Rio de Janeiro: DP&A, 2002. SOARES, Leôncio José Gomes. Educação de jovens e adultos. Rio de Janeiro: DP&A, 2002. SOUZA, Simone de.; FRANCO, Valdeni S.; COSTA, Maria Luisa F. Educação a distância na ótica discente. Educação e Pesquisa, São Paulo, v. 42, n. 1, p. 99-114, jan./mar. 2016 SOARES, Conceição; SANTOS, Edméa. 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DOSSIÊ AMAZÔNIA 37 UNIVERSIDADE FEDERAL DO PARÁ – CAMPUS BRAGANÇA VOLUME – REVISTA DO PROGRAMA DE PÓS GRADUAÇÃO EM LINGUAGENS E SABERES NA AMAZÔN IA M ARÇO NÚMERO 01 38 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Vivemos em tempos de ressignificações, de profundas mudanças, de contestações de toda ordem, de crise da ciência e da sociedade moderna. Época marcada pela transitoriedade, pela busca de novos sentidos e de novas práticas. Práticas que sejam mais próximas da contingência histórica da condição humana e que contribuam para a reconstrução do atual contexto (SILVA, 2003, p.7). Repensar e reconstruir tal contexto é imprescindível; rever velhas ações e práticas pedagógicas que não são coerentes com um cenário que se pretende ser mais humanizado. Portanto, não cabe mais à escola a função exclusiva de transmissão de conteúdo. As novas tecnologias oportunizaram o acesso às informações pelas mais variadas fontes. Assim, se faz necessário ressignificar a educação, especialmente a educação de jovens e adultos (EJA), para que haja coerência com essa nova realidade e, consequentemente, é imprescindível um novo perfil de professor e uma concepção de aprendizagem em consonância com esse projeto. Freire (2007, p.29), ressalta que “o saber se faz através de uma superação constante. O saber superado já é uma ignorância. Todo saber humano tem em si o testemunho do novo saber que já anuncia. Todo saber traz consigo sua própria superação.” Assim, o autor nos apresenta a relativização do saber, do conhecimento. Nesse contexto, a escola precisa reconhecer a transitoriedade das informações e o professor tem um novo papel, mais adequado a essa realidade: o de mediador da aprendizagem. Ele é aquele que cria o ambiente propício para o desenvolvimento dos alunos e para a colaboração mútua. Portanto, esse profissional precisa conhecer bem seus alunos para poder escolher metodologias adequadas ao seu público, buscando garantir a participação ativa desses, enquanto sujeitos de suas aprendizagens, bem com a construção de uma aprendizagem significativa. Uma aprendizagem que faça a diferença na vida dos alunos, que seja crítica e reflexiva, contribuindo para a construção e reconstrução permanente de cada um como cidadão e ser social. Assim, repensar a função da escola, do professor, da aprendizagem representam desafios importantes que estão postos, principalmente quando falamos de EJA. Essa modalidade de ensino que inclui uma parcela da população marcada pela exclusão de direitos e por desigualdades sociais. 1 CONHECIMENTOS PARA A VIDA E A EDUCAÇÃO DE JOVENS E ADULTOS Para aprender, o sujeito elabora hipóteses sobre a realidade, interage com o meio e com os outros, reconstruindo suas percepções e avançando na compreensão desta realidade. Fávero (2007, p. 45) afirma que “o conhecimento é adquirido através de um processo construtivo, no qual a aprendizagem dos sujeitos não é dada a priori e nem mesmo resulta do acúmulo de informações recebidas do meio externo.” Desta maneira, realiza-se um processo dialético de elaboração e reelaboração do conhecimento, no qual os conhecimentos prévios adquiridos ao longo da vida servem como ponto de partida para novas aprendizagens. Assim, aprendemos desde que nascemos e iremos aprender até o fim da vida. Nesse contexto, quando pensamos na EJA, as estratégias de seleção e organização dos conteúdos curriculares, bem como os critérios dessa escolha, devem considerar os conhecimentos, experiências, interesses e expectativas desses jovens e adultos, em consonância com a realidade vivenciada por eles. Souza (2011) acrescenta que: mais do que diagnosticar que saberes os jovens e adultos trazem [...], é preciso saber quem e quantos são, que necessidades de escolarização e de outras formações culturais demandam, que condições socioeconômicas e motivações de aprendizagem apresentam (SOUZA, 2011, p.191). Portanto, a adequação da linguagem e de propostas curriculares diferenciadas para a EJA também merecem destaque quando falamos em metodologia adequada a este público, pois ainda se observa em sala de aula a infantilização da EJA com o uso de expressões e atitudes usadas com crianças. Oliveira (2007), destaca que expressões e atitudes, como: resolver a folhinha, fazer a continha, realizar o dever de casa são totalmente inapropriadas para jovens e adultos, reafirmando uma visão compensatória da EJA e o sentimento de não pertencimento à escola. É preciso reconhecer que esse aluno, jovem ou adulto, retorna aos bancos escolares com uma bagagem já construída, com uma história de vida e conhecimentos acumulados que podem e devem ser valorizados pela escola. O aluno trabalhador é concebido com um ser social que traz experiências de vida e conhecimentos acumulados. Um sujeito fazedor de história que intervém na realidade e que se constrói nas ações coletivas. Um ser integral, cujas dimensões cognitivas, físicas, emocionais, econômicas, políticas, sociais, culturais, éticas e estéticas e espirituais interagem no processo de construção do conhecimento (FÁVERO, 2007, p. 45). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 39 ensino de ciências, em prol de uma educação de jovens e adultos coerente com as demandas atuais da sociedade. NÚMERO 01 Refletir sobre como esses jovens e adultos pensam e aprendem envolve, portanto, transitar pelo menos por três campos que contribuem para a definição de seu lugar social: a condição de “não-crianças”, a condição de excluídos da escola e a condição de membros de determinados grupos culturais (OLIVEIRA, 1999, p. 60). Nesse sentido, a EJA representa uma modalidade de ensino que deve problematizar as questões do cotidiano, indo além de uma simples compensação do tempo perdido e possuir uma proposta mais abrangente, de emancipação social, participativa, cidadã, de formação política para a construção de uma educação mais inclusiva, humanizada, seguindo os ideais de Paulo Freire. Nessa perspectiva, o Ensino de Ciências a ser implementado nessa modalidade deve priorizar questões atuais, que tenham relevância para o seu público, que problematizem o cotidiano e o modo de vida das pessoas, ou seja, pensar no Ensino de Ciências adequado à EJA, através de metodologias ativas de ensino que levem a aprendizagem significativa de seus sujeitos. Com esse propósito, o presente artigo foi elaborado, a partir da observação prática da realidade da sala de aula e de um estudo de vasto material bibliográfico. Trata-se de uma pesquisa bibliográfica, de cunho descritivo, que se propõe a apresentar aspectos relevantes para a prática pedagógica crítica e reflexiva na EJA, buscando alinhar estudos da teoria da aprendizagem significativa como alicerce para a implementação de metodologias ativas no 2 A APRENDIZAGEM SIGNIFICATIVA O conceito de aprendizagem significativa apresentado nesse trabalho tem como referencial o postulado de Ausubel (2003), ao elaborar a teoria da assimilação da aprendizagem e da retenção significativas, a partir dos mecanismos de explicação dos processos psicológicos de aprendizagem e de retenção cognitivas humanas. Assim, a aprendizagem significativa ocorre quando uma nova informação se relaciona com um aspecto relevante da estrutura de conhecimento do aprendiz, produzindo novos significados. Moreira (2012, p. 5) destaca que os “aspectos relevantes da estrutura cognitiva que servem de ancoradouro para a nova informação são chamados subsunçores”. Acrescenta que, à medida que o conhecimento prévio serve de base para a atribuição de significados à nova informação, ele também se modifica, ou seja, os subsunçores vão adquirindo novos significados se tornando mais diferenciados, mais estáveis. Para Ausubel, O conhecimento é significativo por definição. É o produto significativo de um processo psicológico cognitivo (saber) que envolve a interação entre ideias logicamente (culturalmente) significativas, ideias anteriores (ancoradas) relevantes da estrutura cognitiva particular do aprendiz (ou estrutura dos conhecimentos deste) e o mecanismo mental do mesmo para aprender de forma significativa ou para adquirir e reter conhecimentos (AUSUBEL, 2003, p. vi). O autor destaca algumas definições importantes para apreensão de sua teoria. Ele apresenta os tipos de aprendizagem por recepção significativa como sendo: aprendizagem representacional, aprendizagem conceitual e aprendizagem proposicional. A aprendizagem representacional é definida por ele como sendo aquela que se aproxima da aprendizagem por memorização, porém é significativa, pois ocorre sempre que o significado dos símbolos arbitrários se equipara aos referentes (objetos, acontecimentos, conceitos) e tem para o aprendiz o significado, seja ele qual for, que os referentes possuem. A aprendizagem conceitual é uma extensão da representacional e ocorre quando há formação e assimilação de conceitos, possuindo fases sucessivas de aperfeiçoamento. A aprendizagem proposicional é aquela que necessita do conhecimento prévio (de conceitos e símbolos) para atingir a compreensão sobre uma proposição. Moreira; Masini (2006) ratificam a teoria de Ausubel, na qual aquilo que o aluno já sabe é o fator isolado mais importante que influencia a aprendizagem. Assim, o conhecimento prévio é condicionante para a nova aprendizagem, ocorrendo uma interação entre o novo e o já existente, sendo ambos modificados. Dessa forma, Moreira (2012, p. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 40 sinônimo de redução nem simplificação. O aluno da EJA necessita de uma formação de qualidade na Educação Básica, integrada ao mundo do trabalho sempre que possível, respeitando suas especificidades, mas que o permita a prosseguir seus estudos na Educação Superior, se esse for o seu desejo. Portanto, a aprendizagem precisar ser significativa e os conhecimentos construídos devem servir para a melhoria da vida de seus sujeitos. Como aponta Alarcão (2003), o conhecimento tem que ser um bem comum e a aprendizagem ao longo da vida, deve ser mais que um direito, e sim, uma necessidade humana. 1 CONHECIMENTOS PARA A VIDA E A EDUCAÇÃO DE JOVENS E ADULTOS É esse sujeito real que deve receber uma educação coerente com sua realidade, com seus interesses e objetivos; e o currículo e as práticas metodológicas da EJA devem ser selecionados e planejados de acordo com essa realidade. Souza (2011, p.191) ratifica pontuando que “cabe, também, ajustar as propostas pedagógicas às peculiaridades dos contextos locais e dos subgrupos sociais [...].” Portanto, uma educação adequada aos sujeitos da EJA. De acordo com o Parecer CNE/CEB nº 11/2000, que trata das Diretrizes Curriculares Nacionais para a Educação de Jovens e Adultos, “a EJA necessita ser pensada como um modelo pedagógico próprio, a fim de criar situações pedagógicas e satisfazer necessidades de aprendizagem de jovens e adultos”. Assim, não cabe oferecer ao aluno da EJA uma educação aligeirada ou fragmentada; é necessário pensar em um ensino adequado à sua realidade. Porém, essa adequação não é DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 41 A aprendizagem por recepção e a aprendizagem pela descoberta não só possuem basicamente uma natureza e processos essencialmente diferentes como também diferem no que toca aos próprios papéis principais no desenvolvimento intelectual e no funcionamento cognitivo. Essencialmente adquirisse grandes conjuntos de matérias na escola através de aprendizagem por recepção ao passo que os problemas cotidianos se resolvem através da aprendizagem pela descoberta (AUSUBEL, 2003, p. 49). Tendo a aprendizagem significativa como referencial para uma educação que preconiza a participação ativa do aluno na EJA, há de se pensar no papel do professor que vá ao encontro desse postulado. Se levarmos em conta que o público da EJA possui peculiaridades próprias, a aprendizagem por descoberta é, sem dúvida, a mais adequada e que deve ser priorizada pelo professor. p A partir dessa constatação, reforça-se que é imprescindível pensar no aluno da EJA como um sujeito que possui conhecimentos prévios, saberes, experiências e que constrói seus conhecimentos a partir deles. Nesse contexto, Freire (2001, p. 65-66) destaca como um saber necessário à prática educativa “é o que fala do respeito devido à autonomia do ser do educando”. A reflexão sobre quem é esse educando, quais as suas aspirações e expectativas devem ser consideradas de grande relevância, principalmente nessa modalidade, cujos alunos são jovens e adultos com histórias de vida diversas e que, em sua maioria, precisam encontrar sentido no ambiente escolar. Tendo essa premissa, o professor deve fazer das experiências concretas dos alunos a base dos processos de aprendizagem. Essa prática, como aponta Sobreira (2001), é denominada pedagogia orientada no participante, na qual o aluno, assim como o professor, é sujeito-ativo do seu processo de aprendizagem, buscando a construção de significados, ou seja, a aprendizagem significativa. Portanto, quando o professor da EJA se propõe a trabalhar com metodologias ativas, deve-se buscar desenvolver a aprendizagem pela descoberta e evitar a aprendizagem por recepção, pois trazer proposições, problematizar os conteúdos são ações que representam um estímulo a mais para o aluno e oportunizam a aprendizagem significativa. 2 A APRENDIZAGEM SIGNIFICATIVA 6) ressalta que “na aprendizagem significativa, o novo conhecimento nunca é internalizado de maneira literal porque no momento em que passa a ter significado para o aprendiz, entra em cena o componente idiossincrático da significação.” Esse componente é único, pessoal, de cada indivíduo. Outra distinção importante feita por Ausubel (2003) está relacionada aprendizagem por recepção e aprendizagem pela descoberta, que são dois tipos de processos bastante diferentes. O autor destaca que a maioria da instrução na sala de aula está organizada nas linhas da aprendizagem por recepção. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 42 aprende como um educando que possui conhecimentos prévios, saberes e experiências. E quando a referência é a EJA, essa realidade se apresenta de maneira ainda mais marcante, pois este sujeito já possui uma história de vida, que ao serem valorizadas em sala de aula oportunizam seu protagonismo na aprendizagem, contribuindo para a sua permanência e o seu interesse pelos estudos. aprende como um educando que possui conhecimentos prévios, saberes e experiências. E quando a referência é a EJA, essa realidade se apresenta de maneira ainda mais marcante, pois este sujeito já possui uma história de vida, que ao serem valorizadas em sala de aula oportunizam seu protagonismo na aprendizagem, contribuindo para a sua permanência e o seu interesse pelos estudos. p Nesse contexto, as metodologias ativas são caracterizadas como metodologias alternativas ao ensino tradicional porque apresentam uma abordagem diferenciada de apresentação e desenvolvimento do trabalho pedagógico em sala de aula. Contudo, para classificar uma metodologia como viável ao processo de ensino e aprendizagem, é necessário estudar mais a fundo como o aluno aprende, conhecer esse universo e, assim, atender a essas expectativas, que podem ser alcançadas aprofundando o estuda das teorias de aprendizagem. p q p ç p p g Nesse caso, elege-se para esse contexto reflexivo uma parceria das metodologias ativas com o embasamento teórico da aprendizagem significativa, pois acredita-se que o ensino deve culminar com a aprendizagem significativa dos alunos, sujeitos de suas aprendizagens. Essa abordagem vai ao encontro de uma perspectiva transformadora da realidade na qual, segundo Gemignani (2012, p. 3), “a complexidade dos problemas atuais exige novas competências, além do conhecimento específico, tais como: colaboração, conhecimento interdisciplinar, habilidade para a inovação, trabalho em grupo, educação para o desenvolvimento sustentável, regional e globalizado.” Ou seja, uma concepção na qual as questões sociais, culturais, éticas, econômicas, ambientais, políticas são problematizadas para uma tomada de decisão participativa e responsável. Nesse sentido, Auler (2011, p. 93-94) “aponta para a educação, particularmente uma educação em ciências, que contribua para a constituição de valores democráticos e sustentáveis”. O autor (apud Auler 2002, p. 3 AS METODOLOGIAS ATIVAS E SUA INTERLOCUÇÃO COM A EJA Considerando que o mundo se encontra em constante transformação, permeado pela revolução tecnológica, científica e mudanças sociais, a instituição escolar e o ensino, propriamente dito, precisam estar em consonância com essa nova realidade. Pensar em propostas pedagógicas mais adequadas às demandas atuais e às necessidades humanas e sociais, como a sustentabilidade, representam uma mudança de paradigma. Assim, na realidade atual, a discussão de tendências mais adequadas e apropriadas aos processos de ensino e aprendizagem se apresenta de extrema relevância, pois a prática da sala de aula ainda é marcada, em sua maioria, pela passividade do aluno e por metodologias aristotélicas. Nesse sentido, as metodologias ativas se apresentam como uma possível mudança, por meio de estratégias de ensino, que buscam garantir uma maior participação do aluno em sala de aula, destacando a sua liderança no seu processo de aprendizagem, tendo o professor como mediador desse processo. Moran (2018, p. 5) ressalta que “o papel principal do especialista ou docente é o de orientador, tutor dos estudantes individualmente ou nas atividades em grupo, nas quais os alunos são sempre protagonistas”. Segundo Gemignani (2012, p. 14), esse protagonismo do aluno “implica em uma nova visão sobre o ensino e a aprendizagem e representa o deslocamento do foco da dialética para o dialógico, para uma mudança da prática docente, voltada à sensibilização e à conscientização do sujeito para esta transformação”. Nessa perspectiva, é importante pensar no sujeito que 3 Denominação dada pelo próprio autor aos professores de Ensino Fundamental e Médio. O autor argumenta que se deve levar em conta o conhecimento do trabalho dos professores, seus saberes cotidianos. Tal postura desconstrói a ideia tradicional de que os professores são apenas transmissores de saberes produzidos por outros grupos. Sua proposta é que a pesquisa universitária pare de ver os professores de profissão como objetos de pesquisa e que passem a ser considerados como sujeitos do conhecimento, como colaboradores, como copesquisadores. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 43 Nessa perspectiva, Morin (2008, p. 21) ressalta que “a primeira finalidade do ensino foi formulada por Montaigne: mais vale uma cabeça bem-feita que bem cheia”. Assim, ao invés de acumular o saber, o mais importante é dispor, ao mesmo tempo, de uma aptidão para colocar e tratar os problemas e princípios organizadores que permitam ligar os saberes e lhes dar sentido. Dessa forma, o que se objetiva é a construção do conhecimento e não apenas a sua memorização, alcançando, assim, a aprendizagem significativa. Portanto, as metodologias ativas corroboram para se alcançar a aprendizagem significativa, especialmente na EJA, na medida em que colocam o sujeito como atuante e no centro do processo de aprendizagem, representando uma oportunidade de trabalho diferenciado para o professor de ciências. p p Segundo Cachapuz et al. (2001), a didática das ciências na formação de professores deve ser vista como uma disciplina de caráter investigativo, sendo capaz de articular a teoria e a prática, a cultura da investigação e a cultura da ação. Tardif (2002) destaca que o trabalho dos professores de profissão3 deve ser considerado como um espaço prático e específico de produção, de transformação e de mobilização de saberes e, portanto, de teorias, de conhecimentos e do saber-fazer, específicos do trabalho docente. Portanto, o professor de ciências que busca a aprendizagem significativa de seus alunos da EJA deve primar pela seleção e planejamento de conteúdos, estratégias e metodologias que sejam coerentes com esse pressuposto. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 76) se aproxima da proposta freireana quando “defende a necessidade da problematização de construções historicamente realizadas sobre a atividade científico-tecnológica [...]”; uma proposta de educação libertadora, transformadora, emancipatória, na qual a criticidade se faz necessária, quando se reflete sobre a realidade vivida, não havendo espaço para a neutralidade. Portanto, acredita-se que as metodologias ativas se constituem como opção para uma educação coerente com essa conjuntura. Pode-se exemplificar, de acordo com Rocha e Lemos (2014), como pertencentes a essas metodologias: Aprendizagem Baseada em Problemas, Aprendizagem Baseada em Projetos, Instruções por pares (Peer Instruction), Ensino sob medida (Just-In-Time Teaching), Aprendizagem Baseada em Times, Método de Caso, Simulações. Greca; Villagrá (2018) destacam o Ensino por investigação. Todas essas metodologias possuem o objetivo comum de proporcionar uma aprendizagem contextualizada ao aluno, por meio de uma construção do conhecimento. O ensino por investigação, considerado uma metodologia ativa, de acordo com Greca; Villagrá (2018), representa uma opção relevante quando se pretende romper a lógica disciplinar e reconhecer as áreas de conhecimento como integradas; propõe abordagens didáticas em que o aluno trabalhe colaborativamente ou cooperativamente enfrentando problemas, desenvolvendo projetos ou respondendo a questões de seu interesse ou coletivo, após fases ou estágios relativamente flexíveis, guiados ou orientados pelo professor. Essa metodologia didática surge para ser usada, principalmente, no ensino de disciplinas científicas, para ampliar o conhecimento científico dos alunos, promovendo não apenas o conhecimento da ciência, mas também o fazer ciência e o conhecimento sobre ciência. Assim, as atividades de investigação científica buscam desenvolver nos alunos habilidades para formular perguntas e encontrar formas de coletar dados, por meio de observação e medição para respondê-las, analisar e interpretar dados e participar da discussão dos resultados e o processo para alcançá-los; sendo uma metodologia que leva ao conhecimento e compreensão do mundo natural e artificial (GRECA; VILLAGRÁ, 2018). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 44 Nesse contexto, há que se refletir, ainda, sobre o trabalho experimental no ensino de ciências, pois a experimentação desperta um forte interesse nos alunos, devido ao seu caráter lúdico e interativo, essencialmente vinculado aos sentidos. Porém, é necessário superar a concepção de simples prática de laboratório em situações de ensino, sustentada pela aplicação do Método Científico, de influência positivista. Bachelard (apud Giordan, 1999, p. 46) destaca o papel do erro no progresso da ciência, rompendo como a linearidade da sucessão “fenômeno corretamente observado/medido → interpretação inequívoca”, destacando que essa concepção obstrui o pensamento reflexivo e corrobora as explicações imediatas. Dessa forma, a experimentação coerente com uma proposta pedagógica que promova aprendizagem significativa deve permitir as possibilidades de erro e acerto, utilizando-os como estratégia de ensino. Nessa conjuntura, a democratização do ensino de ciências ainda é necessária, principalmente quando nos referimos à modalidade EJA. É preciso um bom ensino de ciências para todos e que seja desmistificada a ideia de que a ciência está à disposição de apenas alguns privilegiados. Essa premissa necessita ser priorizada, principalmente quando se acredita no papel da educação como mecanismo de inclusão social. Assim, o ensino de ciências na EJA representa uma oportunidade de desenvolvimento da chamada alfabetização ou letramento científico, de acordo com Chassot (2003), contribuindo para a inclusão desses sujeitos e com a popularização da ciência. Nesse contexto, os conhecimentos científicos e tecnológicos devem instrumentalizar os alunos para atuarem como cidadãos, participando do desenvolvimento da humanidade. Chassot (2003, p. 91) acrescenta a ideia de que a ciência seja encarada como uma linguagem. Assim, para ele, “ser alfabetizado cientificamente é saber ler a linguagem em que está escrita a natureza.” Essa leitura do mundo natural, segundo ele, nos ajuda a entender o mundo em que vivemos, como também contribui para a inclusão social, sendo “facilitadora do estar fazendo parte do mundo” (CHASSOT, 2003, p. 93). Santos (2007, p. 478) ressalta que “os grandes domínios da educação científica estão centrados na compreensão do conteúdo científico e na compreensão da função social da ciência [...], estando estes interrelacionados e imbricados”. 4 O ENSINO DE CIÊNCIAS NA EDUCAÇÃO DE JOVENS E ADULTOS Ao fazer uma retrospectiva histórica, se percebe uma evolução no ensino de ciências, ao longo dos anos, sendo permeado por diversos movimentos, que refletem diferentes objetivos da educação, modificados em função de transformações no âmbito da política e economia nacional e mundial. Porém, esse processo tem sido marcado por avanços e retrocessos, erros e acertos, no qual o que se busca é atender aos objetivos de cada sociedade, bem como às necessidades do momento. Nesse sentido, à medida que a ciência e a tecnologia foram evoluindo e se tornando essenciais para o desenvolvimento econômico, cultural e social das nações, o ensino das Ciências também foi crescendo de importância em diversos níveis. Alguns autores, como Krasilchik (2000), Cachapuz (2005), Santos (2007), destacam que vários estudos mostraram a necessidade de uma reavaliação crítica do ensino de ciências nas escolas. Nesse sentido, é preciso que ocorram alterações profundas no papel da escola e reformulações curriculares, especialmente nas disciplinas científicas. Krasilchik (2000) pontua a incoerência entre o disposto nas reformas educacionais, especialmente no ensino das ciências, e a realidade. A autora enfatiza que os resultados das pesquisas nessa área ainda não atingiram os centros de decisão, o que ocasiona falhas na preparação e avaliação dos currículos, nos projetos de aperfeiçoamento dos docentes e nas relações entre os elementos que interagem nas escolas. Cachapuz et al. (2005, p. 39) ressalta que a forma como o ensino vem sendo implementado, transmite uma percepção equivocada da ciência e dos conhecimentos científicos, “visões empobrecidas e distorcidas que criam o desinteresse, quando não a rejeição, de muitos estudantes e se convertem num obstáculo para a aprendizagem”. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 De acordo com o autor, não se pode pensar a educação científica de forma neutra; como também não é possível questionar a função social do conhecimento científico, sem a compreensão do seu conteúdo. Mais recentemente, Lederman et al. (2018) realizaram uma pesquisa colaborativa em vários países do mundo, incluindo o Brasil, para avaliar como a investigação científica é compreendida por alunos do ensino fundamental. Ao analisar as respostas dos estudantes brasileiros, percebeu-se que eles podem conceituar alguns aspectos da investigação científica, mas não conseguem identificá-los em situações reais. Esse resultado reflete a falta de ênfase curricular nacional sobre a investigação científica no ensino de ciências do ensino fundamental e a ausência de experiência em investigar nas salas de aula de ciências. Esse resultado não foi muito diferente dos demais países participantes da pesquisa, pois, de um modo geral, os resultados mostraram, predominantemente, que estudantes no segundo segmento do ensino fundamental têm muito pouco entendimento sobre o que é investigação científica, apesar de ser considerado um objetivo altamente valorizado da educação científica em todo o mundo e de ser um componente significativo da alfabetização científica. Nessa perspectiva, percebe-se que os currículos escolares ainda precisam superar a fragmentação e especialização do saber científico. Tornar a educação científica uma cultura científica é desenvolver valores estéticos e de sensibilidade, popularizando o conhecimento científico pelo seu uso social como modos elaborados de resolver problemas humanos. Para isso, torna-se relevante o uso de meios informais de divulgação científica, como textos de jornais e revistas e programas televisivos e radiofônicos em sala de aula. Além disso, visitas programadas DOSSIÊ AMAZÔNIA 45 a espaços não-formais de educação, como museus de ciência, jardins zoológicos, jardins botânicos, planetários, centros de visita de instituições de pesquisa e de parques de proteção ambiental e museus virtuais, entre outros, são importantes estratégias para inculcar valores da ciência na prática social (SANTOS, 2007, p. 487). O desenvolvimento dessa cultura científica é extremamente relevante na Educação de jovens e adultos, já que estes sujeitos têm acesso a muitos desses meios de comunicação e também podem comparecer, de forma independente, a espaços não-formais, desde que sejam incentivados, tenham interesse e/ou percebam sua importância. Porém, apesar de estarmos no século XXI, ainda existe a necessidade de se superar o cientificismo, ou seja, a supervalorização do conhecimento científico em detrimento às demais áreas do conhecimento humano. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Como destacado por Santos (1991), a crise do paradigma dominante se apresenta como sendo, não só profunda, como também irreversível, visto que o paradigma vigente não dá mais conta das complexidades de nossa realidade. A visão holística e transdisciplinar do paradigma emergente supera uma visão fragmentada e disciplinar. Nessa perspectiva, é mister perceber o ser humano como um ser complexo, inserido numa sociedade em constante transformação, bem como repensar as funções da escola e planejar um ensino mais adequado e coerente com os objetivos dos alunos para o qual essa educação se destina. q j p q ç Nesse contexto, a interdisciplinaridade e a contextualização devem ser priorizadas na elaboração do currículo escolar na EJA, favorecendo uma maior interlocução entre os conteúdos trabalhados e a realidade dos alunos. Em se tratando do ensino das Ciências, essa prática se torna extremamente relevante, pois as disciplinas científicas estão intimamente relacionadas e, planejadas adequadamente, despertam o interesse do aluno e a sua participação mais efetiva. Assim, no que tange à EJA, a interdisciplinaridade e a contextualização representam uma oportunidade de se obter uma aprendizagem significativa. A interdisciplinaridade leva o alunado a perceber a inter-relação entre os conteúdos curriculares, rompendo com a divisão das disciplinas; a busca pela contextualização é realizada quando se trabalha com questões atuais, da vivência dos alunos. Ao trazer para a sala de aula a realidade vivida pelos alunos, estes se sentem mais motivados e participam das aulas com mais empenho. Essa atitude aumenta a autoestima dos alunos e contribui para uma melhor aprendizagem, o que na EJA é de extrema relevância. É É importante destacar, ainda, uma prática que, cada vez mais, tem sido incorporada ao ensino de ciências: a aprendizagem colaborativa. É necessário criar oportunidades não somente para realizar experimentos em equipe, mas também para a colaboração entre equipes. A formação de um espírito colaborativo de equipe pressupõe uma contextualização socialmente significativa para a aprendizagem, tanto do ponto de vista da problematização – temas socialmente relevantes, como também da organização do conhecimento científico – temas epistemologicamente significativos (GIORDAN, 1999, p. 6). Quando o processo de ensino de ciências é desenvolvido nessa perspectiva, o professor caminha para a promoção de uma aprendizagem significativa, promovendo a interação entre os alunos, a troca de experiências, contextualizando e dando sentido ao conhecimento. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 É digna de nota a capacidade que tem a experiência pedagógica para despertar, estimular e desenvolver em nós o gosto de querer bem e o gosto da alegria sem a qual a prática educativa perde o sentido. É esta força misteriosa, às vezes chamada vocação, que explica a quase devoção com que a grande maioria do magistério nele permanece, apesar da imoralidade dos salários. E não apenas permanece, mas cumpre, como pode, seu dever. Amorosamente [...] (FREIRE, 2001, p. 161). Nessa perspectiva, é necessário competência técnica e sensibilidade para poder intervir de forma consciente e planejada, visando a melhoria da qualidade do processo educativo e atendendo as demandas apresentadas pelos alunos da EJA. Portanto, o uso de metodologias ativas no ensino de ciências, adequado à realidade dessa modalidade de ensino, se apresenta como estratégia eficaz para aprendizagem significativa desses sujeitos da EJA, favorecendo uma formação humana e educacional, coerente com seus anseios e suas reais necessidades. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Portanto, deve-se estimular no ensino de ciências da EJA a prática em sala de aula de desenvolver o processo educativo, a partir de concepções prévias dos alunos e seus contextos, oportunizando que os saberes desses jovens e adultos sejam compartilhados e valorizados. Esses saberes, muitas vezes, acabam sendo desmistificados e, outras vezes, ratificados por fazerem parte da cultura popular. Segundo Pozo (1998), para a inserção de um novo assunto, a utilização de técnicas, como: entrevistas, questionários e situações-problemas devem levar o professor a conhecer as concepções alternativas (ou concepções prévias) dos alunos, auxiliando-o no seu planejamento; NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 46 bem como oportunizar espaços para que os alunos manifestem suas concepções alternativas e propiciar discussões que favoreçam a integração de ideias, concepções e o trabalho colaborativo. bem como oportunizar espaços para que os alunos manifestem suas concepções alternativas e propiciar discussões que favoreçam a integração de ideias, concepções e o trabalho colaborativo. Independente da estratégia adotada, para verificar qual o nível de conhecimento que os alunos trazem para a sala de aula, o professor deverá elaborar um roteiro de atividades, por meio da inserção de um problema que faz parte do cotidiano do aluno. Este problema deverá abordar indiretamente os aspectos mais importantes sobre o conteúdo e, ao mesmo tempo, fazer com que o conteúdo seja compatível com o grau de conhecimento em que o aluno se encontra (OLIVEIRA, 2005, p. 239). Cabe destacar, outra questão relevante apontada por Moran (2018, p. 9) “a combinação de aprendizagens personalizadas, grupais e tutoriais no projeto pedagógico é poderosa para obter os resultados desejados”. Nesse contexto, fica evidente a importância do papel do professor no planejamento e organização das atividades de ensino na EJA e, consequentemente, a formação do mesmo frente à essa realidade. Se ao professor é atribuído o papel de organizador do processo ensino aprendizagem, interlocutor entre os alunos e o conhecimento, sua formação deve ser permeada pela reflexão, ou seja, pelo repensar contínuo do ser e fazer pedagógico, de forma crítica, humana e responsável, buscando a parceria com seus pares e o desejo permanente de fazer a diferença na vida dos alunos. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 47 É necessário reorientar as estratégias educativas, em busca de um novo modelo de aprendizagem das ciências, mediante um processo de investigação científica, que leve a reconstrução de conhecimentos e que pode ser alcançado, a partir de situações problemáticas de interesse dos alunos. Romper com a abordagem disciplinar do currículo e propor um currículo integrado, exige uma formação adequada dos professores e o desenvolvimento de novos recursos de ensino (livros, materiais, software etc.). O magistério exige comprometimento, estudo, questionamento, reflexão e pesquisa. Assim, a formação do professor deve ser realizada numa perspectiva técnica (não alienada), prática e crítica, emancipatória, na qual se busque respostas para as indagações e necessidades reais dos alunos e da sociedade. Assim, pensar em aprendizagem significativa no ensino de ciências na EJA representa almejar um ensino significativo, em uma escola que faça sentido para o aluno trabalhador e para a sociedade; concebida como um organismo vivo, em constante desenvolvimento, norteada pelo objetivo maior de educar, que se concretiza através de um projeto educativo crítico. As reflexões aqui apresentadas não representam apenas um discurso inovador, mas uma concepção de aprendizagem e proposta de metodologias adequadas ao contexto atual; e que, de fato, colocam o aluno da EJA como sujeito protagonista do ato educativo; sujeito este capaz de colaborar para as transformações que a sociedade tanto necessita. É papel da educação contribuir para um país e um mundo melhor, em que a tolerância, a igualdade, a justiça social e o respeito pelos outros constituam valores humanos imprescindíveis. LARCÃO, Isabel. Professores reflexivos em escola reflexiva. São Paulo: Cortez, 2003 AULER, Décio; SANTOS, Widson Luiz Pereira dos. CTS e educação científica: desafios, tendências e resultados de pesquisa. Brasília: Editora UnB, 2011. AULER, Décio; SANTOS, Widson Luiz Pereira dos. CTS e educação científica: desafios, tendências e resultados de pesquisa. Brasília: Editora UnB, 2011. AUSUBEL, David Paul. Aquisição e retenção de conhecimentos: uma perspectiva cognitiva. Tradução Lígia Teopisto. 1. ed. Lisboa: Paralelo Editora, 2003, 226 p. Título original: The acquisition and retention of knowledge: a cognitive view. ISBN 972-707-364-6 AUSUBEL, David Paul. Aquisição e retenção de conhecimentos: uma perspectiva cognitiva. Tradução Lígia Teopisto. 1. ed. Lisboa: Paralelo Editora, 2003, 226 p. Título original: The acquisition and retention of knowledge: a cognitive view. ISBN 972-707-364-6 REFERÊNCIAS ALARCÃO, Isabel. Professores reflexivos em escola reflexiva. São Paulo: Cortez, 2003. 5 CONSIDERAÇÕES FINAIS Este artigo não tem a pretensão de esgotar o assunto, e sim apresentar algumas reflexões sobre como a educação de jovens e adultos pode ser ressignificada, na busca de uma participação ativa do aluno, para que ele se torne protagonista e sujeito de sua aprendizagem. Repensar e reconstruir permanentemente a prática pedagógica é primordial no exercício docente, especialmente na EJA, uma modalidade de ensino que possui uma especificidade em relação aos seus sujeitos: jovens e adultos que já possuem as marcas da exclusão social. Assim, o professor da EJA deve promover um ambiente acolhedor em sala de aula, no qual o aluno se sinta pertencente àquele espaço educativo. As metodologias ativas são vistas como uma oportunidade de desenvolver nos alunos da EJA, um conjunto de habilidades de pensamento, raciocínio, trabalho em equipe, pesquisa, comunicação e criatividade, de modo que possam usar essas habilidades em todas as áreas em que atuarem. Nesse sentido, implementar o ensino de ciências a partir de metodologias ativas e priorizar a aprendizagem significativa representam um compromisso com esses sujeitos e com as demandas emergentes do mundo atual. CHASSOT, Áttico. Alfabetização científica: uma possibilidade para a inclusão social. Revista Brasileira de Educação, ANPEd, n. 26, p.89-100, 2003. Disponível em: BRASIL. Parecer CNE/CEB nº 11/00. Diretrizes Curriculares Nacionais para a Educação de Jovens e Adultos. Brasília: MEC, 2000. BRASIL. Parecer CNE/CEB nº 11/00. Diretrizes Curriculares Nacionais para a Educação de Jovens e Adultos. Brasília: MEC, 2000. CACHAPUZ, António; CARRASCOSA ALÍS, Jaime; GIL-PÉREZ, Daniel; TERRADES, Isabel Martinéz . A emergência da didática das ciências como campo específico de conhecimento. Revista Portuguesa de Educação, Minho, v. 14, n. 1, p. 155-95, 2001. Disponível em: https:www.researchgate.net/publication/26464941_A_emergencia_da_didactica_das_ciencias _como_campo_especifico_de_conhecimento. Acesso em: 29 set. 2020. p https:www.researchgate.net/publication/26464941_A_emergencia_da_didactica_das_ciencias _como_campo_especifico_de_conhecimento. Acesso em: 29 set. 2020. CACHAPUZ, António; CARVALHO, Ana Maria Pessoa de; GIL-PEREZ, Daniel; PRAIA, João.; VILCHES, Amparo (Org.) A necessária revolução do ensino de ciências. São Paulo: Cortez, 2005. CHASSOT, Áttico. Alfabetização científica: uma possibilidade para a inclusão social. Revista Brasileira de Educação, ANPEd, n. 26, p.89-100, 2003. Disponível em: NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 48 https:www.scielo.br/scielo.php?pid=S1413- 24782003000100009&script=sci_abstract&tlng=pt. Acesso em: 27 set.2020. 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DOSSIÊ AMAZÔNIA 49 OLIVEIRA, Inês Barbosa. Reflexões acerca da organização curricular e das práticas pedagógicas da EJA. Educar, Curitiba, n. 29, p. 83-100, 2007. Disponível em: https:www.scielo.br/pdf/er/n29/07.pdf. Acesso em: 28 set. 2020. OLIVIERA, Marta Kohl de. Jovens e adultos como sujeitos de conhecimento e aprendizagem. Revista Brasileira de Educação, Rio de Janeiro, p.59-73, set. 1999. OLIVEIRA, Silmara Sartoreto. Concepções alternativas e ensino de biologia: como utilizar estratégias diferenciadas na formação inicial de licenciados. Educar, Curitiba, n. FREIRE, Paulo. Pedagogia da Autonomia. São Paulo: Paz e Terra, 2001. 26, p. 233- 250, 2005. Disponível em: https:www.scielo.br/pdf/er/n26/n26a16.pdf. Acesso em: 28 set. 2020. POZO, Juan Ignácio. A aprendizagem e o ensino de fatos e conceitos. In: COLL, Cesar. et al. Os conteúdos na reforma. Porto Alegre: Artes médicas, 1998. p. 17-71. ROCHA, Henrique Martins; LEMOS, Washington Macedo. Metodologias ativas: do que estamos falando? Base conceitual e relato de pesquisa em andamento. IX Simpósio Pedagógico e Pesquisas em Educação, 2014. Disponível em: https:www.aedb.br/wp- content/uploads/2015/05/41321569.pdf. Acesso em: 29 set. 2020. SANTOS, Boaventura Souza. Um discurso sobre as Ciências. Porto Alegre: Melhoramentos, 1991. SANTOS, Boaventura Souza. Um discurso sobre as Ciências. Porto Alegre: Melhoramentos, 1991. SANTOS, Wildson. Luiz. Pereira dos. Educação científica na perspectiva de letramento como prática social: funções, princípios e desafios. Revista Brasileira de Educação, ANPED, Rio de Janeiro, v. 12, n.36, p. 474-550, 2007. Disponível em: https:www.scielo.br/pdf/rbedu/v12n36/a07v1236.pdf. Acesso em: 28 set.2020. SILVA, Janssen Felipe da. Avaliação do ensino e da aprendizagem numa perspectiva formativa reguladora. In: ESTEBAN, Maria Teresa.; HOFFMANN, Jussara; SILVA, Janssen Felipe da. (org.) Práticas avaliativas e aprendizagens significativas em diferentes áreas do currículo. Porto Alegre: Mediação, 2003, p. 7-18. SOBREIRA, Henrique Garcia. Anotações éticas e estéticas sobre a educação do educador. In: RAMOS-DE-OLIVEIRA, Newton; ZUIN, Antônio Álvaro Soares; PUCCI, Bruno. (Org.) Teoria crítica, estética e educação. Campinas: Autores associados, Unimep, 2001, p. 161- 75. SOBREIRA, Henrique Garcia. Anotações éticas e estéticas sobre a educação do educador. In: RAMOS-DE-OLIVEIRA, Newton; ZUIN, Antônio Álvaro Soares; PUCCI, Bruno. (Org.) Teoria crítica, estética e educação. Campinas: Autores associados, Unimep, 2001, p. 161- 75. SOUZA, Marta Lima de. Alfabetização e aprendizagem ao longo da vida: armadilhas e refúgios. In: SOUZA, José Santos.; SALES, Sandra Regina. (Org.) Educação de Jovens e Adultos: políticas e práticas educativas. Rio de Janeiro: NAU Editora, 2011. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 51 – – M 202 202 REVISTA DO PROGRAMA DE PÓS- GRADUAÇÃO EM LINGUAGENS E 2318 - M 2318 2318 - CAMPUS BRAGANÇA VOLUME NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 52 currículo por Tema Gerador, bem como do uso de produções que situam essa experiência no município pesquisado, desde uma experiência piloto em 2009 até o retorno em 2015 na Rede Municipal de Educação de Tracuateua. currículo por Tema Gerador, bem como do uso de produções que situam essa experiência no município pesquisado, desde uma experiência piloto em 2009 até o retorno em 2015 na Rede Municipal de Educação de Tracuateua. Na escuta das vozes dos técnicos pedagógicos que estiveram na Secretaria Municipal de Educação (SEMED), visualizou-se as dificuldades, os avanços e os recuos estratégicos marcados, sobretudo, pela conjuntura política nacional brasileira desses últimos anos. A questão que se apresenta é saber: de que maneira a percepção se fez presente nas práticas educacionais de jovens e adultos, em particular no processo de implantação do currículo via Tema Gerador em Tracuateua-PA? O ponto de partida para responder a essa indagação reside em reconstruir o caminho iniciado no ano de 2009 por ser ele o marco de referência de um projeto piloto em uma única escola de Tracuateua. Nessa caminhada, apontamos de que forma ocorreu este tipo de organização curricular, seus pressupostos epistemológicos, os avanços e desafios na materialização desse currículo. Para dar conta da apresentação desse percurso, organizamos o trabalho a partir de uma introdução que apresenta os elementos essenciais que propusemos a refletir. Em seguida, adentramos no item que trata da concepção de Paulo Freire como base teórica e prática para a implementação da EJA no Brasil, no qual apresentamos suas ideias acerca da educação. Por fim, traçamos uma análise do currículo via Tema Gerador implantado em Tracuateua em 2015- 2017. Nas considerações finais, sinalizamos aspectos importantes desse currículo que ora toma lugar central como projeto de educação libertadora, ora é silenciado pela resistência de sujeitos que estão à frente da dinâmica educacional nacional. q A conjuntura política nacional e local, a partir dos anos de 2009, foram fundamentais para os atores educacionais de Tracuateua experimentarem uma organização curricular diferente das que estavam em curso. Essa conjuntura fez surgir um desejo de mudança nos sujeitos que reivindicavam outras pedagogias, com existência e resistência transformadora, a partir de um currículo dialógico e participativo. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 53 se intimamente associado à tomada de consciência da situação real vivida pelos educandos. Nesse contexto, o educador Paulo Freire preocupado com a situação do analfabetismo e, mais ainda, com a opressão das pessoas, deveriam atuar a serviço da libertação de homens e mulheres, acreditando na sua capacidade de criação e de crítica. se intimamente associado à tomada de consciência da situação real vivida pelos educandos. Nesse contexto, o educador Paulo Freire preocupado com a situação do analfabetismo e, mais ainda, com a opressão das pessoas, deveriam atuar a serviço da libertação de homens e mulheres, acreditando na sua capacidade de criação e de crítica. E, assim, na cidade de Angicos em 1962, estado do Rio Grande do Norte, Freire constrói uma proposta de educação de adultos cujo resultado foi a alfabetização de 300 trabalhadores rurais em apenas 45 dias. Uma educação que tinha como base a libertação dos sujeitos oprimidos, capaz de despontar a confiança na pessoa humana e na sua capacidade de educar-se como sujeito histórico e social. A educação de Freire, visava uma postura proativa, dialogal, horizontal, participante e crítica para todos os sujeitos educacionais envolvidos com a alfabetização de adultos. Uma experiência de educação disseminada em várias partes do país, cujo desejo era ainda maior, com o plano elaborado no governo Goulart em 1964. Esse plano objetivava a implantação de 20 mil Círculos de Cultura em todo o território brasileiro. O Círculo se constituía em um grupo de trabalho cujo interesse seria o debate da linguagem no contexto de uma prática social livre e crítica. Os educadores do Círculo de Cultura eram chamados de coordenadores e os seus papéis eram dialogar com os alfabetizandos oferecendo-lhes os instrumentos para uma alfabetização além das letras. Essa atitude de diálogo proporcionava de fato uma educação libertadora e não simplesmente a domesticação dos sujeitos, visto que o diálogo é uma relação de ida e volta, de intertrocas, ou seja, uma relação de dois sujeitos interlocutores. Devemos reforçar no âmbito da proposta de Freire, a prática libertadora de educação. Esta se corporifica a partir do processo de construção de conhecimento, diferentemente da concepção bancária ou tradicional, a qual prioriza a transmissão de conteúdo, ora pelo professor, ora pelo aluno. A prática libertadora de educação se faz pelo diálogo crítico, na troca de experiências. 1 A CONCEPÇÃO DE PAULO FREIRE COMO BASE TEÓRICA E PRÁTICA PARA A IMPLEMENTAÇÃO DA EJA NO BRASIL As concepções teóricas que embasam a educação em Paulo Freire são resultado de uma construção marcada pelo contexto histórico e fruto das diferentes experiências pelas quais ele passou. Foram estas experiências que o ajudaram a construir conceitos essencialmente necessários à elaboração e consolidação de sua proposta educacional na década de 1960. Reportamo-nos ao pensamento de Paulo Freire quando ele diz que a educação vigente na época não encorajava o homem a debater as problemáticas do momento. Isso acontecia por falta de embasamento teórico capaz de despertar o gosto pelo conhecimento, invenção e pesquisa. Diante da transição da sociedade brasileira, Freire se via angustiado e esperava ser ele, o educador que poderia contribuir incitando os sujeitos a reivindicarem mudanças nas condições objetivas de existência. Almejava uma educação diferente daquela que até então existia. Uma educação crítica capaz de mudar o pensamento, que saísse da transitividade ingênua para uma proposta dialógica crítica. Freire entendia que era necessária uma educação para o protagonismo, capaz de colocar os sujeitos em diálogo permanente com seus pares, pois o sentido da democracia implicava participação, mudança, tomar partido, debater ideias. Nas palavras desse teórico, “uma educação que possibilitasse ao homem a discussão corajosa de sua problemática” (FRERE, 1983, p. 89) permitindo-os ver que o mundo não estava acessível a todos. Freire (1983) se debruça sobre a alfabetização por ser ela o campo inicial e ao mesmo tempo, o tema da maior relevância social e política no Brasil. O aprendizado deveria encontrar- DOSSIÊ AMAZÔNIA 113). A alfabetização abre os trabalhos do Círculo de Cultura, e é também o início da conscientização. Ao educador cabe registrar fielmente o vocabulário dos alfabetizandos, selecionar algumas palavras básicas que aparecem com frequência no seu universo vocabular, garantindo que estas sejam relevantes e carregadas de sentido e, sobretudo, que apresentem complexidade fonêmica. p Freire (1983) anuncia as fases de estruturação de sua proposta de educação apresentando cinco passos: Freire (1983) anuncia as fases de estruturação de sua proposta de educação apresentando assos: p 1 – Levantamento do universo vocabular dos grupos com quem se trabalha; 2 – Escolha das palavras selecionadas do universo vocabular pesquisado, a partir de critérios, (riqueza fonêmica, dificuldades fonéticas e teor pragmático da palavra); q p g p 3 – Criação de situações existenciais típicas do grupo com quem vai se trabalhar; 4 – Elaboração de fichas roteiros que auxiliem os coordenadores de debate no seu trabalho, estas vão servir apenas como subsídios para os mesmos; p p 5 – Feitura de fichas com a decomposição das famílias fonêmicas correspondentes aos vocábulos geradores. É interessante destacar algumas palavras geradoras dos Círculos de Cultura que Freire acompanhou nessa sua experiência, como: favela, chuva, arado, terreno, comida, batuque, poço, bicicleta, trabalho, tijolo, riqueza. É interessante destacar algumas palavras geradoras dos Círculos de Cultura que Freire acompanhou nessa sua experiência, como: favela, chuva, arado, terreno, comida, batuque, poço, bicicleta, trabalho, tijolo, riqueza. Fazemos menção à primeira palavra geradora pesquisada e aplicada no Estado do Rio de Janeiro, a palavra FAVELA, quando da organização do Círculo de Cultura. Naquela ocasião, o uso de fotografia ampliava os debates, ao passo que faziam emergir os problemas de habitação, alimentação, vestuário, saúde e educação. Os participantes descobriram a favela como situação problemática, percebendo como ela se formava enquanto texto/contexto. Outras experimentações foram evidenciadas, por exemplo, o grupo de alfabetizandos começaram a criar palavras com as combinações a sua disposição. Essa experiência de educação de Freire nos mostra que a alfabetização e a conscientização jamais se separam. É possível ler as palavras e ler o mundo concomitantemente. No entanto, o referido autor, alerta que o maior desafio reside em fazer os educandos se perceberem como sujeitos ou não do processo educativo. A tomada de consciência possibilita abrir os olhos para aquilo que julgam “normal”, “natural”, desnaturalizando as condições de opressão, de pobreza, de fome, de exploração, de analfabetismo. DOSSIÊ AMAZÔNIA Um processo que escapa à polaridade professor/aluno, visto que se ramifica pelo diálogo, pois ambos têm muito a ensinar e muito o que aprender em uma concepção dialética de conhecimento. Freire (1983) informa que com menos de seis meses os resultados do seu trabalho de alfabetização eram surpreendentes. Os sujeitos não alfabetizados começaram a demostrar a tomada de consciência, reconhecendo-se não apenas sujeitos no mundo, sem propósito, mas sentindo-se parte dele. É É importante destacar que a prática pedagógica de Paulo Freire, revela o respeito à liberdade dos educandos que nunca são chamados de analfabetos, mas de alfabetizandos. O fato de chamá-los desta forma evidencia o caráter processual em que ocorre a tomada de consciência, cujo desejo se reveste de esperança de que eles possam sair de sua condição de não alfabetizados para sujeitos alfabetizados, leitores das palavras e do mundo. A educação como uma prática libertadora mostra-se mais democrática do que o ensino tradicional. Nessa perspectiva, o papel do professor e do aluno são colocados em discussão, visto que o professor não é o detentor do conhecimento e, tampouco, se julga transmissor daquilo que julga saber aos alunos. Estes não são considerados apenas assimiladores de conteúdos de forma passiva e receptiva, no qual o conhecimento em nada contribui para/na construção de um sujeito crítico. Nesse sentido, a educação em uma perspectiva libertadora considera tanto professores quanto alunos sujeitos protagonistas do processo de ensino/aprendizagem. Considera-os como sujeitos cognoscentes da realidade em uma prática emancipatória, ainda que, o conhecimento se apresente de forma diferenciada. Com isso, o conhecimento pode ser produzido de diferentes formas, seja por meio dos saberes, das experiências historicamente acumuladas, das histórias de vida. O fato é que a Pedagogia Libertadora, não cabe apenas nos espaços tradicionais de educação chamados NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 54 escolas, ela viabiliza ações que vão possibilitar uma reflexão em uma perspectiva de mudança na sociedade como um todo. Assim, pensar a educação não restringindo-a ao sentido meramente pedagógico, mas também como “um ato político!” (FREIRE, 1986, p. 113). escolas, ela viabiliza ações que vão possibilitar uma reflexão em uma perspectiva de mudança na sociedade como um todo. Assim, pensar a educação não restringindo-a ao sentido meramente pedagógico, mas também como “um ato político!” (FREIRE, 1986, p. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 55 Jovens e Adultos. Com um projeto pedagógico piloto inovador, mostrou-se capaz de atender os educandos desenvolvendo sua criticidade. Permanentemente reinventado e reelaborado, o projeto via Tema Gerador de base freiriano, longe de ser perfeito, mostra os avanços e suas dificuldades ao longo dos anos de implementação. O destaque que queremos dar a essa experiência, reside na perspicácia de alguns protagonistas, agentes do processo educativo que estiveram como condutores em seus contextos, seja a escola, o sistema de ensino ou a comunidade. DOSSIÊ AMAZÔNIA Embora interrompido pela ditadura militar, observa-se que a proposta pedagógica de Paulo Freire para a educação de Jovens e Adultos é aplicável até hoje em nossa educação, sofrendo reinvenções e reelaborações. Quanto ao problema de analfabetismo, verifica-se que o perfil descompromissado da educação escolar com as classes trabalhadoras questionado por Paulo Freire na década de 1960, ainda prevalece nos dias atuais. Podemos dizer que os militares impediram, em grande medida, a concretização do que ficou conhecido como proposta de Alfabetização de Adultos e imprimiram uma pedagogia tecnicista que respondia às necessidades do processo de internacionalização da economia brasileira na década de 1960 (FREIRE, 1986). No entanto, as lutas pela redemocratização do país no final da década de 1970, trouxeram as propostas pedagógicas de Paulo Freire para os sistemas educativos atuais, cujo desejo é o de uma educação não fragmentada pelas artimanhas do autoritarismo. Movente como os sonhos de Freire, Tracuateua a partir dos anos 90 veio experimentando a pedagogia libertadora em seus currículos escolares para a Educação de 2 CURRÍCULO VIA TEMA GERADOR E A EXPERIÊNCIA DO MUNICÍPIO DE TRACUATEUA-PA Tabela 1- Demonstrativo da demanda escolar na rede municipal de Tracuateua desde a sua implantação em 1997 MUNICIPIO DE TRACUATEUA Nº DE ALUNOS DA ZONA URBANA Nº DE ALUNOS DA ZONA RURAL ANO LETIVO Nº DE ESCOLAS Nº DE ALUNOS DA REDE MUNICIPAL EJA EJA TOTAL ZONA URBANA ZONA RURAL EJA SÉRIES INICIAIS SÉRIES FINAIS SÉRIES INICIAIS SÉRIES FINAIS 1997 49 1 48 0 0 0 0 0 1998 55 1 54 166 0 166 0 0 1999 59 2 57 334 0 334 0 0 2000 59 2 57 434 0 186 0 248 2001 59 2 57 893 32 191 427 243 2002 58 2 56 1012 36 194 479 303 2003 54 2 52 725 31 96 407 191 2004 54 2 52 602 42 181 260 119 2005 61 4 57 565 0 361 0 204 Tabela 1- Demonstrativo da demanda escolar na rede municipal de Tracuateua desde a sua imp em 1997 monstrativo da demanda escolar na rede municipal de Tracuateua desde a sua implantação NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 56 2006 64 5 59 466 0 302 0 164 2007 56 4 52 472 0 338 0 134 2008 67 5 62 461 21 255 23 162 2009 59 5 54 376 22 191 21 142 2010 60 5 55 531 19 217 116 179 2011 58 5 53 533 23 198 104 208 2012 59 5 54 517 34 169 108 206 2013 58 5 53 668 38 182 209 239 2014 60 5 55 558 31 163 148 216 2015 59 5 54 329 23 139 30 137 2016 59 5 54 241 19 77 45 100 2017 59 5 54 188 15 101 30 42 Fonte: Sistema Educacenso / MEC/INEP – Consulta a Matrícula / SEMED, 2017 Os dados mostram que as matrículas na Educação de Jovens e Adultos tem diminuído drasticamente nos últimos anos. Isso nos leva a refletir sobre o futuro dessa modalidade de educação em Tracuateua forjando um cenário de perguntas: Quais os motivos que levam os alunos a evadirem da escola? O que o município tem feito para evitar a evasão escolar? Longe de ter respostas para essas questões, queremos evidenciar os caminhos percorridos em aproximadamente uma década de educação municipal. 3 https://pt.scribd.com/document/107190461/os-temas-geradores-na-formacao-de-educadoras-e-educadores-e-na- alfabetizacao-de-jovens-e-adultoS 2 CURRÍCULO VIA TEMA GERADOR E A EXPERIÊNCIA DO MUNICÍPIO DE TRACUATEUA-PA Diante das escutas realizadas com os técnicos educacionais da Secretaria Municipal de Educação, sempre houve preocupação acerca da educação municipal e os seus rumos. Uma delas, diz respeito à organização pedagógica das escolas em seus níveis e modalidades de ensino. Percebeu-se na polissemia das vozes desses sujeitos um desejo materializado em ato, o de buscar outras perspectivas de currículos que não se distanciassem da realidade dos seus educandos e que, sobretudo, primasse pela dialogicidade e pela construção coletiva. Ressaltamos que nos últimos anos, o trabalho pedagógico mostra-se promissor desse ato coletivo, do fazer com o outro, envolvendo diferentes sujeitos da escola, como os professores, alunos, gestores, técnicos pedagógicos, profissionais de apoio e comunidade Considera-se na experiência relatada sobre o projeto via Tema Gerador, que todos os sujeitos são importantes, agindo como construtores da educação de Tracuateua. Nas vozes dos técnicos pedagógicos, as resistências por parte de alguns grupos eram presentes, mas o que se coloca é antes, o esforço para materializar uma proposta que necessitava, em sua essência, do outro como sujeito de voz. Foi nessa tentativa, diante da grande evasão dos alunos da Educação de Jovens e Adultos, que Tracuateua experimentou em 2009 outra organização curricular, o currículo via Tema Gerador. No que compete a essa evasão escolar, podemos visualizar e compreender melhor analisando a Tabela a seguir, que traz um levantamento dos últimos 20 anos. 2 CURRÍCULO VIA TEMA GERADOR E A EXPERIÊNCIA DO MUNICÍPIO DE TRACUATEUA-PA Talvez as respostas estejam imbricadas na conjuntura política, cujo cenário nem sempre esteve a favor da educação libertadora. Portanto, iniciemos falando da ocasião em que o projeto via Tema Gerador foi implantado em uma escola piloto de Tracuateua. Na ocasião em que se colocou em prática o currículo por Tema Gerador em 2009, havia apenas três escolas dentro do quantitativo de 59 de toda a Rede Municipal que ofertavam essa modalidade. E foi em uma delas que o projeto foi implantado sob a orientação e assessoria de professores da Universidade Federal do Pará - UFPA. Em um texto produzido por estes professores (NEVES; MARCIEL, 2009) que encontra- se disponível em endereço eletrônico3, eles descrevem como se deu essa primeira experiência, os seus desdobramentos e a organização da proposta. Dentre os objetivos almejados estava o desenvolvimento de uma formação comprometida com a vida das pessoas e com a história do município de forma que o resultado fosse a transformação. Os formadores/autores chamam esta experiência de teia do conhecimento. A teia direcionava o trabalho educativo, tomando como ponto de partida a realidade dos alunos, pois só assim eles estariam preparados para lidarem com as problemáticas sociais. A construção da teia se fazia via processo formativo diante das falas significativas. Estavam em cenas educadores, educadoras e a escolarização dos Jovens e Adultos. Para fazer a formação dos educadores era preciso se colocar no contexto onde atuavam para transformar aquela realidade. Diante disso, a equipe técnica pedagógica da escola formada pelo diretor e coordenadores pedagógicos tinham um papel central enquanto articuladores e orientadores da proposta. Todos eram convidados a assumirem o lugar de formadores nos seus contextos. Empoderados das discussões, os polos eram frutificados com debates, discussões e proposições. Essa produção coletiva revestia-se pela importância dada pelos sujeitos à essa construção. DOSSIÊ AMAZÔNIA 57 Russo (2011, p. 504) destaca o trabalho do diretor e coordenador pedagógico, responsabilizando-os pela: Russo (2011, p. 504) destaca o trabalho do diretor e coordenador pedagógico, responsabilizando-os pela: [...] mediação para que o ensino se realize com o máximo de eficácia e de eficiência possível em cada caso concreto, isto é, com os recursos humanos, materiais e financeiros disponíveis em cada escola. 2 CURRÍCULO VIA TEMA GERADOR E A EXPERIÊNCIA DO MUNICÍPIO DE TRACUATEUA-PA O trabalho desses agentes tem efeito direto no processo de ensino aprendizagem porque tem como finalidade criar condições favoráveis à sua realização, à preparação adequada dos docentes e dos discentes, à sua participação no processo ou, ainda, à correção de desvios que possam contribuir para o fracasso do processo. Assim, acreditar que eles poderiam empoderar-se para construir condições favoráveis ao ensino dos alunos. Para os formadores da UFPA, o conhecimento não seria apenas uma doação daquilo que julgavam possuir, mas uma ferramenta importante na construção da compreensão da realidade e na conquista da escolarização de Jovens e Adultos. Os professores que lançaram essa proposta no município se reportam a teia enquanto perspectiva que possibilitaria desnaturalizar as situações, no qual os sujeitos estão em movimento para alcançar a autonomia. Uma experiência que se traduz no trabalho coletivo entre educadores, educadoras, rede de educação e apoio pedagógico. As etapas da construção e escolha do tema gerador nascia da pesquisa da realidade sobre a história e o fazer dos sujeitos do processo, neste caso, os alunos da EJA da escola Raimundo Pinheiro de Melo4. Os autores descrevem que durante o processo de formação em 2009 percorreram dois caminhos, conforme mostra a narrativa a seguir: O primeiro foi fazer a pesquisa a partir das histórias de vida dos educandos e educandas, alunos da Escola RPM, num processo de escuta, diálogo e reflexão. Este material foi organizado em Teia de conhecimento tanto a formação dos/as educadores/as quanto para organização da estrutura curricular da EJA. Para este levantamento, os sujeitos da EJA, coordenados pelos/as educadores/as, registram em todas as turmas, através do desenho, o que era significativo em sua história de vida. Depois socializaram as suas histórias. Neste processo, os/as educadores/as buscaram registrar o que havia de comum nas histórias de educandos e educandas e, a partir daí, encontramos o tema gerador e os eixos temáticos que possibilitarão a reflexão sobre o contexto em que os sujeitos da EJA vivem e no qual se inserem também os/as educadores/as dessa modalidade de ensino. [...]. O segundo caminho articulado ao primeiro, foi organizado no sentido de que os/as educadores/as possam desenvolver pesquisa em suas turmas, utilizando–se da metodologia de projetos. Cabe ressaltar que um projeto não é apenas um plano de trabalho ou um conjunto de atividades bem organizadas. Há muito mais na essência de um bom projeto. 4 Escola da rede municipal de educação, localizada no Bairro Água-Fria. Naquele momento, atendia crianças da Educação Infantil, do ensino fundamental e da Educação de Jovens e Adultos. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 58 Este foi um recorte de como se deu essa experiência em 2009, uma vez que pouco foram os achados para nos permitir reconstruir o início de uma trajetória que pensamos ser a mais indicada para o trabalho com a Educação de Jovens e Adultos. Uma experiência que gerou muitas expectativas por parte dos professores, pois eles não estavam sozinhos, mas podiam contar com a parceria de outras instituições, de outros sujeitos, como os gestores, coordenadores pedagógicos e alunos. p g g Durante todo o ano de 2009 foram ofertadas formações docentes sobre o que significava trabalhar com os temas geradores. De certa forma, havia o reconhecimento de que a educação necessitava dos sujeitos, professor, aluno, gestor, coordenador. Um trabalho árduo, mas que potencializava a educação libertadora. As formações constantes direcionadas pela Rede Municipal de Educação e o apoio da gestão escolar colaboraram na caminhada dos professores rumo a autonomia na elaboração e construção das suas práticas. Houve a redescoberta por parte dos alunos da sua importância e centralidade dentro do processo educativo. Um projeto novo baseado nos princípios da “democracia, da solidariedade e da esperança” (ESTÊVÃO, 2013, p. 24) que foi freado com as mudanças no campo político de Tracuateua a partir das eleições municipais. Refletimos que a política partidária tem mudado drasticamente os rumos da educação do município. Os discursos ensaiados por quem ocupa os cargos de gestão nas secretarias reside em não continuar os projetos de gestões anteriores, sob a alegação de quererem deixar sua marca, construir outra história. O problema disso, é que a história não pode ser apagada, pois a memória, sobretudo a coletiva, se encarrega de avivar aquilo que muitos querem deixar adormecido. Em 2015 e 2016 o projeto retorna para a Rede Municipal, dessa vez não apenas para a EJA, mas para todos os níveis e modalidades de educação. Orientado pelos mesmos professores que estiveram em 2009, o currículo via Tema Gerador se dividiu entre os críticos e os adeptos. Para este início foram realizadas formações intensas e periódicas com a equipe técnica da Secretaria Municipal de Educação. O objetivo dessas formações era aproximar os docentes e técnicos daquilo que seria a base de sua atuação, a epistemologia de Paulo Freire como subsídio teórico e prático na construção de um currículo que tomasse como ponto de partida, o lugar e a história dos sujeitos. 2 CURRÍCULO VIA TEMA GERADOR E A EXPERIÊNCIA DO MUNICÍPIO DE TRACUATEUA-PA Esta metodologia pretende possibilitar elementos teórico/ metodológicos aos/as educadores/as, ou seja, a forma que procedemos no levantamento dos Temas Geradores e na organização da programação deve servir de referência para suas práticas em suas turmas de escolarização. Não posso investigar o pensar dos outros, referido ao mundo, se não penso. Mas não penso autenticamente se os outros também não pensam. Simplesmente não posso pensar pelos outros nem para os outros, nem sem os outros (NEVES; MARCIEL, 2009. p. 4). Sobre as escolhas dos Temas Geradores, Neves e Marciel (2009) indicam que eles partiam das necessidades e não das vontades pessoais dos sujeitos do processo. Por isso, as necessidades compunham um patamar que precisavam ser entendidas teoricamente, para que o planejamento e o processo de tomada de decisão (escolhas) fossem coerentes com a realidade. As orientações eram de que a escolha do tema para trabalhar o bimestre fossem resultado da vontade do coletivo, tomando como referência os alunos, a comunidade e a sociedade. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Nós partirmos das experiências vividas acreditando que a formação dos professores, a ideia do planejamento ela tem que estar correlacionada ao processo de formação dos professores, onde o próprio texto que a gente trabalha é um texto que discute a categoria da ideia da formação no exercício. Nós trabalhamos com a visão de que o planejamento não pode ser algo estanque. Essa é uma das coisas que a gente pauta. (Professora Joana D’arc, docente da UFPA, comunicação oral, Secretaria Municipal de Educação, 08, fevereiro de 2018). As formações sempre focavam nas diretrizes desse currículo, incitando o diálogo com a realidade dos sujeitos. O diálogo possibilitaria o desenvolvimento de uma consciência crítica e transformadora. O texto de apoio utilizado para aprofundar as discussões teóricas foi uma tese de doutorado de Silva (2004), na qual o autor fundamenta as concepções deste currículo. O referido autor vai buscar em Freire (1992) e Dussel (2000) as bases epistemológicas de um currículo ético e crítico que parte das falas significativas no processo de problematização da realidade para trabalhar com práticas contextualizadas. Com isso, ele assume a importância do outro no processo de construção da educação. Freire (1987, p. 49) indica o caminho que devemos fazer para chegar a essas falas significativas: O que temos de fazer, na verdade, é propor ao povo, através de certas contradições básicas, sua situação existencial, concreta, presente, como problema que, por sua vez, DOSSIÊ AMAZÔNIA 59 o desafia e, assim, lhe exige resposta, não só no nível intelectual, mas no nível da ação. o desafia e, assim, lhe exige resposta, não só no nível intelectual, mas no nível da ação. As contradições presentes nas situações existenciais do povo, surgiria mediante a pesquisa sociocultural, a qual revelaria as situações significativas e conflituosas, uma vez que “é na realidade mediatizadora, na consciência que dela tenhamos educadores e povo, que iremos buscar o conteúdo programático da educação (FREIRE, 1987, p. 50). O conteúdo da educação seria aquele que surge das vozes do povo diante da realidade que vivem. Um conteúdo vivo, dinâmico e em construção. Nesta perspectiva, ele confronta com os modelos anti-dialógicos que chegam sem nenhum questionamento. Freire, educador que defende o caráter dialógico da educação, vem dizer que é pelo diálogo que se chega ao “universo temático do povo ou o conjunto de seus temas geradores” (FREIRE, 1987, p. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 50), como explicita a fala da professora Joana quando diz “Nós tínhamos um formato de diálogos com os professores e gestores e eles ficavam responsáveis em realizar as formações com os professores das escolas”. (Joana D’arc, docente da UFPA, comunicação oral, Secretaria Municipal de Educação, 08, fevereiro de 2018). O diálogo ocorria entre os sujeitos da escola sempre com a intenção de chegar aos temas de maior relevância para serem trabalhados. De 2015 a 2016 foram dois anos de debate, estudos e construções com o mesmo grupo que iniciou o projeto em 2009. Novamente, as mudanças no cenário político provocaram (re) laborações no campo curricular das escolas de Tracuateua. Desta vez, os temas geradores se restringem apenas a modalidade da Educação de Jovens e Adultos. A fala de uma coordenadora que esteve responsável pela EJA na Secretaria Municipal de Educação, reflete a preocupação em permanecer com o trabalho que vinha sendo desenvolvido em anos anteriores: [...] eu não queria que adotassem outro currículo, eu não queria que fosse feito de outra forma, eu queria que dessem continuidade a um trabalho que a gente já tinha construído no decorrer dos anos anteriores, essa era a minha maior preocupação (Esp. Danubia Ribeiro, técnica da SEMED, comunicação oral, Secretaria Municipal de Educação, 05, janeiro de 2018). Permanecer nessa perspectiva curricular na modalidade de EJA foi um desafio, uma vez que a rotatividade dos professores de um nível para outro ou mesmo a saída deles da função docente tem provocado quebras na dinâmica formativa e prática nas escolas. Há ainda, o fato de que nem todos os professores se alinham bem ao projeto por Tema Gerador, optando em seguir os conteúdos de ensino das matrizes curriculares. Diante disso, fica explícito que Tracuateua foi audaciosa em buscar construir um projeto coletivo de educação, no qual o foco eram os sujeitos e suas problemáticas. Uma educação para a mudança privilegiando a coparticipação, a dialogicidade, a humanização. Sem deixar de refletir nos desafios encontrados, vemos essa tentativa como uma possibilidade para alcançar graus mais elevados de democracia e autonomia. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 60 As bases epistemológicas que ajudaram na construção desse projeto nas escolas de Tracuateua, tomaram como ponto de partida a concepção de que a escola não pode servir como depósito de conhecimento, mas é preciso senso crítico para enfrentar os problemas da atualidade. Uma educação que problematiza as situações existenciais, tornando as pessoas sujeitos de sua história. Em diálogo permanente é possível chegar ao conteúdo da educação que emerge das vozes dos sujeitos nos seus lugares de vida e de cultura. Nesses termos, é imperativo para qualquer projeto de EJA o diálogo como prática de liberdade, de autonomia, de mudança, de esperança. A partir dos pressupostos freireanos, o município de Tracuateua tem buscado nesses anos em que o projeto esteve em vigor, construir uma educação problematizadora que fosse capaz de revelar a identidade dos seus sujeitos, agora vistos como protagonistas de história, da sua própria história. Apesar dos grandes desafios, é possível frisar que os ganhos são evidentes, ainda que não tenham contemplado todos, uma vez que para isso é preciso vontade, responsabilidade e compromisso político associado a uma boa dose de utopia. DUSSEL, Henrique. Ética da Libertação na idade da globalização e da exclusão. Petrópolis: Vozes, 2000. DUSSEL, Henrique. Ética da Libertação na idade da globalização e da exclusão. Petrópolis: Vozes, 2000. ESTÊBÃO, Carlos Vilar. A qualidade da educação: suas implicações na política e na gestão pedagógica. RBPAE – v.29, n. 1, p.15-26, 2013. Disponível em: http://seer.ufrgs.br/index.php/rbpae/article/view/42818. Acesso em: 12 ago. 2020. BRASIL, Ministério da Educação. INEP. Município- Resultado do Censo Escolar. 2017. BRASIL, Ministério da Educação. INEP. Município- Resultado do Censo Escolar. 2017. BRASIL, Ministério da Educação. INEP. Município- Resultado do Censo Escolar. 2017. 3 CONSIDERAÇÕES FINAIS Trazer para o centro da discussão a efetivação de experiências freireanas realizadas em um município da região bragantina não é um exercício fácil de realizar. A implantação do currículo via Tema Gerador não foi tão simples, mas muito promissor. Foi preciso ir ao encontro de vozes que desde o começo foram imprescindíveis na provocação sobre o ato de planejar, de pensar a escola e a educação. Necessário ainda, foi o encontro com as ideias de Paulo Freire enquanto base epistemológica na materialização de um currículo que considera os sujeitos como verdadeiros autores desse projeto. Fato que verbera o sentido mais autêntico da educação. Uma educação partilhada com os sujeitos que dela se fazem produto e processo. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 FREIRE, Paulo. Educação como prática da liberdade. 14ª ed. Rio de Janeiro: Paz e terra, 1983. FREIRE, Paulo. Educação como prática da liberdade. 14ª ed. Rio de Janeiro: Paz e terra, 1983. FREIRE; Paulo; SHOR; Ira. Medo e Ousadia: o cotidiano do professor. Trad. LOPES, Adriana. Rio de Janeiro: Paz e Terra, 1986. FREIRE, Paulo. Pedagogia do oprimido. 17ª. ed. Rio de Janeiro: Paz e Terra, 1987. FREIRE, Paulo. Pedagogia da Esperança. Rio de Janeiro: Paz e Terra, 1992. NEVES, Joana Dárc; MARCIEL, Rogerio. Os temas geradores na formação de educadoras e educadores na escolarização de jovens e adultos. Disponível em: http://pt.scribd.com/document/107190461/os-temas-geradores-na-formacao-de-educadoras-e- educadores-e-na-alfabetizacao-de-jovens-e-adultoS. Acesso em: 19 nov. 2019. RUSSO, Miguel Henrique. Trabalho e administração da escola: desenvolvimento e apropriação do sentido que assumem no processo de produção pedagógica. RBPAE – v.27, n.3, p. 361-588, set./dez. 2011. Disponível em: http://seer.ufrgs.br/rbpae/article/view/26416. Acesso em: 18 nov. 2019. SILVA, Antônio Fernando Gouvêa da. Construção do currículo na Perspectiva Popular crítica: das falas significativas às práticas contextualizadas. Tese (Doutorado em Educação) SILVA, Antônio Fernando Gouvêa da. Construção do currículo na Perspectiva Popular crítica: das falas significativas às práticas contextualizadas. Tese (Doutorado em Educação) Programa de pós-graduação em educação: currículo. Pontifícia Universidade Católica de São Paulo. São Paulo, 2004. Disponível em: http://websmed.portoalegre.rs.gov.br/escolas/quintana/tese_gouvea.pdf. Acesso em: 12 ago. 2020. rograma de pós-graduação em educação: currículo. Pontifícia Universidade Católica de S aulo. São Paulo, 2004. Disponível em: DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 61 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 63 REVISTA DO PROGRAMA DE P VOLUME IX – GRADUAÇ SABERES NA AMAZÔNIA 4 O Termo Amazônia Marajoara é entendido como o arquipélago da ilha do Marajó, localizado no estado do Pará, compreendido por 15 municípios do território. O recorte espacial é o município de Salvaterra, concebido da necessidade geográfica de entender os diferentes movimentos sociais, políticos, ideológicos, religiosos, econômicos e culturais que constituíram as diferentes visões acerca do Marajó. O termo produz uma representação dos amazônidas presentes no arquipélago do Marajó, como uma forma de reproduzir a identidade marajoara a partir dos diversos recortes que caracterizam a Amazônia (PACHECO, 2010). INTRODUÇÃO Este estudo analisa a disciplina de Língua Inglesa (LI) da Educação de Jovens e Adultos (EJA) na perspectiva da Educação Escolar Quilombola (EEQ) na comunidade quilombola de Vila União do município de Salvaterra, da Amazônia Marajoara4. Pesquisas recentes na área da educação brasileira apontam a necessidade de descortinar olhares sob as práticas socioeducativas para uma ação pedagógica inclusiva e mais próxima das identidades dos alunos da EJA (ARROYO, 2011; PENNYCOOK, 1998; RAJAGOPALAN, 2003). Por essa razão, propusemos a entender quais os desafios existentes no ensino de Língua Estrangeira (LE) – LI em uma turma de EJA do Ensino Fundamental - Anos Finais de uma escola quilombola do município de Salvaterra da Amazônia Marajoara. O ensino de LI no Brasil tem se tornado uma tarefa desafiadora para professores e alunos no âmbito da educação pública, sobretudo para a EJA. Em linhas gerais, pesquisas de British Council (2015) apontam que o ensino da LI na escola pública apresenta desafios estruturais e funcionais. Nesse interim, percebe-se que há falta de materiais didáticos voltados para a realidade dos educandos, ausência de um ambiente propício para a aprendizagem em LI, carga horária insuficiente para o ensino do idioma e professores com baixa proficiência em LI, e até mesmo com escasso conhecimento linguístico do idioma, e tais questões têm implicado indiretamente na qualidade do ensino aprendizagem de LI na escola pública. p q p g p Na EJA essa realidade atravessa tempos e espaços que dificultam a qualidade do ensino da LI para jovens e adultos. O ensino do idioma apresenta-se bastante complexo e desafiador para muitos estudantes da EJA, uma vez que boa parte deles não são alfabetizados em sua língua materna, dificultando o aprendizado na LI. Além disso, a maioria dos professores de LI que atuam na EJA não possuem formação voltada para o ensino do idioma nessa modalidade de ensino, e são habituados a ensinarem o idioma a partir de suas crenças pedagógicas (BRITISH COUNCIL, 2015; ARROYO, 2011). p g g O ensino da LI é indispensável para o desenvolvimento de qualquer educando no mundo globalizado, caracterizando uma condição essencial para a inclusão nas sociedades modernas, definida por uma sociedade assentada por diversas identidades sociais. Na Amazônia Marajoara, essa diversidade é marcada pela presença de comunidades indígenas, ribeirinhas, camponesas e quilombolas, nas quais precisam compreender a importância do aprendizado do idioma, para assim terem mais chances de inclusão nesse mundo globalizado. UNIVERSIDADE FEDERAL DO PARÁ – CAMPUS BRAGANÇA NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 64 field to reach the quality of the teaching of EL in the EEQ proposal. The need for continuing education of IL teachers for ethnic-racial relations is na emergency need, as the data indicated that the challenges are mostly associated with the pedagogical practices of the EL teacher. Keywords: School Education. Quilombola. EJA. English Language. Keywords: School Education. Quilombola. EJA. English Language. Data de submissão: 29.09.2020 Data de aprovação: 26.12.2020 Data de submissão: 29.09.2020 Data de aprovação: 26.12.2020 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 65 conforme estabelece as Diretrizes Curriculares para a Educação Escolar Quilombola (DCNEEQ). As comunidades quilombolas possuem um saber peculiar, desenvolvem dinâmicas temporais de vinculação a um espaço físico que se torna território coletivo, resultantes das múltiplas formas de relações integradas à natureza, constituído por conhecimentos gerados e transmitidos pela tradição (BRANDÃO, 2010). g p ç Neste estudo, a configuração da EJA na EEQ é fundamentada em uma memória coletiva que permeia tempos e espaços, e diversos elementos que caracterizem a cultura quilombola, as línguas remanescentes, práticas culturais, produção de trabalho, acervos e repertórios orais. As tradições e demais elementos que configuram o patrimonial cultural das comunidades quilombolas do Brasil, em especial para sua territorialidade. (BRASIL, 2012). Nesse bojo, apontamos a necessidade de dialogar e analisar as interfaces da LI na EJA, na perspectiva da EEQ, saber como os alunos quilombolas marajoaras da EJA constroem seus conhecimentos a partir da relação de cultura, linguagem e sociedade. A EJA é uma modalidade de ensino que vem se consolidando no Brasil há alguns anos, amparada por lei e voltada para pessoas que não tiveram acesso ao ensino regular na idade apropriada, com foco no processo de alfabetização, reingresso e permanência do aluno nas salas de aulas. Entretanto, esta modalidade de ensino acarreta diversas implicações que impedem sua total eficiência no ensino, como: idade, nível de conhecimento, preconceito, exclusão e estereótipos (grifo nosso) (BRASIL, 2000). Nesse sentido, o ensino da LI nesta modalidade para a EEQ, tem um papel importante na formação interdisciplinar e intelectual dos alunos quilombolas da EJA, quando contribui para a construção da cidadania e da sua própria identidade, favorecendo a participação na sociedade brasileira, sobretudo no mundo globalizado. A EJA na EEQ da Amazônia Marajoara, apresenta grandes desafios em seu processo de escolarização para os alunos quilombolas. No município de Salvaterra, esse cenário apresenta fortes implicações no aprendizado da LI para os alunos da EJA. Este estudo buscou dialogar sobre o ensino da LI na EJA da Escola Municipal de Ensino Infantil e Fundamental Quilombola Maria Lúcia Ledo Carvalho, da comunidade quilombola de Vila União do município de Salvaterra-Marajó-PA, no sentindo de compreender as diferentes práticas pedagógicas do professor de LI da EJA no âmbito da EEQ, considerando DCNEEQ, Estrutura Curricular Pedagógica da EEQ da Rede Municipal de Salvaterra e as Diretrizes Curriculares Nacionais para a Educação de Jovens e Adultos (DCNEJA). DOSSIÊ AMAZÔNIA Desta maneira, apresentamos na primeira seção deste estudo, o percurso metodológico, na segunda seção abordamos o contexto histórico da identidade quilombola marajoara, e em seguida discutimos a concepção da EJA na EEQ, e o contexto da EJA a partir de alunos quilombolas da Amazônia Marajoara. Ao longo do trabalho, discorremos sobre o ensino da LI na EJA e por final apresentamos nossas considerações finais. 5 É também denominada de tradição interpretativista ou hermenêutica-dialética (BORTONI-RICARDO, 2008). INTRODUÇÃO Na Amazônia Marajoara os quilombos são entendidos como povos ou comunidades tradicionais, “[...] grupos culturalmente diferenciados que se reconhecem como tais; [...] possuidores de formas próprias de organização social; detentores de conhecimento, tecnologias, inovações e práticas geradas e transmitidas pela tradição” (BRASIL, 2012, p. 4), 1 PERCURSO METODOLÓGICO Considerando o objetivo da nossa pesquisa e suas características peculiares, buscou-se a compreensão dos significados produzidos pelos sujeitos: os alunos e professores de LI no contexto social em que atuam. Optamos pela abordagem qualitativa de cunho interpretativista5, a qual visa analisar os significados que permeiam o ensino da LI na EJA da EEQ da Amazônia Marajoara. A abordagem qualitativa é considerada interpretativa e procura descrever os “[...] padrões característicos da vida dos participantes” (MOITA LOPES, 1994, p. 23-24). A abordagem qualitativa busca “[...] a compreensão da realidade humana vivida socialmente” a NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 66 partir dela, trabalha-se com a vivência, a experiência, a compreensão dos resultados da ação humana objetiva, do “[...] ponto de vista onde as práticas e as coisas são inseparáveis” (MINAYO, 1994, p. 23-24). partir dela, trabalha-se com a vivência, a experiência, a compreensão dos resultados da ação humana objetiva, do “[...] ponto de vista onde as práticas e as coisas são inseparáveis” (MINAYO, 1994, p. 23-24). O paradigma interpretativista pode auxiliar de forma significativa no desenvolvimento de uma compreensão das escolhas e decisões dos participantes em seu tratamento do ensino e aprendizagem em LI. Para Bortoni-Ricardo (2008), esta perspectiva surgiu como uma alternativa para interpretar ações e significados que as pessoas atribuem a tais ações na vida social. Utilizamos da pesquisa bibliográfica como ponto de partida para mapear a produção intelectual da área e verificamos que a pesquisa qualitativa ajuda na compreensão e interpretações dos fenômenos sociais inseridos no contexto do ensino de LI da EJA na EEQ. Em face disso, podemos reiterar que a pesquisa qualitativa-interpretativista é a mais adequada para este tipo de estudo, uma vez que ela busca descrever o que os sujeitos fazem em seus contextos, e como percebem as suas ações, e como constroem a tecitura do seu mundo social. O estudo foi realizado na Escola Municipal de Ensino Infantil e Fundamental Quilombola Maria Lúcia Ledo Carvalho, situada na comunidade quilombola de Vila União, com aproximadamente 115 km de distância do centro urbano da cidade de Salvaterra, Marajó- Pará. 1 PERCURSO METODOLÓGICO Na pesquisa de campo, foram realizadas algumas visitas ao lócus da pesquisa (Figura 1), a fim de conhecer a realidade escolar e entender o processo socioeducativo dos alunos quilombolas da EJA, além disso foram observadas algumas aulas de LI na EJA, para saber como é trabalhada a cultura quilombola marajoara a partir de seu contexto. Figura 1 – Escola Municipal de Ensino Infantil e Fundamental Maria Lúcia Ledo Carvalho Fonte: Acervo Pessoal dos Pesquisadores, 2020 O Corpus de Análise deste estudo se constituiu na observação e na aplicação de questionário semiestruturado, que foram aplicados com (=1) professora de LI da EJA e com (=18) alunos da 3ª Etapa da EJA (6º e 7º anos) com um universo de alunos pertencentes a uma faixa etária de 23 a 43 anos de idade, sendo uma turma composta por 11 homens e 7 mulheres. Com o objetivo de analisar as práticas pedagógicas da professora de LI acerca do ensino da LI na EJA da EEQ, os questionários permitiram compreender se as aulas de LI eram articuladas com a proposta da EJA da EEQ na perspectiva das relações étnico-raciais, e perceber como se configura o ensino da LI na EJA da EEQ. Para análise dos dados, consideraram-se as respostas mais expressivas dos alunos e da professora de LI tendo como escopo as representações do ensino da LI na EJA da EEQ, com vista à construção positiva da identidade quilombola marajoara. Figura 1 – Escola Municipal de Ensino Infantil e Fundamental Maria Lúcia Ledo Carvalho Fonte: Acervo Pessoal dos Pesquisadores, 2020 Figura 1 – Escola Municipal de Ensino Infantil e Fundamental Maria Lúcia Ledo Carvalho Fonte: Acervo Pessoal dos Pesquisadores, 2020 Figura 1 – Escola Municipal de Ensino Infantil e Fundamental Maria Lúcia Ledo Carvalho Fonte: Acervo Pessoal dos Pesquisadores, 2020 O Corpus de Análise deste estudo se constituiu na observação e na aplicação de questionário semiestruturado, que foram aplicados com (=1) professora de LI da EJA e com (=18) alunos da 3ª Etapa da EJA (6º e 7º anos) com um universo de alunos pertencentes a uma faixa etária de 23 a 43 anos de idade, sendo uma turma composta por 11 homens e 7 mulheres. 2 NAS TRILHAS DOS QUILOMBOS DE SALVATERRA: DA RESISTÊNCIA A SOBREVIVÊNCIA DOS JOVENS QUILOMBOLAS DA AMAZÔNIA MARAJOARA A presença de negros e africanos nas regiões brasileiras, estiveram sempre marcadas pela exploração da mão de obra barata e pela vida compulsória nas relações econômicas no período colonial no Brasil. Na Amazônia Marajoara, esse percurso iniciou-se durante o período de criação da Companhia Geral do Comércio do Grão-Pará e do Maranhão em 1755. A presença de negros e africanos na região se intensificou cada vez mais com o aumento da produção agrícola na região Norte e, assim, os negros e africanos foram trazidos compulsoriamente da sua terra natal para a região amazônica (LISBOA, 2012). Grande parte dessa população formada de negros e africanos, e até indígenas foram direcionadas para a Amazônia Marajoara, com a finalidade de trabalhar nas lavouras, fazendas e criatórios, que gradativamente se organizavam para suas atividades econômicas. A concentração maior dos negros e africanos foi na região do criatório, formada pelas fazendas de gado e búfalos, localizadas nos municípios de Soure, Salvaterra, Cachoeira do Arari, Santa Cruz do Arari e Chaves (LISBOA, 2012). Ao longo desse período, evidenciou-se uma relação subalterna e compulsória em que os negros e africanos viviam para atender as atividades econômicas impostas pelas autoridades da Coroa Portuguesa. Essa relação estabelecia uma ordem e orientação para que os oficiais acabassem com os antigos mocambos e aldeias (DIAS, 2016). Quanto à questão subalterna e compulsória de negros e africanos nessa porção da região da Amazônia Marajoara, Baena (2004) considera que haviam 31 negros e africanos em Salvaterra na década de 1830. O autor também apresenta dados historiográficos do século XVIII que estão vinculados à atual formação de territórios quilombolas nessa região. Baena descreve a “roça São Macário”, no século XVIII, “aderente” à fazenda São Lourenço ou Paracauari que pertenceu aos padres mercedários e, no ano de 1794, foi-lhes sequestrada. Essa informação é um elo importante dos povoados negros de Salvaterra, pois nas terras dessa ordem surgem as terras de Santa Mercês, ocupadas pelos “pretos velhos” de Bacabal, do Bairro Alto. (ACEVEDO, 2009, p. 214). Nesse interim, Salvaterra é considerada a cidade da Amazônia Marajoara que apresenta maior número de comunidades quilombolas. De acordo com os dados da Fundação Cultural dos Palmares (FCP), o município de Salvaterra apresenta 17 comunidades quilombolas: Bacabal, Santa Luzia, Rosário, Campina, Vila União, Valentim, Boa Vista, Deus Ajude, Bairro Alto, Caldeirão, Pau Furado, São Benedito Salvá, Mangueiras, Providencia, Siricarí. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 67 Portanto, foram analisadas e consideradas no questionário, as assertivas mais expressivas, tendo como parâmetro os significados da LI da EJA na EEQ, pela professora de LI e pelos alunos quilombolas. As demais assertivas foram comentadas no decorrer da análise, conforme a relação existente entre o ensino da LI e a EEQ e os seus desdobramentos na EJA. 1 PERCURSO METODOLÓGICO Com o objetivo de analisar as práticas pedagógicas da professora de LI acerca do ensino da LI na EJA da EEQ, os questionários permitiram compreender se as aulas de LI eram articuladas com a proposta da EJA da EEQ na perspectiva das relações étnico-raciais, e perceber como se configura o ensino da LI na EJA da EEQ. Para análise dos dados, consideraram-se as respostas mais expressivas dos alunos e da professora de LI tendo como escopo as representações do ensino da LI na EJA da EEQ, com vista à construção positiva da identidade quilombola marajoara. O Corpus de Análise deste estudo se constituiu na observação e na aplicação de questionário semiestruturado, que foram aplicados com (=1) professora de LI da EJA e com (=18) alunos da 3ª Etapa da EJA (6º e 7º anos) com um universo de alunos pertencentes a uma faixa etária de 23 a 43 anos de idade, sendo uma turma composta por 11 homens e 7 mulheres. Com o objetivo de analisar as práticas pedagógicas da professora de LI acerca do ensino da LI na EJA da EEQ, os questionários permitiram compreender se as aulas de LI eram articuladas com a proposta da EJA da EEQ na perspectiva das relações étnico-raciais, e perceber como se configura o ensino da LI na EJA da EEQ. Para análise dos dados, consideraram-se as respostas mais expressivas dos alunos e da professora de LI tendo como escopo as representações do ensino da LI na EJA da EEQ, com vista à construção positiva da identidade quilombola marajoara. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 68 como coletiva e legítima, associada pelo vínculo com a terra, modo de vida, produção e cultura. Essa relação buscou ampliar as políticas públicas de ações afirmativas, como saúde e educação. Segundo Acevedo (2009), o município é eminentemente quilombola: como coletiva e legítima, associada pelo vínculo com a terra, modo de vida, produção e cultura. Essa relação buscou ampliar as políticas públicas de ações afirmativas, como saúde e educação. Segundo Acevedo (2009), o município é eminentemente quilombola: Quilombolas de Salvaterra produziram um processo de territorialização que experimenta mudanças devido à existência de condições limitantes à sua existência, aos modos de produzir e de organizar a vida social. As cercas construídas pelos fazendeiros, cujo primeiro significado é de identificação e de relação com um “dono, proprietário”, privatizam os recursos (igarapés, lagos) e estabelecem impedimento, coerção, restrição de deslocamentos, de gozo da liberdade de movimentar-se livremente no território. (ACEVEDO, 2009, p. 215). Segundo os dados do Centro de Estudos e Defesa do Negro no Pará (CEDENPA), Instituto de Colonização e Reforma Agrária (INCRA), Instituto de Terras do Pará (INTERPA) e Coordenação das Associações Remanescentes de Quilombo do Pará (MALUNGU), as comunidades quilombolas de Salvaterra começaram a requerer a titulação e certificação de suas terras desde 2000. O município de Salvaterra é eminentemente quilombola, e suas comunidades estão concentradas no espaço rural, e foi a partir de 2004 que as comunidades começaram a ser certificadas perante aos Órgãos Competentes. ç p g p A partir de vários movimentos de reivindicações para o reconhecimento de suas terras, foram que as comunidades quilombolas de Salvaterra começaram a se organizar e se estruturar de maneira mais sólida. Doravante, as comunidades começaram a lutar por seus direitos, dentre eles o de construir uma sociedade que respeitasse sua identidade e cultura, surge então, o sentimento de construção de escolas nas comunidades quilombolas de Salvaterra. A EEQ foi implantada no município de Salvaterra em 2013, através do Parecer nº 392/13 e a Resolução nº 418/213, emitido pelo Conselho Estadual de Educação do Estado do Pará (CEE/PA), com a implementação das Estruturas Curriculares para a EEQ do Ensino Fundamental no município de Salvaterra. Art. 9º: A Educação Escolar Quilombola compreende: I – escolas quilombolas II – escolas que atendem estudantes oriundos de territórios quilombolas. I – escolas quilombolas 2 NAS TRILHAS DOS QUILOMBOS DE SALVATERRA: DA RESISTÊNCIA A SOBREVIVÊNCIA DOS JOVENS QUILOMBOLAS DA AMAZÔNIA MARAJOARA Segundo Arruti (2017), o conceito de quilombo é amplo e complexo, e permeia por discussões referentes ao uso comum da terra e da identidade étnica das populações quilombolas. Nesse sentido, o conceito de quilombo neste estudo remete à variação de identidade quilombola, ancestralidade, a vinculação com a terra ligada a coletividade do grupo social. Para Almeida (2005), esse sentido possui uma relação com áreas doadas, repassadas ou conquistadas por um grupo ou uma pessoa de origem negra que possua documentação legalizada. Esse percurso referente à definição de quilombo foi o que demarcou os territórios das comunidades quilombolas de Salvaterra, e caracterizou a identidade quilombola marajoara NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 A necessidade de implantar a EEQ no município surgiu a partir das reivindicações das lideranças das comunidades quilombolas de Salvaterra, que ora foram representados pelo Movimento Quilombola de Salvaterra (MQS), que lutaram pela construção e/ou readaptação de escolas em Comunidades Remanescentes Quilombolas (CQR), e que atendessem as peculiaridades da população quilombola marajoara, reafirmando o compromisso e implementação da Lei nº 10.639/2003. A EEQ possui um contexto histórico de institucionalização sob a conquista do Movimento Negro e Quilombola no Brasil, que possibilitou o acesso ao ensino básico da população negra, como reconhecimento do direito à educação. Uma luta que foi enfrentada e conquistada como proposta desde o processo de implementação da Lei nº. 10.639/03, sendo referenciada no Plano Nacional de Implementação das Diretrizes Curriculares Nacionais para a Educação das Relações Étnico-raciais e para o Ensino de História e Cultura Afro-brasileira e Africana. Diante de um panorama de reivindicações do Movimento Negro e Quilombola acerca do direito a educação, diversas discussões e atividades foram organizadas pelo Grupo de Trabalho para Educação Quilombola, junto ao Conselho Nacional de Educação (CNE). Nesse sentido, no ano de 2012, as DCNEEQ’s, foram elaboradas e aprovadas pela Câmara de Educação Básica do Conselho Nacional de Educação. Dessa forma, o ensino é destinado às populações quilombolas como política pública educacional é concebida como: DOSSIÊ AMAZÔNIA 69 Parágrafo Único: Entende-se por escola quilombola aquela localizada em território quilombola. (BRASIL, 2012, p. 18). Parágrafo Único: Entende-se por escola quilombola aquela localizada em território quilombola. (BRASIL, 2012, p. 18). Ao compreender que a EEQ concebe o território como uma categoria principal na modalidade de ensino, esta define como essa política pública educacional deve ser adotada na educação básica. Nesse sentido, a definição de quilombo no Brasil, transita por diferentes momentos e processos históricos de ancestralidade das populações negras no país, buscando não recair sobre uma conotação que naturaliza esse termo somente ao processo de exploração dos negros em séculos passados. De acordo com a DCNEEQ, a EJA é uma modalidade legalmente amparada na EEQ, com uma proposta pedagógica flexível, com finalidades e funções específicas, levando em consideração os conhecimentos das experiências de vida dos jovens e adultos quilombolas, atendendo às realidades socioculturais e interesses das comunidades quilombolas. A proposta pedagógica deve ser contextualizada, levando em consideração seus tempos e espaços, às suas questões históricas, sociais, políticas, culturais e econômicas das comunidades quilombolas (BRASIL, 2012). As DCNEEQ’s, no art. nº. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 23 preconizam que a oferta da EJA no Ensino Fundamental da EEQ, deva substituir a oferta regular dessa etapa da Educação Básica na EEQ, independentemente da idade: § 4º Na Educação Escolar Quilombola, as propostas educativas de EJA, numa perspectiva de formação ampla, devem favorecer o desenvolvimento de uma Educação Profissional que possibilite aos jovens, adultos e idosos quilombolas atuar nas atividades socioeconômicas e culturais de suas comunidades com vistas ao fortalecimento do protagonismo quilombola e da sustentabilidade de seus territórios. (BRASIL, 2012, p. 11). § 4º Na Educação Escolar Quilombola, as propostas educativas de EJA, numa perspectiva de formação ampla, devem favorecer o desenvolvimento de uma Educação Profissional que possibilite aos jovens, adultos e idosos quilombolas atuar nas atividades socioeconômicas e culturais de suas comunidades com vistas ao fortalecimento do protagonismo quilombola e da sustentabilidade de seus territórios. (BRASIL, 2012, p. 11). A EJA na EEQ, portanto, vai além dos sujeitos tradicionalmente compreendidos como adultos e fora da escola, estabelecendo diferentes conexões e trajetórias daquela que, por algum motivo, foi estabelecida e considerada como “padrão” ou norma na sociedade, trazendo em seu escopo, também, grupos sociais que possuem cultura e produção de conhecimentos peculiares, como indígenas, ribeirinhos, camponeses, imigrantes entre outros. De uma forma ou de outra, a EJA da EEQ se configura com identidades que são socialmente ignoradas, silenciadas e invisibilizadas, tidas como irregulares indesejáveis e fora do padrão, que sofrem tentativas de “normalização” (SKLIAR, 2006, p. 23), e apagamento quando silenciam suas características peculiares em prol de um padrão assumido como único. A Constituição Federal Brasileira de 1988, declarou a obrigatoriedade e o acesso à educação a toda a população brasileira. Nesse sentido, 3 EDUCAÇÃO DE JOVENS E ADULTOS E A EDUCAÇÃO ESCOLAR QUILOMBOLA A EJA passou a ser reconhecida em vários países devido às conferências organizadas pela a Organização das Nações Unidas para a Educação, Ciência e Cultura (UNESCO) nos anos 1940. A partir de então, surgiu no Brasil um movimento de mobilização nacional, no sentido de diagnosticar metas e ações para a EJA (SCHNEIDER; FONSECA, 2013). Contudo, a educação formal e escolar de jovens e adultos só começou a ter condições reais de existência e execução como política pública a partir de 1990 (HADDAD; DI PIERRO, 2000), sendo solidificada e reconhecida como modalidade com a criação da Lei de Diretrizes e Bases da Educação (LDB nº. 9.394/96). A Constituição Federal Brasileira de 1988, declarou a obrigatoriedade e o acesso à educação a toda a população brasileira. Nesse sentido, 70 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 [...] educação é direito de todos e dever do Estado e da família, será promovida e incentivada com a colaboração da sociedade, visando ao pleno desenvolvimento da pessoa, seu preparo para o exercício da cidadania e sua qualificação para o trabalho. (BRASIL, 1988, p. 63). Dessa maneira, todos os cidadãos brasileiros têm direito a educação pública, independentemente de suas desigualdades sociais. O Estado é obrigado a ofertar e ampliar vagas para todos os níveis da educação básica, incluindo a EJA (BRASIL, 2000). A LDB nº. 9.394/96 é o documento basilar que ampara legalmente as diretrizes para a EJA na educação brasileira. Nesse sentido, o art. nº. 37, da Lei nº. 9.394/96, considera que “A educação de jovens e adultos será destinada àqueles que não tiveram acesso ou continuidade de estudos no ensino fundamental e médio na idade própria” (BRASIL, 1996) Destarte, Arroyo (2011), adensa que para atender os processos socioeducativos a EJA não se pode tomar apenas os documentos oficiais e as políticas públicas que abalizam e orientam essa modalidade de ensino, mas compreender como os sujeitos se constituem e se organizam: A Educação de Jovens e Adultos tem de partir, para sua configuração como um campo específico, da especificidade dos sujeitos concretos, históricos que vivenciam esses tempos da vida – juventude e vida adulta – e da especificidade dos sujeitos concretos históricos que vivenciam esses tempos. (ARROYO, 2011, p. 22). A EJA possui uma longa trajetória no Brasil, galgada de lutas e enfrentamos em face das grandes desigualdades sociais que assolam a sociedade brasileira. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 71 educação, visto que o preconceito e discriminação difundidos de maneira (in)consciente, são entendidos como uma barbárie, caracterizando uma violência simbólica, e que por sua vez deve ser combatido com uma intervenção pedagógica. A presença de alunos quilombolas nas salas de aula nessa modalidade de ensino, não tem sido suficiente para a garantia e realização de um trabalho pedagógico de qualidade. Ensinar na EJA da EEQ exige a aceitação do novo e o abandono de qualquer forma de preconceito e discriminação. O ensino da LI nessa modalidade de ensino é considerado um sistema muito complexo, por entender que a fase da vida dos alunos exige da EJA demandas educativas específicas, características diferentes de aprendizado, práticas adequadas de trabalho e representações distintas acerca da idade escolar. O professor de LI da EJA na EEQ deve (des)construir seus conceitos e pensamentos no seu cotidiano escolar, considerando as pluralidades de seu alunado. Diante disso, o educador de LI da EJA deve oferecer um ensino que valorize os saberes, as tradições e o patrimônio cultural das comunidades quilombolas e fortalecer a implementação da Lei nº 10.639/2003 sobre o ensino das relações étnico-raciais na educação brasileira. Além dos aspectos apontados até aqui, a relação de ensinar e aprender a LI na EJA perpassa o reconhecimento de múltiplos saberes e a relação dialógica de Freire entre “educador-educando” e “educando-educador” que trazem saberes diversos para dialogar (FREIRE, 1987, p. 46) e aprender um com o outro. Na próxima seção discorreremos sobre o contexto em que se insere a EJA da Amazônia Marajoara, sob as luzes da EEQ. 3 EDUCAÇÃO DE JOVENS E ADULTOS E A EDUCAÇÃO ESCOLAR QUILOMBOLA “[...] a história da EJA transitou à margem da construção do Sistema Escolar: campanhas, movimentos sociais, Organizações Não Governamentais – ONG’s, Igrejas, Sindicatos e Voluntários” (ARROYO, 2011, p. 43). No âmbito das políticas públicas, a EJA passou por diversas transições e tensões, até chegarmos aos dias atuais. Arroyo (2011, p. 19) já asseverava que este é “[...] um campo ainda não consolidado nas áreas de pesquisa, políticas públicas e diretrizes educacionais, da formação de educadores e intervenções pedagógicas”. É É preciso considerar neste estudo que os alunos quilombolas marajoaras da EJA, especificamente das escolas quilombolas de Salvaterra, possuem vivências, experiências e conhecimentos que os diferem dos alunos da educação regular. Entretanto, é indispensável descortinar os olhos e focar nos desafios existentes quando se almeja uma educação de qualidade no âmbito da Amazônia Marajoara. Sendo necessário proporcionar reflexões sobre os desafios da EJA na EEQ no campo do ensino da LI, a fim de que possam avançar gradualmente no mundo do conhecimento e do trabalho, respeitando suas especificidades, onde a escola e os professores passem a refletir ainda mais sobre o ensino da LI para estes alunos tanto na vida escolar quanto na vida social. A EJA atende principalmente a população mais carente da sociedade brasileira, que se viu durante muito tempo excluída do processo educacional por múltiplas causas. Diante de uma realidade marcada por desigualdades sociais (BRASIL, 2000), as questões étnico-raciais e a EJA, nunca andaram separadas, percebemos que os negros sempre estiveram presentes nessa modalidade de ensino, especialmente por fazer parte das populações subalternas, silenciadas e negligenciadas, neste caso das comunidades quilombolas que por várias vezes foram excluídas do processo educacional. Pensar a realidade da EJA é pensar a realidade dessas pessoas excluídas e marginalizadas pela sociedade e pela educação, pois desde quando a EJA se constituiu, os alunos jovens e adultos sempre foram considerados pobres, desempregados e excluídos (ARROYO, 2011). Os métodos de combate às desigualdades sociais na EJA devem ter lugar priorizado nos seus processos educativos, principalmente sob o olhar dos profissionais da NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 72 De acordo com os documentos curriculares legais da educação brasileira, o ensino da LI deve priorizar um aprendizado em multiletramentos e competências comunicativas. No município de Salvaterra, os documentos curriculares que abalizam a EEQ no âmbito do ensino da LI, tem uma proposta curricular dividida em quatro eixos em sua redação, e que devem ser aplicados, articulados e trabalhados a todos os conteúdos dos diversos componentes curriculares elencados na matriz curricular, são eles: Memória e Cultura; Saberes e Tecnologia; Relações Ambientais; Promoção e Igualdade Racial e de Gênero. (SALVATERRA, 2017). O documento orienta ainda que os conteúdos devem contemplar a amplitude da diversidade cultural das comunidades quilombolas e da sociedade afro- brasileira. Nesse sentido, o documento apresenta uma perspectiva de trabalho galgada no caráter interdisciplinar, no qual constitui o campo de estudos teóricos dessa proposta curricular. Para isso, discute aspectos não somente culturais, mas políticos, econômicos, geográficos, pedagógicos e éticos, como conteúdos inseridos à pluralidade cultural, necessários para a construção de uma identidade nacional. O documento orienta também a seleção dos conteúdos que devem ser articulados na proposta da EEQ, buscando contemplar a amplitude da diversidade cultural das comunidades quilombolas e da sociedade afro-brasileira. Segundo o documento, o ensino da LI faz parte juntamente com os conteúdos de Português e Literatura: 1- Textos Leituras, interpretação e produções diversas; concursos de frases, poesias, músicas, paródias; 1.1 Contadores de histórias locais (narrativas, contos e causos); 1.2 Provérbios populares; 1.3 Poetas, poesias, e contos, causos e lendas; 1.4 Falares africanos x língua colonizadora; 1.5 Os países e os idiomas falados por eles; 1.6 As influências estrangeiras. (SALVATERRA, 2017, p. 3). 1- Textos Leituras, interpretação e produções diversas; concursos de frases, poesias, músicas, paródias; 1.1 Contadores de histórias locais (narrativas, contos e causos); 1.2 Provérbios populares; 1.3 Poetas, poesias, e contos, causos e lendas; De acordo com a proposta do ensino da LI na EEQ, a ideia apresentada descortina a imagem que temos sobre o ensino da LI difundida na educação básica, sobre imperialismo linguístico cultural estadunidense e/ou britânico no currículo escolar, uma vez que o ensino da LI sustenta um processo de expansão ideológica e de dominação e hegemonia no mundo. Rajagopalan (2003, p. 4 O ENSINO DA LÍNGUA INGLESA NA PERSPECTIVA DA EDUCAÇÃO ESCOLAR QUILOMBOLA EM SALVATERRA-PA Diante das reflexões apresentadas até aqui, é emergente e relevante compreender como as práticas pedagógicas para o ensino da LI no âmbito da EJA da EEQ são desenvolvidas nas comunidades quilombolas da Amazônia Marajoara. De acordo com as DCNEEQ, a proposta da LI na EJA da EEQ deve ser de natureza interdisciplinar, envolvendo os saberes tradicionais das comunidades quilombolas. Esses saberes devem valorizar identidade, ancestralidade, memória, religiosidade e os saberes culturais a partir de uma matriz africana para que, assim, o ensino da LI atinja as perspectivas interculturais no âmbito da EEQ. O processo de ensino aprendizagem em LI devem contemplar conteúdos que levem a refletir a realidade local quilombola, de modo em que os alunos quilombolas os internalizem criticamente a partir da sua realidade social, imbuído de fortalecer sua própria identidade. É É importante compreendermos que o contexto histórico da Língua Estrangeira (LE) na educação brasileira sofreu uma perda gradativa para o ensino da LI na educação básica, na qual houve pouca regulamentação curricular, e que ocasionou uma série de desafios para o ensino do idioma no Brasil e, consequentemente, esses desdobramentos inviabilizaram a qualidade do ensino da LI na escola pública. Todavia, em 2017, através da Lei nº 13.415/2017, a LI tornou-se a LE obrigatória a partir do 6º ano do Ensino Fundamental. Doravante, em 2018 foi homologada a Base Nacional Curricular Comum (BNCC) para o Ensino Fundamental, um documento de natureza normativa com parâmetros e diretrizes para a educação brasileira, e a partir de 2020 esse novo documento curricular passou a ser implementado na educação básica. No âmbito da LI, a BNCC prescreve em suas diretrizes um ensino galgado na globalização, possibilitando ao aluno o acesso aos saberes linguísticos necessários para o seu desenvolvimento crítico e intelectual. O documento considera a LI como língua franca, e é concebida como multiletramento, possibilitando o aluno de circular e interagir em diferentes semioses e linguagens verbais, desenvolvendo a competência comunicativa em LI por meio do seu engajamento discursivo (BRASIL, 2017). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 73 neoliberal, de modo a favorecer a coesão social dos grupos quilombolas historicamente (in)visibilizados e silenciados. A LI como instrumento de prática social, é abordada pelo seu histórico hegemônico, sobreposta na cultura branca (britânica e estadunidense), é um instrumento ideológico nas relações de poder no mundo globalizado. Dessa forma, o ensino da LI na EJA da EEQ deve descortinar e incorporar outros planos da diversidade cultural apresentado pelo idioma, a partir da cultura afro-anglófona6. Dessa forma, o ensino da LI na EJA da EEQ tem um papel de descortinar o cânone da supremacia das culturas hegemônicas anglófonas (britânicas e estadunidenses), diluídas e legitimadas no currículo e nas práticas pedagógicas dos professores de LI. Para isso, realizamos na próxima seção um recorte para compreendermos como a LI é trabalhada e articulada na EJA da EEQ em Salvaterra, sobretudo nos desdobramentos da identidade quilombola marajoara dos alunos EJA em seu ambiente escolar. 6 Territórios e nações que têm o inglês como língua materna no continente Africano. Seus principais núcleos encontram-se: África do Sul, Gâmbia, Libéria, Serra Leoa, Zâmbia, Zimbábue. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 17) considera que “o ensino de língua de maneira bem geral, em especial de línguas estrangeiras, faz parte de uma política linguística em vigor num determinado país”, devendo o ensino da LI ir para além do que preconiza os estudos linguísticos, sucumbindo aspectos sociopolíticos e coordenadas geográficas, nas quais influenciam indiretamente o comportamento linguístico do cidadão quanto políticas públicas adotadas por seus Governos. Nesse sentido, evidencia-se o panorama histórico e hegemônico do ensino da LI, descortinando seus mecanismos de legitimação e descentralização das culturas consideradas hegemônicas nas de aulas LI (cultura britânica e estadunidense), rompendo com os paradigmas tradicionais legitimados no currículo escolar. Essa ruptura, abre caminhos para uma perspectiva intercultural no ensino da LI, na qual valorize a diversidade cultural deste idioma, reconhecendo a heterogeneidade e outras interfaces do uso da LI de outros países anglófonos, nesse caso de afro-anglófonos para o ensino da LI na EEQ. Dentro do contexto e da configuração da EEQ, o ensino da LI deve apresentar interfaces que contemplem uma matriz africana e que possibilite (re)pensar uma prática pedagógica de ensino antirracista e inclusiva. O professor de LI da EJA da EEQ deve pensar em uma perspectiva de ensino intercultural, que busque questionar o modelo político vigente NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 74 na educação brasileira, e que estão fortemente influenciadas nas formações acadêmicas alinhadas a um currículo eurocêntrico e norte americano. O inglês difundido na educação brasileira ainda possui resquícios de uma matriz curricular embranquecida (britânica e estadunidense), constituindo uma supremacia linguística e hegemônica, que naturaliza o ensino numa perspectiva colonial. Nesse sentido, podemos evidenciar o campo complexo e desafiador para o ensino da LI para alunos da EJA na EEQ, e (re)pensar a necessidade emergencial para uma concepção de aprendizagem que compreenda a língua como cultura viva na EEQ. Sobre isso, Rajagopalan (2003) adensa que há uma necessidade de compreender o processo de aprendizagem em língua estrangeira como parte integrante do processo (re)definição de identidade cultural do aluno(a). Considerando esse panorama complexo e desafiador do ensino de LI para alunos da EJA na EEQ, questionamos da professora de LI como ela trabalha para manter os alunos motivados nas suas aulas, no sentido de evitar a evasão escolar: É um desafio extremamente grande e complexo em trabalhar com alunos da EJA da EEQ, em virtude que muitos deles possuem famílias e atividades de subsistência, tendo que muitas das vezes se ausentarem da escola para cuidar de suas atividades na agricultura e na pesca. Eu sempre procuro entender essa realidade e ajudar na medida do possível, garantindo aos alunos que no retorno de suas atividades façam suas atividades pendentes, e procuro relacionar suas histórias de vida nas aulas de LI a partir de história de povos afro-anglófonos que sofreram durante muito tempo em busca de melhores condições de vida. (PROFESSORA DE LÍNGUA INGLESA). Diante do relato da professora, podemos perceber que os desafios no ensino da LI passam a ser o fio condutor em suas aulas, levando-a a questionar e repensar seus métodos de ensino e abordagens como elementos neutros e absolutos, descobrindo seus horizontes por uma busca de novas epistemologias e por uma visão mais crítica da produção do conhecimento. 5 O ENSINO DA LÍNGUA INGLESA NA PERSPECTIVA DA EDUCAÇÃO ESCOLAR QUILOMBOLA: DESAFIOS DE UMA PROFESSORA DE LÍNGUA INGLESA E DE ALUNOS QUILOMBOLAS MARAJOARAS NA EDUCAÇÃO DE JOVENS E ADULTOS Aqui iniciamos um recorte analítico do ensino da LI na EJA na perspectiva da EEQ em Salvaterra, no sentido de compreender como a LI é trabalhada e articulada com os saberes tradicionais dos alunos quilombolas marajoaras. Buscamos evidenciar, por meio de questionários direcionados a professora de LI e aos alunos quilombolas da EJA, como é trabalhada a cultura quilombola marajoara e afro-brasileira nas aulas de LI. Ademais, foi evidenciado como a identidade dos alunos quilombolas marajoaras sofrem influências e os resquícios hegemônicos de culturas esbranquiçadas para o ensino da LI na EJA da EEQ de Salvaterra. Durante a pesquisa de campo, percebemos que as propostas pedagógicas da professora de LI na sala de aula da EJA na EEQ se assemelham a uma perspectiva de ensino urbanocêntrico, onde os alunos quilombolas da EJA ainda estão (in)visibilizados quanto a valorização dos seus saberes culturais nas aulas de LI. A perspectiva de ensino da professora de LI ainda apresenta resquícios coloniais de cultura esbranquiçada e hegemônica, dificultando um ensino adensado na matriz africana e afro-brasileira. De acordo com as entrevistas realizadas com a professora de LI, ela apontou que seu maior desafio para ensino de LI na EJA da EEQ, seria a falta de formação continuada para as relações étnico-raciais. Para ela, a capacitação nas relações étnico-raciais seria o ponto de partida para compreender melhor o processo de ensino aprendizagem da LI na EJA da EEQ. Nesse sentido, foi questionado à professora como ela busca trabalhar os elementos da cultura quilombola marajoara ou afro-brasileiras em suas aulas, e ela considerou: Eu sinto dificuldades em trabalhar elementos da cultura quilombola marajoara nas aulas de inglês, na minha formação acadêmica não tive nenhuma formação para as relações étnico-raciais, sempre aprendemos o inglês na perspectiva americana e britânica, para assim ensinar aos nossos alunos. (PROFESSORA DE LÍNGUA INGLESA.) A partir do enunciado da professora, podemos perceber o quanto que a hegemonia e supremacia cultural da LI presente em suas práticas pedagógicas atravessam tempos e espaços DOSSIÊ AMAZÔNIA 75 relacioná-los a sua prática pedagógica, voltando-se contra a perpetuação das desigualdades sociais, como também o imperialismo linguístico padronizado (estadunidense e britânico) para o ensino da LI difundido no currículo e nas práticas pedagógicas dos professores de LI da educação brasileira. ç No questionário aplicado aos alunos da EJA da EEQ, evidenciamos uma turma bastante heterogênea e com grandes pluralidades. Diante dessa realidade, percebemos que a LI é considerada uma disciplina bastante complexa para os alunos. A falta de conhecimentos linguísticos em sua língua materna dificulta o aprendizado da LI. Nesse sentido, foi questionado a eles se as aulas de inglês eram relacionadas com a cultura quilombola marajoara ou afro-brasileira. De acordo com os enunciados dos alunos, eles consideraram: Nossas aulas de inglês são bem divertidas e legais, a professora as vezes trabalha a cultura africana nas aulas por meio da história de personagens negros brasileiros, mas eu não consigo entender muito essa relação. (ALUNO DA EJA, 22 ANOS DE IDADE). Uma vez a professora trabalhou os nomes das frutas locais da nossa comunidade em inglês, eu achei interessante porque nós conseguimos aprender o nome dos nossos alimentos em inglês, eu até ensinei para meus familiares. (ALUNA DA EJA, 38 ANOS DE IDADE). Eu não consigo aprender inglês e nunca vi nenhuma pessoa da minha comunidade falar o inglês bem, na verdade eu nunca vi uma pessoa negra falar inglês igual os turistas estrangeiros que vem visitar Salvaterra. Eu acho difícil, a professora tenta nos ajudar, mas é muito difícil. (ALUNO DA EJA, 24 ANOS DE IDADE). Diante dos relatos apresentados pelos alunos, é possível evidenciar de forma triangular a relação dos ideais de “raça branca” à concepção e valorização cultural da LI difundida na escola pública, consubstanciada no ensino da cultura hegemônica e embraquecida, na qual o professor apropria-se da cultura de determinado país (geralmente Estados Unidos ou Inglaterra) para o ensino da língua estrangeira (MOITA LOPES, 2005, grifo nosso). Nesse sentido, a professora de LI da EJA da EEQ, acaba (re)produzindo estereótipos e noções de verdade que valorizam culturas hegemonias em detrimento à cultura dos alunos quilombolas, principalmente quando o ensino da LI é abalizado na cultura estadunidense e/ou americana. Nesse sentido, é possível evidenciar as influências hegemônicas e coloniais que a escola tem em relação à identidade dos alunos quilombolas marajoaras, e a própria professora de LI da EJA da EEQ. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Essa visão, levou a professora a compreender a necessidade de um olhar mais sensível e (de)colonial no ensino da LI da EJA, voltado a conhecer melhor os seus estudantes, e ao mesmo tempo buscar pedagogias mais sensíveis às suas necessidades e realidades, isso evidencia uma relação dialógica e horizontal em que o professor quanto alunos aprendem simultaneamente (MIGNOLO, 2014; KUMARAVADIVELU, 2016). Com base nas questões apresentadas pela professora, é importante compreendermos a ideia de (de)colonialidade empregada neste estudo. Para discutir esse conceito, recorremos a Mignolo (2014, p. 105), que entende como um “movimento de resistência teórico e prática, político e epistemológico, à lógica da modernidade/colonialidade”. Por isso, a perspectiva (de)colonial propõe uma ação de enfrentamento à visão colonial, ela permite desenvolver um pensamento crítico por outras visões, e que possam apontar para nossas regionalidades e tradições, como enfrentamento ao pensamento colonial eurocêntrico. As perspectivas adotadas pela professora de LI, corroboraram para a desconstrução da imagem hegemônica e cultural do ensino da LI na educação brasileira, e permitiu visibilizar outros protagonismos para o ensino do idioma. As culturas afro-anglófonas foram cânones para a desconstrução e reconstrução das abordagens culturais difundidas no ensino da LI na EEQ, e serviu para validar e fortalecer a implementação da Lei. nº 10.639/03 nas aulas de LI, sobretudo a EEQ. Logo, é de fundamental importância legitimar as contribuições políticas, culturais, sociais e linguísticas dos povos afro-brasileiro, para a formação socioeducativa dos alunos quilombolas da EJA, sobretudo no âmbito do ensino da LI a partir das contribuições culturais de povos afro-anglófonos. Dessa forma, o trabalho do professor de LI da EJA na EEQ, deve voltar-se para a importância da implementação Lei nº 10.639/03 e de uma proposta pedagógica interdisciplinar, comprometida em (re)conhecer seus princípios norteadores e NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 76 para além das salas de aulas. O professor perante os seus alunos, são submetidos a um discurso, e à uma relação de poder, em virtude que maioria dos alunos desconhecem determinados temas e pensamentos, acreditam geralmente naquilo que é difundido pelo professor (MOITA LOPES, 2002; PENNYCOOK, 1998; APPLE, 2002, grifo nosso) para além das salas de aulas. O professor perante os seus alunos, são submetidos a um discurso, e à uma relação de poder, em virtude que maioria dos alunos desconhecem determinados temas e pensamentos, acreditam geralmente naquilo que é difundido pelo professor (MOITA LOPES, 2002; PENNYCOOK, 1998; APPLE, 2002, grifo nosso) A partir desses apontamentos, foi questionado à professora se durante a graduação ela havia participado e/ou recebido formação acadêmica de algum curso sobre EEQ? Ela considerou: “Não, na universidade nós nunca tivemos nenhuma disciplina que pudesse trabalhar a educação quilombola e nem mesmo as relações étnico-raciais, eu até desconhecia a Lei 10.639/03 antes de trabalhar na escola quilombola”. Podemos observar no relato da professora, a falta de formações e discussão na academia voltada para as relações étnico-raciais, e esse panorama inviabiliza atender a proposta da EEQ, sobretudo para o ensino da LI. Segundo a professora, suas práticas pedagógicas buscam correlacionar os elementos da cultura quilombola marajoara e afro-brasileira em suas aulas, utilizando textos e vocabulários do cotidiano da comunidade, sendo que os textos geralmente são de sua autoria e que sempre buscaram trazer para dentro de sala de aula os saberes da comunidade quilombola de Vila União. Para isso, precisamos romper com a ideia de ensino tradicional da LI, ancorado e difundido por princípios coloniais, homogêneos, esbranquiçados e elitizados. Temos que romper com o ensino as desigualdades sociais, para assim atendermos a proposta da EEQ, sobretudo no âmbito da EJA. Pennycook (1998) e Silva (2009), apontam que romper com o ensino tradicional não é uma tarefa fácil, pois as concepções de mundo no fazer docente precisam ser reavaliadas às novas concepções que propõem reflexões e estudos, de maneira a ressignificar novas concepções e que façam mais sentidos para novas propostas e práticas pedagógicas. DOSSIÊ AMAZÔNIA Nesse sentindo, Pennycook (1998), Moita Lopes (2002) Ferreira (2006) asseveram que o papel do professor tem função maior de educar cidadãos, formar sujeitos conscientes e críticos para que esses vivam e interajam na sociedade com uma postura de questionamentos, rupturas e resistência. O ensino da LI tem desdobramentos agravantes na EEQ, com a valorização do ensino de matriz embranquecida e hegemônica (estadunidense e britânica). Essa cosmovisão é perceptível quando analisamos por exemplos os livros didáticos de inglês, e por evidenciar uma corrente positiva e tendenciosa pela supremacia cultural e hegemônica da LI. Desse modo, o professor de LI, ainda possui uma visão holística pela língua perfeita e colonial, exigindo do aluno o uso da língua “correta” que contemple padrões linguísticos culturais hegemônicos (britânico e estadunidense). Para Apple (2002) e Pennycook (1998), essa relação traduz a ideia de poder-ensino que configura a escola contemporânea, quer seja brasileira ou estrangeira, instituindo a educação como uma relação de poder na sociedade. Ao se pensar na formação de professores nas universidades, existe uma relação que excede as paredes da academia. O reconhecimento do “poder” existente no engajamento discursivo do professor influencia diretamente e inconscientemente a relação professor/aluno DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 77 cultura quilombola a partir de outros protagonismos negros, que levem em consideração as culturas afro-anglófonas para o aprendizado do idioma. O ensino da LI na EJA da EEQ da Amazônia Marajoara, precisa inserir em seu horizonte, a necessidade de ensinar inglês de uma maneira interdisciplinar e contextualizada, no âmbito das especificidades locais da cultura quilombola marajoara. Dada a importância do tema, torna-se necessário aprofundar mais estudos do ensino da LI na EJA da EEQ, pois é perceptível que o desafio maior está na forma como a LI é ensinada e difundida nessa modalidade de ensino, e também na capacitação de professores de LI para desenvolverem competências necessárias para contemplar a proposta da EJA na EEQ. REFERÊNCIAS ACEVEDO, Rosa Elizabeth. Quilombolas na ilha do Marajó: Território e organização política. In: GODOI, Emília Pietravesa; MENEZES, Marilda Aparecida de. Diversidade do campesinato: expressões e categorias: construções identitárias e sociabilidades. v 1. São Paulo: Editora UNESP, 2009. ACEVEDO, Rosa Elizabeth. Quilombolas na ilha do Marajó: Território e organização política. In: GODOI, Emília Pietravesa; MENEZES, Marilda Aparecida de. Diversidade do campesinato: expressões e categorias: construções identitárias e sociabilidades. v 1. São Paulo: Editora UNESP, 2009. ALMEIDA, Maria Geralda. Fronteiras, Territórios e identidades. Revista ANPEGE, ano 2, 2005. APPLE, Michael. W. Freire and the politics of race in education. International Jornal of Leadership in Education, April-June, vol 6, no. 2. Wiscousin: Masison-USA, 2002. Disponível em: https://www.tandfonline.com/doi/abs/10.1080/13603120304821. Acesso em: 18 ago. 2020. ARROYO, Miguel. Currículo, território em disputa. Petrópolis, Rio de Janeiro: Vozes, 2011. 6 CONSIDERAÇÕES FINAIS A EJA na história na educação brasileira é marcada por profundas desigualdades sociais, políticas e econômicas. Na EEQ, essa modalidade apresenta grandes desafios para o ensino da LI, por entender um conjunto complexo de epistemologias que visam articular saberes das comunidades tradicionais, afro-brasileiras às práticas pedagógicas do professor de LI. Este estudo permitiu compreender e refletir sobre os desdobramentos da LI na identidade quilombola marajoara para os alunos da EJA. A partir do referencial teórico, foi possível descortinar novos olhares para o ensino da LI na EJA da EEQ. Nesse bojo, percebemos que os alunos da EJA internalizam o aprendizado da LI a partir de uma ideia ainda fragmentada. Essa inflexão evidenciou a impetuosa supremacia da LI difundida a partir de culturas hegemônicas e brancas (estadunidense e britânica) nas práticas pedagógicas dos professores de LI da educação básica da EEQ. Percebemos que o maior desafio do professor de LI é trabalhar as relações étnico- raciais e validar a Lei nº. 10.639/03 nas aulas de LI da EJA na EEQ. A partir dos resultados, foi possível perceber que um dos maiores desafios existentes para o ensino de LI na EJA está na carência de formação continuada para professores que atuam nessa modalidade de ensino. A falta de apoio pedagógico e materiais didáticos voltados para as especificidades dos alunos quilombolas têm sucumbindo a qualidade do aprendizado em LI para alunos da EJA na Amazônia Marajoara. Mesmo com todos os desafios evidenciados até aqui, ainda assim, a LI tem um papel importante para esses alunos e também para a comunidade Quilombola de Vila União. Através da LI, os alunos poderão tomar conhecimento sobre outras culturas e a sua própria ARROYO, Miguel. Currículo, território em disputa. Petrópolis, Rio de Janeiro: Vozes, 2011. ARRUTI, José M. Conceitos, normas e números: uma introdução à educação escolar quilombola. 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Altera a Lei no 9.394, de 20 de dezembro de 1996, que estabelece as diretrizes e bases da educação nacional, para incluir no currículo oficial da Rede de Ensino a obrigatoriedade da temática "História e Cultura Afro-Brasileira", NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 78 e dá outras providências. Diário Oficial da União, Brasília, 2003. Disponível em: http://www.planalto.gov.br/ccivil_03/leis/2003/l10.639.htm. Acesso em: 22 ago. 2020. e dá outras providências. Diário Oficial da União, Brasília, 2003. Disponível em: http://www.planalto.gov.br/ccivil_03/leis/2003/l10.639.htm. Acesso em: 22 ago. 2020. e dá outras providências. Diário Oficial da União, Brasília, 2003. Disponível em: http://www.planalto.gov.br/ccivil_03/leis/2003/l10.639.htm. Acesso em: 22 ago. 2020. BRASIL. Diretrizes Curriculares Nacionais para a Educação Escolar Quilombola. Resolução nº 08/2012. CNE/CEB. Brasília, 2012. Disponível em: http://www.educadores.diaadia.pr.gov.br/arquivos/File/pdf/diretrizes_nacionais_educacao_esc olar_quilombola.pdf. Acesso em: 25 ago. 2020. BRASIL. Lei nº 9.394, de 20 de dezembro de 1996. Estabelece as diretrizes e bases da educação nacional. Lei de diretrizes e bases da educação nacional. Disponível em: https://www2.camara.leg.br/legin/fed/lei/1996/lei-9394-20-dezembro-1996-362578- publicacaooriginal-1- pl.html#:~:text=Estabelece%20as%20diretrizes%20e%20bases%20da%20educa%C3%A7%C 3%A3o%20nacional.&text=%C2%A7%201%C2%BA%20Esta%20Lei%20disciplina,trabalh o%20e%20a%20pr%C3%A1tica%20social. Acesso em: 20 ago. 2020. pl.html#:~:text=Estabelece%20as%20diretrizes%20e%20bases%20da%20educa%C3%A7%C 3%A3o%20nacional.&text=%C2%A7%201%C2%BA%20Esta%20Lei%20disciplina,trabalh o%20e%20a%20pr%C3%A1tica%20social. Acesso em: 20 ago. 2020. BRASIL. Base Nacional Curricular Comum. Brasília, 2017. Disponível em: http://basenacionalcomum.mec.gov.br/images/BNCC_EI_EF_110518_versaofinal_site.pdf. Acesso em: 22 ago. 2020. BRANDÃO, Carlos Rodrigues. A comunidade tradicional. In: Cerrado, Gerais, Sertão: comunidades tradicionais dos sertões roseanos (Relatório de Pesquisa). Montes Claros, 2010, p. 347-361. BRITISH COUNCIL. O ensino de inglês na educação básica brasileira: elaborado como exclusividade para o British Council pelo Instituto de Pesquisas. Plano CDE. São Paulo: British Council Brasil, 2015. Disponível em: https://www.britishcouncil.org.br/sites/default/files/estudo_oensinodoinglesnaeducacaopublic abrasileira.pdf. Acesso em: 27 ago. 2020. DIAS, Joel Santos. “Confuso e intrincado labirinto”: Fronteira, território e poder na Ilha Grande de Joanes (séculos XVII e XVIII). 2016. ARROYO, Miguel. Currículo, território em disputa. Petrópolis, Rio de Janeiro: Vozes, 2011. Tese (Doutorado em História) - Universidade Federal do Pará, Instituto de Filosofia e Ciências Humanas, Programa de Pós- Graduação em História, Belém, 2016. FERREIRA, Aparecida de Jesus. Formação de professores: raça/etnia. Cascavel: Coluna do Saber, 2006. FERREIRA, Aparecida de Jesus. Formação de professores: raça/etnia. Cascavel: Coluna do Saber, 2006. REIRE, Paulo. Pedagogia do oprimido. 17 ed. Rio de Janeiro: Paz e Terra, 1987. FREIRE, Paulo. Pedagogia do oprimido. 17 ed. Rio de Janeiro: Paz e Terra, 1987. HADDAD, Sérgio; DI PIERRO, Maria Clara. Escolarização de jovens e adultos: observações específicas sobre juvenilização da EJA e os dois grupos conflitantes existentes, 2000. KUMARAVADIVELU, B. The Decolonial Option in English Teaching: Canthe Subaltern Act? TESOL Quarterly, v. 50, n. 1, 2016. LISBOA, Pedro Luiz Braga. A terra dos Aruã: uma história ecológica do arquipélago do Marajó. Belém, Museu Paraense Emilio Goeldi, 2012. DOSSIÊ AMAZÔNIA 79 MINAYO, Maria Cecília de Souza. Ciência, técnica e arte: o desafio da pesquisa social. In: Pesquisa social: teoria, método e criatividade. 18 ed. Petrópolis: Vozes, 1994. MIGNOLO, Walter. Educación y decolonialidad: aprender a desaprender para poder reaprender. [Entrevista cedida a] Facundo Giuliano e Daniel Berisso. Revista del IICE, Buenos Aires, n. 35, 2014. MOITA LOPES, Luiz Paulo da. Identidades Fragmentadas: a construção de raça, gênero e sexualidade na sala de aula. Campinas: Mercado de Letras, 2002. MOITA LOPES, Luiz Paulo da. Pesquisa interpretativa em linguística aplicada: a linguagem como condição e solução. Delta, São Paulo, v. 10, n. 2, 1994. MOITA-LOPES, Luiz Paulo da. Inglês no mundo contemporâneo: Ampliando oportunidades sociais por meio da educação. Texto básico apresentado no simpósio Inglês no mundo contemporâneo: ampliando oportunidades sociais por meio da educação. São Paulo: Centro Brasileiro Britânico. 25- 26 de abril de 2005. PACHECO, Agenor Sarraf. As Áfricas nos Marajós: Visões, fugas e redes de contatos. In: SCHAAN, D. P.: MARTINS, C. P. (org). Muito além dos campos: arqueologia e história da Amazônia Marajoara. Belém: GKNORONHA, 2010. PENNYCOOK, Alastair. A linguística aplicada nos anos 90: em defesa de uma abordagem crítica. In: SIGNORINI, Inês CAVALCANTI, M. C. Linguística aplicada e transdicilinariedade. Campinas: Mercado de Letras, 1998. RAJAGOPALAN, Kanavillil. Por uma linguística crítica: linguagem, identidade e a questão ética. São Paulo: Parábola, 2003. SALVATERRA (Estado do Pará). Proposta da Educação Escolar Quilombola de Salvaterra. Secretaria Municipal de Educação de Salvaterra. 2017. SKLIAR, Carlos. A inclusão que é “nossa” e a diferença que é do “outro”. In: RODRIGUES, D. (Org.) Inclusão e educação: doze olhares sobre a educação inclusiva. [S.l.]: Summus Editorial, 2006. SCHNEIDER, Sônia Maria; FONSECA, Maria da Conceição Ferreira Reis. Esse é o meu lugar... Esse não é o meu lugar: inclusão e exclusão de jovens e de adultos na escola. Educ. Soc., Campinas, v. 34, n. 122, 2013. SILVA, Paula de Almeida. Reflexões sobre a raça e racismo em sala de aula: um estudo de caso com duas professores de inglês negras. 2009. – CAMPUS BRAGANÇA FREIRE, Paulo. Pedagogia do oprimido. 17 ed. Rio de Janeiro: Paz e Terra, 1987. Dissertação (Mestrado em Letras) – Universidade Federal de Goiás, Goiânia, 2009. REVISTA DO PROGRAMA DE PÓS - SABER ES NA AMAZÔ NIA UNIV ERSIDA DE FEDERAL D O PARÁ As práticas comunitárias são processos de construções sociais diretamente associadas ao contexto socioeconômico, político e cultural. São elas que habilitam o indivíduo para os fluxos da memória e do corpo, de forma singular e coletiva, cujas pesquisas e pesquisadores são conexões comunitárias que se deslocam sobre as vivências conjuntas de espaços, modos de vida e de aproximações humanas na relação entre indivíduos e modos de saber-fazer por entre comunidades, um cruzamento de corpos plurais. Os artigos presentes neste dossiê estão atravessados pela cultura, numa diversidade de expressões possíveis das práticas sociais que os movimentam. O artigo Narrativas de crianças sobre o saber/fazer em festas amazônicas: O caso da marujada de São Benedito e São Sebastião em Tracuateua/Pa apresenta um estudo que identifica saberes e fazeres inerentes a festa da marujada em Tracuateua. Os saberes aqui são vivenciados e partilhados pelo movimento entre marujos e marujas de diferentes gerações por meio da escuta, da observação e da oralidade. A voz e as percepções das crianças são aqui valorizadas, mostrando como é possível dar significado ao mundo a partir de diferentes ‘lugares de fala’, neste caso reconhecendo as crianças como sujeitos de direitos, como é proposto pela convenção dos direitos da criança da UNICEF, adotada pela Assembleia Geral das Nações Unidas em 20 de novembro de 1989: Todas as crianças têm “o direito de exprimir livremente a sua opinião sobre as questões que lhe respeitem, sendo devidamente tomadas em consideração as opiniões da criança, de acordo com a sua idade e maturidade” (Artigo 12º) (UNICEF, 2019, p. 13)6. Ainda tratando-se de saberes de crianças o artigo Os saberes da tradição da comunidade Segredinho na percepção das crianças propõe-se refletir sobre os saberes da tradição apreendidos por meio da pesca artesanal no Lago do Segredo a partir da percepção das crianças da comunidade Segredinho/Capanema-PA. Saberes referentes à pesca artesanal adquiridos por intermédio da transmissão e da observação dos mais experientes na atividade são repassados de geração para geração. A pesca no Lago do Segredo além de ser uma atividade econômica congrega aspectos míticos, simbólicos e da tradição. 6 UNICEF. Convenção sobre os direitos da criança e protocolos facultativos. Comité português para a UNICEF, 2019. Disponível em: https://www.unicef.pt/media/2766/unicef_convenc-a-o_dos_direitos_da_crianca.pdf O artigo intitulado Saberes e práticas socioambientais na pesca artesanal do caranguejo-uçá na Amazônia bragantina (Pontinha do Bacuriteua-Pa) traz um conjunto de saberes que os tiradores aprenderam com as gerações anteriores e que são colocados em práticas ao movimentarem-se em seus locais de trabalho, diariamente. Seguindo o campo dos saberes, as ricas etnozoologias e etnotaxonomias de pescadores artesanais sobre tartarugas-marinhas e aves-costeiras locais revelam um conhecimento aprofundado sobre estes animais que não são de interesse para o autossustento. E ainda, a consciência dos pescadores quanto à importância dos animais para os ecossistemas são etnoconhecimentos importantes para o entendimento das relações humanos-animais, bem como para ações de preservação e manejo localmente referenciadas. Este estudo está apresentado no artigo Etnoconhecimentos sobre animais de pescadores artesanais na Amazônia costeira paraense. O ofício de benzer como produção de conhecimento no município de Tracuateua – Pa – Amazônia – Brasil estimula a reflexão sobre o benzimento enquanto prática cultural, sinaliza a importância de diálogos entre os diferentes saberes, independentemente de sua origem. Na parte de seção livre o dossiê apresenta quatro trabalhos, são eles: Na parte de seção livre o dossiê apresenta quatro trabalhos, são eles: Os espaços de representação segundo a Geografia Crítica discute a partir de um estudo bibliográfico com base em vários autores da geografia e de outros campos do conhecimento, busca-se entender como essa forma de ver o espaço contribui para uma nova forma de traçar caminhos e entender a realidade de maneira mais profunda. conhecimento, busca-se entender como essa forma de ver o espaço contribui para uma nova forma de traçar caminhos e entender a realidade de maneira mais profunda. A interpretação da velhice da antiguidade até o século XXI aborda fragmentos históricos a partir da interpretação de alguns filósofos sobre a velhice, a destacar para a construção de um envelhecimento digno, participativo e ativo na sociedade, foco das Universidades- UNATIs e UMA, da Gerontologia e Geriatria, - áreas da Medicina que estuda e trata o envelhecimento humano respectivamente. Alimentação e religiosidades no romance menina que vem de Itaiara de Lindanor Celina. A narrativa da obra retrata a cultura patriarcal da época, apresentando valores e costumes religiosos locais. Uma memória literária rica em conhecimento sobre alimentação e religiosidades e os diversos processos de cura. Da construção do currículo à aprendizagem significativa: a prática docente de Geografia na educação de jovens e adultos tem como objetivo mostrar a realidade do contexto educacional contemporâneo da Educação de Jovens e Adultos (EJA), buscando entender os próprios alunos como sujeitos e ao mesmo tempo objetos no processo de ensino- aprendizagem. Dessa forma, o artigo alerta para a relevância de se trabalhar com os conteúdos a partir das práticas socioespaciais dos discentes, pois assim, os estudantes podem se (re) conhecer dentro de sua realidade, possibilitando a aprendizagem significativa e tornando o discente um sujeito do processo didático-pedagógico em Geografia. j p p g g g Como nos lembram Boavida e Amado (2006)7, no estudo de problemas sociais da ciência “importa não desperdiçar a experiência de vida e o senso comum, de modo a não haver um afastamento dos problemas reais da humanidade e de se proporcionar uma autoemancipação através do conhecimento” (p. 139). É também fundamental que nos dediquemos a consolidar as dimensões ética e política da ciência, usando-a como arma como as opressões e as desigualdades (Vieira, 2019)8, e como meio de tornar visíveis pessoas, grupos e problemáticas esquecidos pela chamada ciência mainstreaming. Na parte de seção livre o dossiê apresenta quatro trabalhos, são eles: Com toda a legitimidade, as pessoas participantes nos estudos integrantes dos vários artigos que compõem este dossiê sentiram-se certamente recompensadas por partilharem os seus saberes e percepções com alguém que, no papel de investigador/a, quis aprender com eles e elas e recorreu ao seu estatuto de privilégio enquanto cientista para recolher dados e traduzir para linguagens formais, inteligíveis para a comunidade científica, toda uma riqueza simbólica que de outra forma seria inatingível. Os ganhos são, por isso, mútuos. Imprensa da Universidade de Coimbra, 2006. 8 VIEIRA, C. C. Investigação, conhecimento científico e responsabilidade social: reflexões a partir das Ciências Sociais e Humanas. Exedra – Revista Científica da ESEC. Número temático EIPE 2019, 28-37, 2019. Disponível em: http://exedra.esec.pt/wp-content/uploads/2020/01/03-EIPE2019.pdf 7 BOAVIDA, J. E AMADO, J. Ciências da Educação. Epistemologia, Identidade e Perspectivas. Coimbra: Imprensa da Universidade de Coimbra 2006 As organizadoras Dra. Cristina Maria Coimbra Vieira Professora Associada - Faculdade de Psicologia e de Ciências da Educação Universidade de Coimbra e Centro de Investigação em Educação de Adultos e Intervenção Comunitária (CEAD) da Universidade do Algarve, Portugal. E-mail: vieira@fpce.uc.pt. Dra. Norma Cristina Vieira Professora do Programa de Pós-Graduação Mestrado em Linguagens e Saberes da Amazônia (PPLSA), Universidade Federal do Pará (UFPA), Campus de Bragança (CBRAG). E-mail: normacosta@ufpa.br. 83 DOSSIÊ AMAZÔNIA VOLUME IX – NÚMERO 01 – M INTRODUÇÃO Este artigo teve como objetivo analisar as narrativas de crianças sobre os saberes e fazeres em festas amazônicas com o recorte para a festividade da marujada de São Benedito e São Sebastião em Tracuateua/PA. Para isso, foi necessário discutir as categorias teóricas como: cultura, educação, saberes e infância sobre novos olhares, numa perspectiva de que a cultura é uma “teia de significados” que são construídos nas relações sociais dos homens; a educação como uma “fração dessa cultura” e que pode ser desenvolvida em diferentes lugares com diversos grupos sociais. A infância, foi analisada à luz da “sociologia da infância”, compreendendo a criança como protagonista de suas vivências. Brandão (2002, p. 09) destaca que: “Não há uma única forma, nem um único modelo de educação, a escola não é o único lugar em que ela acontece e talvez nem seja o melhor; o ensino não é sua única prática e o professor não é seu único praticante”. A educação para o autor é uma maneira do modo de vida dos diversos grupos sociais que criam e recriam sua cultura. Sendo assim, a educação também pode ser encontrada nos diversos saberes populares perpassando por um processo socioeducativo. De acordo com Geertz (2014, p. 04), o entendimento sobre a cultura deve partir do contexto de que: “O homem é um animal amarrado a teias de significados que ele mesmo teceu” e que vem criando e recriando suas maneiras de viver, de acordo com suas necessidades, pois é necessário compreender que a cultura não se explica uniformemente pelas bases naturais, mas, principalmente por suas bases sociais condicionadas a vida do homem às suas formas de viver e interpretar o mundo. O autor ainda menciona que os fenômenos culturais que pertencem a sociedade, são penetrados por teias de signos e significados, tecidas pelos próprios homens e mulheres em suas ações, numa relação dinamicamente interpretativa e, por isso, entende que a cultura precisa de uma base teórica que supra o discurso descritivo e superficial sobre ela. Como ele mesmo ressalta: O conceito de cultura que eu defendo, é essencialmente semiótico. Acredito [...] que o homem é um animal amarrado a teias de significados que ele mesmo teceu, assumo a cultura como sendo essas teias e suas análises; portanto, não como uma ciência experimental em busca de leis, mas como uma ciência interpretativa à procura de significados. (GEERTZ, 2014, p. 04). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 84 (twelve) years of age, participating in the party. The methodological path was characterized by a qualitative approach with research techniques such as conversation circles, observation, field diaries, photographic records and the dynamics with drawings. Thus, based on the diversity of knowledge constructed in the daily life of the marujada, knowledge and actions inherent to the party were identified, experienced and shared by / by the movement between sailors and sailors of different generations through listening, observation and orality. (twelve) years of age, participating in the party. The methodological path was characterized by a qualitative approach with research techniques such as conversation circles, observation, field diaries, photographic records and the dynamics with drawings. Thus, based on the diversity of knowledge constructed in the daily life of the marujada, knowledge and actions inherent to the party were identified, experienced and shared by / by the movement between sailors and sailors of different generations through listening, observation and orality. Key Words: Culture. Knowledge. Marujada. Child narratives. Key Words: Culture. Knowledge. Marujada. Child narratives. Data de submissão: 20.10.2020 Data de aprovação: 26.12.2020 Data de submissão: 20.10.2020 Data de aprovação: 26.12.2020 O conceito de cultura que eu defendo, é essencialmente semiótico. Acredito [...] que o homem é um animal amarrado a teias de significados que ele mesmo teceu, assumo a cultura como sendo essas teias e suas análises; portanto, não como uma ciência experimental em busca de leis, mas como uma ciência interpretativa à procura de significados. (GEERTZ, 2014, p. 04). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 85 são produtos culturais manufaturados”, ou seja, são tecidas pelas mãos dos sujeitos, sendo que para a compreensão dessas expressões ou desses signos é necessário compreender o significado e chegar até “os conceitos específicos das relações entre eles” (GEERTZ, 2014, p. 36,37). É por isso que pretendemos compreender o que pensam as crianças sobre a marujada de Tracuateua/PA, e depois, analisar os saberes que perpassam essa festa e, consequentemente analisar as relações de aprendizagem desses saberes com os intérpretes dessa pesquisa. Outro ponto importante para essa pesquisa são as contribuições sobre cultura do historiador inglês Thompson (1995, p. 19), uma vez que ele, também concebe a cultura numa perspectiva discursiva como fenômeno social, interpretando os processos sociais nos quais, e pelos quais, "as formas simbólicas permeiam o mundo social", de modo crescente e generalizado. g Para o autor conhecer a cultura na reconstrução das experiências das pessoas comuns; é compreender o passado à luz de nossas próprias experiências e da experiência dos outros, considerando a subjetividade do homem. Por meio dessa subjetividade os fenômenos culturais como os comportamentos, os valores, as condutas, os costumes, enfim, a cultura do homem poderia ser analisada, ou melhor, as culturas, no sentido de que ela se refere a uma realidade específica. Por isso Thompson (1995), diz que o estudo dos fenômenos culturais poderia ser pensado e analisado numa visão histórica e social: Pode ser pensado como o estudo das maneiras como expressões significativas de vários tipos são produzidas, construídas e recebidas por indivíduos situados em um mundo sócio histórico. Pensado dessa maneira, o conceito de cultura se refere a uma variedade de fenômenos e a um conjunto de interesses que são, hoje, compartilhados por estudiosos de diversas disciplinas, desde a sociologia e antropologia até a história e a crítica literária. (THOMPSON, 1995, p.165). Ao analisar o termo cultura, Thompson (1995) apresenta três tipos de conceitos construídos ao longo da história para apresentar sua própria visão a respeito dessa concepção cultural, tais como: a clássica, a descritiva e a simbólica. A clássica faz referência à cultura como um processo intelectual ou espiritual que se diferencia sob certos aspectos da civilização. A descritiva, seria um variado conjunto de valores, crenças, costumes, convenções, hábitos e práticas características de uma dada sociedade específica ou de um período histórico (THOMPSON, 1995, p.166). INTRODUÇÃO A essência dessa semiótica, defendida por Geertz (2014) analisa a cultura como um conjunto de sistemas que poderia se chamar teias de significados, e nessas teias passam os mais diferentes tipos de produção cultural. Através da compreensão desses significados e sua inter- relação, pode se constituir uma ciência interpretativa ou obter conceitos mais definidos de cultura. As expressões da criança de forma geral, como: “As ideias, os valores, e as emoções DOSSIÊ AMAZÔNIA E a simbólica, por sua vez, culminaria nos “fenômenos culturais [...] são fenômenos simbólicos e o estudo da cultura está essencialmente interessado na interpretação dos símbolos e da ação simbólica” (THOMPSON, 1995, p.166). Nesses três tipos de conceitos de cultura apresentados, podemos perceber que eles culminam nas práticas sociais desenvolvidas pelos homens no decorrer de sua história, que ao longo do tempo vai sendo moldada de sentidos e significados. Assim, a festa da marujada em Tracuateua/PA está imersa nesse aspecto simbólico, por ser palco de uma manifestação que traz, na sua história, inúmeros sentidos e significados inscritos em seus saberes e fazeres, por isso deve ser vista como um elemento cultural que está condicionada às práticas sociais que revelam, por meio dos saberes, a vivência e a experiência desse grupo. Aqui vamos considerar o que defende Charlot (2013, p. 162) quando apresenta a ideia de heterogeneidade do saber. Para ele, essa ideia é fundamental na relação do saber, uma vez que não existe uma única forma de aprender, e não existem carências na aprendizagem. O que existe são outras “formas de se relacionar com o mundo, outro tipo de vínculo com o mundo, outra forma de entrar no processo de aprender” Brandão (2002) assinala seu conceito de cultura afirmando que: Somos o que criamos para efemeramente nos perpetuarmos e transformarmos a cada instante. Tudo aquilo que criamos a partir do que nos é dado, quando tomamos as coisas da natureza e as recriamos como os objetos e os utensílios da vida social, NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 86 representa uma das múltiplas dimensões daquilo que, em uma outra, chamamos de cultura. (BRANDÃO, 2002, p. 22). representa uma das múltiplas dimensões daquilo que, em uma outra, chamamos de cultura. (BRANDÃO, 2002, p. 22). Nas análises de Brandão (2007), a sociedade está estruturada em códigos sociais de inter-relação entre seus membros e entre os de outra sociedade. Estão imersos em costumes, princípios, regras de modos de ser estabelecidos em leis escritas ou não, de forma a agir sobre a vida e o crescimento da sociedade, tanto no sentido de suas forças produtivas como no desenvolvimento de seus valores culturais. DOSSIÊ AMAZÔNIA Nesse sentido será importante pesquisar o saber/fazer da festa da marujada para a criança, pois estas serão vistas e ouvidas como indivíduos que dão significados para as suas práticas desenvolvidas nessa manifestação, uma vez que elas vivem e compartilham suas experiências nesse contexto da festa. Sendo assim, as crianças serão percebidas como agentes participativos da sociedade em que vivem e, portanto, como homens e mulheres que dividem suas “experiências e suas concepções”. Trazer a criança para a pesquisa científica como protagonista de sua vivência e história, é compreender a sociologia da infância, que apresenta novos discursos e conceitos acerca da criança, como produtora de cultura e, concebida como forma específica de construção de conhecimento, comunicação e expressão (SARMENTO, 1997). A Sociologia da Infância propõe o estabelecimento de uma distinção analítica no seu duplo objeto de estudo: as crianças como atores sociais, nos seus mundos de vida, e a infância, como categoria social do tipo geracional, socialmente construída. A infância é relativamente independente dos sujeitos empíricos que a integram, dado que ocupa uma posição estrutural. (SARMENTO, 1997, p. 07). Na perspectiva da Sociologia da Infância, as crianças apresentam expressões interpretativas e atitudes que ajudam a compreender elementos de transformações e maneiras de ver e viver das sociedades, pois é um campo de estudo que se propõe a construir a infância como “objeto sociológico”, assim como ela “compreende a criança como objeto de investigação, contribuindo para o próprio conhecimento não apenas da infância, mas como uma categoria social” (ALVES, 2014, p. 37). Cohn (2005) aborda em suas ideias que a infância é uma fase de construção de aprendizagens em que a criança é um ser ativo e possuidor de um papel importante nas relações sociais, visto que ela é “atuante é aquela que tem um papel ativo na constituição das relações sociais em que se engaja, [...] não sendo, portanto, passiva na incorporação de papéis e comportamentos sociais” (CONH, 2005, p. 28). Nessa perspectiva e discussão a criança é um sujeito que interage com sua realidade, um ser que, ao participar das interações e relações por meio da comunicação com seus pares, vai construindo a sua identidade e a sua subjetividade. DOSSIÊ AMAZÔNIA Para uma análise consistente das narrativas de crianças sobre o fazer/saber da marujada, o artigo está dividido em quatro seções: a primeira consiste nas escritas introdutórias levando em consideração a apresentação do objeto de estudo em uma perspectiva teórica com as categorias cultura, educação, saberes e infância. A segunda, faz uma breve análise do caminho percorrido e as técnicas de pesquisa utilizadas com recorte para a marujada de Tracuateua-PA. A terceira, mostra as principais características da festa da marujada destacando sua origem. A quarta, trata das narrativas das crianças sobre os saberes e fazeres inscritas na festa da marujada de Tracuateua/PA. 1 O CAMINHAR DA PESQUISA: PERCURSO METODOLÓGICO O referido estudo sobre narrativas de crianças sobre festas amazônicas fez um recorte para obtenção de dados por meio da festa da marujada de Tracuateua/PA. Este é um 87 DOSSIÊ AMAZÔNIA município localizado na região do salgado, especificamente na mesorregião Nordeste do Pará, pertencente a microrregião bragantina. Territorialmente contém uma área de 936,1 km². Teve sua origem com a construção da ferrovia Belém-Bragança por volta de 1908. município localizado na região do salgado, especificamente na mesorregião Nordeste do Pará, pertencente a microrregião bragantina. Territorialmente contém uma área de 936,1 km². Teve sua origem com a construção da ferrovia Belém-Bragança por volta de 1908. Figura 1- Mapa de localização do município de Tracuateua- PA na Amazônia Legal Fonte: Elaborado por Pinheiro (Janeiro/2016) Tracuateua conta com aproximadamente 32.000 habitantes. Distribuídos entre a zona urbana e rural do município. Estas unidades locais, limita-se a leste com o município de Bragança; ao sul com o município de Santa Luzia do Pará; ao norte com o oceano Atlântico; a oeste com os municípios de Capanema e Quatipuru. Está localizado a 196 quilômetros da capital do estado do Pará – Belém. Foi desmembrado de Bragança em 1994, mas só se tornou município dois anos depois, em 1996. Elevado à categoria de município continuando com a denominação de Tracuateua, pela lei estadual nº 5858, de vinte e nove de novembro de mil novecentos e noventa e quatro (29/09/1994), desmembrado de Bragança (IBGE, 2016). O município concentra muitos encantos festivos, dentre eles está o círio de Nossa Senhora de Nazaré que expressa a manifestação de fé das famílias católicas e a festa da marujada, outra manifestação de devoção e fé que encanta e contagia os participantes, e os que somente apreciam. Essa festa da Marujada é realizada e apresentada no salão conhecida como “Templo ou Salão da Marujada”, localizado no centro da cidade ao lado da praça principal, conforme figura abaixo: Figura 1- Mapa de localização do município de Tracuateua- PA na Amazônia Legal Fonte: Elaborado por Pinheiro (Janeiro/2016) Figura 1- Mapa de localização do município de Tracuateua- PA na Amazônia Legal Fonte: Elaborado por Pinheiro (Janeiro/2016) Tracuateua conta com aproximadamente 32.000 habitantes. Distribuídos entre a zona urbana e rural do município. Estas unidades locais, limita-se a leste com o município de Bragança; ao sul com o município de Santa Luzia do Pará; ao norte com o oceano Atlântico; a oeste com os municípios de Capanema e Quatipuru. 1 O CAMINHAR DA PESQUISA: PERCURSO METODOLÓGICO Está localizado a 196 quilômetros da capital do estado do Pará – Belém. Foi desmembrado de Bragança em 1994, mas só se tornou município dois anos depois, em 1996. Elevado à categoria de município continuando com a denominação de Tracuateua, pela lei estadual nº 5858, de vinte e nove de novembro de mil novecentos e noventa e quatro (29/09/1994), desmembrado de Bragança (IBGE, 2016). Tracuateua conta com aproximadamente 32.000 habitantes. Distribuídos entre a zona urbana e rural do município. Estas unidades locais, limita-se a leste com o município de Bragança; ao sul com o município de Santa Luzia do Pará; ao norte com o oceano Atlântico; a oeste com os municípios de Capanema e Quatipuru. Está localizado a 196 quilômetros da capital do estado do Pará – Belém. Foi desmembrado de Bragança em 1994, mas só se tornou município dois anos depois, em 1996. Elevado à categoria de município continuando com a denominação de Tracuateua, pela lei estadual nº 5858, de vinte e nove de novembro de mil novecentos e noventa e quatro (29/09/1994), desmembrado de Bragança (IBGE, 2016). q g ç O município concentra muitos encantos festivos, dentre eles está o círio de Nossa Senhora de Nazaré que expressa a manifestação de fé das famílias católicas e a festa da marujada, outra manifestação de devoção e fé que encanta e contagia os participantes, e os que somente apreciam. Essa festa da Marujada é realizada e apresentada no salão conhecida como “Templo ou Salão da Marujada”, localizado no centro da cidade ao lado da praça principal, conforme figura abaixo: q g ç O município concentra muitos encantos festivos, dentre eles está o círio de Nossa Senhora de Nazaré que expressa a manifestação de fé das famílias católicas e a festa da marujada, outra manifestação de devoção e fé que encanta e contagia os participantes, e os que somente apreciam. Essa festa da Marujada é realizada e apresentada no salão conhecida como “Templo ou Salão da Marujada”, localizado no centro da cidade ao lado da praça principal, conforme figura abaixo: NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 88 Figura 2 - Localização do salão da Marujada em Tracuateua/PA DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 89 presença do outro, respeitando-o como pessoas e cidadão”. Nisso também nos preocupamos com o que defende Kramer (1994, p. 32), pois, a decisão de participar é uma opção da criança “que não deve ser pressionada [...] e que definem em comum acordo com o pesquisador, os nomes, fictícios ou não, a fim de resguardar a privacidade/identificação”. Figura 2 - Localização do salão da Marujada em Tracuateua/PA g ç j Fonte: Elaborado por Pinheiro (Janeiro/2016) Fonte: Elaborado por Pinheiro (Janeiro/2016) A partir do recorte da pesquisa teve-se a preocupação de seguir os princípios da Abordagem Qualitativa (MINAYO, 2015), por acreditar que, além de responder questões bem particulares, ela trabalha com o sentido e o significado, nos mais variados universos. A partir do recorte da pesquisa teve-se a preocupação de seguir os princípios da Abordagem Qualitativa (MINAYO, 2015), por acreditar que, além de responder questões bem particulares, ela trabalha com o sentido e o significado, nos mais variados universos. Por considerar que os intérpretes deste estudo são as crianças, isso sinaliza um maior dinamismo para coleta dos dados. Dessa forma, procuramos não delimitar a pesquisa a uma única técnica de coleta de dados, para isso escolhemos: a) a observação participante; b) a roda de conversa; c) o diário de campo; d) o registro fotográfico e d) a técnica do desenho. Esta última somente como uma dinâmica de aproximação com as crianças e não como técnica de análise. Assim, tais técnicas possibilitaram a compreensão dos processos ritualísticos, bem como da circulação de saberes vivenciados na marujada (LAVILLE; DIONNE, 1999, p.176). ç j p Para o tratamento e análise dos dados, foi utilizada a técnica de análise de conteúdo, que trata de um meio de recursos metodológicos que se aperfeiçoam sempre e que se aplicam a diferentes falas e discursos. Esta técnica permite o tratamento das informações com objetivos bem definidos e que servem para desvelar o que está oculto no texto. A Análise de Conteúdo “atua sobre a fala [...]. Ela descreve, analisa e interpreta as mensagens/enunciados de todas as formas de discurso, procurando ver o que está por detrás das palavras” (BARDIN, 1994, 58). É importante lembrar que essa pesquisa também se preocupa com o que diz respeito aos seus aspectos éticos, para isso procuramos nos assegurar e obedecer ao que rege a Resolução 196/96, da Organização Mundial da Saúde – OMS, garantindo o anonimato em relação aos nomes verdadeiros dos sujeitos no Consentimento Livre e Esclarecido (TCLE) para as crianças e para os seus responsáveis, deixando claro os procedimentos no que diz respeito aos sujeitos menores de idade, os quais fazem parte deste estudo. Nesse sentido nos alertam Oliveira e Motta Neto (2011, p. 13) ao abordarem os cuidados éticos na pesquisa, pois “assumir responsabilidade na e com a pesquisa é assumir a A grande prosperidade da região de Bragança e entorno, ocasionada pela lavoura de produtos agrícolas, notadamente mandioca, arroz, feijão, tabaco, milho e arroz a produção de gado e peixe, propiciou um grande fluxo de escravos para a mão de obra entre os séculos XVIII e XIX. Sendo assim, a região bragantina foi notada por representar uma fronteira com a Província do Maranhão, com a entrada de escravos dali e de seus engenhos, de suas lavouras, de suas minas de ouro. É natural assim nesse contexto que as tensões entre senhores e escravos fossem mais frequentes e para controlar essas tensões existiras estratégias como as permissões de criação de irmandade entre os negros pelos seus senhores. (FERNADES, 2011, p.58-59). 2 A FESTA DA MARUJADA: PRINCIPAIS CARACTERÍSTICAS DE SUA ORIGEM E TRADIÇÃO A marujada é uma festa de caráter religioso que se faz presente em algumas partes do Brasil, no estado do Pará, como Bragança, Tracuateua, Augusto Correa, Primavera e Quatipuru com algumas características semelhantes como os rituais das danças, a louvação aos santos e a indumentária; assim como existem aspectos que as diferenciam em seus significado que é constatado no estudo de Amorim (2008, p. 26), e ainda em um estudo realizado pelo Instituto de Artes do Pará, pelo autor Silva (2006), a existência da festa da marujada também no município de Ananindeua. j p Nos estudos de Silva (1981) intitulado “Contribuições ao Estudo do Folclore Amazônico na Zona Bragantina” a Marujada de Bragança, como culto religioso, está ligada a Irmandade de São Benedito no Pará, caracterizada como festa religiosa. Ela teria seu marco histórico e sua origem, em Bragança/PA, provavelmente “no século XVIII (1798), com a permissão dos senhores brancos aos seus escravos para exaltar e apreciar São Benedito” (SILVA, 1981, p. 32). Essa permissão dada aos escravos realizaria os seus rituais em louvor à São Benedito, formando assim a organização de uma irmandade e, partir daí acontece a primeira festa e ocorre o primeiro compromisso com o Estatuto da referida irmandade. A origem dessa festa é vinculada à um contexto histórico da colonização de grande concentração de quilombos que se estendiam de Bragança a Ourém, no estado do Pará. Desse contexto resultaram muitas manifestações de origens negras africanas, pois em Bragança e seus arredores concentravam atividades agrícolas a qual proporcionou um fluxo de mão de obra de escravos para tal região. A grande prosperidade da região de Bragança e entorno, ocasionada pela lavoura de produtos agrícolas, notadamente mandioca, arroz, feijão, tabaco, milho e arroz a produção de gado e peixe, propiciou um grande fluxo de escravos para a mão de obra entre os séculos XVIII e XIX. Sendo assim, a região bragantina foi notada por representar uma fronteira com a Província do Maranhão, com a entrada de escravos dali e de seus engenhos, de suas lavouras, de suas minas de ouro. É natural assim nesse contexto que as tensões entre senhores e escravos fossem mais frequentes e para controlar essas tensões existiras estratégias como as permissões de criação de irmandade entre os negros pelos seus senhores. (FERNADES, 2011, p.58-59). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 90 seus irmãos com a imagem do Santo em comemoração a legitimidade de sua irmandade. A partir daí essa festa foi sendo repassada de geração a geração fazendo louvores ao santo padroeiro, ganhando uma nova roupagem e renovando e incluindo alguns rituais sem perder, é claro, sua originalidade negra e africana. seus irmãos com a imagem do Santo em comemoração a legitimidade de sua irmandade. A partir daí essa festa foi sendo repassada de geração a geração fazendo louvores ao santo padroeiro, ganhando uma nova roupagem e renovando e incluindo alguns rituais sem perder, é claro, sua originalidade negra e africana. A marujada é caracterizada como uma dança de caráter religioso, formada por homens e mulheres, chamados de marujos e marujas, que dançam em devoção ao Santo. Nesse sentido, Silva (1997), apresenta a marujada como dimensão simbólica com ritual de dança apresentada na festa de São Benedito como um momento que privilegia a construção da identidade de um grupo especifico, no contexto cultural da festa e na sociedade em que esta manifestação está presente. Sobre a origem da marujada de Tracuateua, nos informou o presidente da Associação de São Benedito e São Sebastião de Tracuateua (AMSSSBT) e vice capitão da marujada, que nesse município, essa festa foi fundada em junho de 1946, pelo vereador e comerciante, José Olegário Pinheiro, conhecido como José Maranhense. O mesmo apreciava muito essa festa e solicitou ao presidente da Irmandade de Bragança, que fosse permitido uma apresentação no ano de 1946 na vila de Tracuateua. Com o pedido concedido, a comunidade de Tracuateua, recebeu pela primeira vez uma apresentação dos marujos e marujas de Bragança. No entanto, no ano seguinte, a irmandade de Bragança não enviou seus marujos e marujas para outra apresentação, sob alegação de falta de recursos, foi então que a comunidade tracuateuense decidiu formar e fundar sua própria festa da marujada nos mesmos moldes da apresentação Bragantina com devoção a São Benedito e São Sebastião. Esse último faz parte da Festividade porque é Santo Padroeiro do Município de Tracuateua. E assim se realiza no município as homenagens aos santos padroeiros nos dias 19 e 20 de janeiro. Sobre a origem da marujada de Tracuateua, Reis (2015, p. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 14) destaca as contribuições do historiador Antônio Jorge da Silva, ao descrever que a marujada nesse município tem relações com os seguintes aspectos: [...] proveio do fato de os irmãos portugueses Augusto e Antônio Pio, ao virem do Rio de Janeiro, trouxeram o culto de São Sebastião, realizado no dia 20 de Janeiro, para a localidade de Tracuateua. [...] o vereador José Olegário Pinheiro pediu à marujada de Bragança que dançasse durante os festejos de São Sebastião. A partir daí, ficou a prática de “a Marujada de Bragança” dançar no dia 19 de janeiro, antes da festa do santo padroeiro, São Sebastião. Porém, houve um ano em que a Marujada de Bragança não pode participar da festividade de Tracuateua por falta de condução para os marujos. Como já estava tudo organizado, José Olegário pediu a um grupo de dez marujos, residentes na cidade, mas que faziam parte da irmandade de Bragança, que os substituísse. Após esse contratempo, a comunidade resolveu fundar sua própria irmandade aos moldes da bragantina. Assim, em 9 de junho de 1946, foi criada a Marujada de Tracuateua. (REIS, 2015, p.14). Nesse sentido, podemos perceber e analisar que a origem da marujada de Tracuateua tem influências da marujada de Bragança, visto que essa manifestação cultural se movimenta nas espacialidades da microrregião bragantina articulando e desenvolvendo as práticas culturais de sua tradição, o que de alguma forma, mantém essa festa viva e presente culturalmente. 2 A FESTA DA MARUJADA: PRINCIPAIS CARACTERÍSTICAS DE SUA ORIGEM E TRADIÇÃO A grande prosperidade da região de Bragança e entorno, ocasionada pela lavoura de produtos agrícolas, notadamente mandioca, arroz, feijão, tabaco, milho e arroz a produção de gado e peixe, propiciou um grande fluxo de escravos para a mão de obra entre os séculos XVIII e XIX. Sendo assim, a região bragantina foi notada por representar uma fronteira com a Província do Maranhão, com a entrada de escravos dali e de seus engenhos, de suas lavouras, de suas minas de ouro. É natural assim nesse contexto que as tensões entre senhores e escravos fossem mais frequentes e para controlar essas tensões existiras estratégias como as permissões de criação de irmandade entre os negros pelos seus senhores. (FERNADES, 2011, p.58-59). É, portanto, neste cenário de inquietude e aflição entre senhores e pessoas escravizadas que surge a Festa da Marujada em Bragança/PA. Contexto no qual eram expressas grades tensões entre senhores escravizados, sendo a legalização da irmandade de São Benedito uma permissão e forma de controlar tais inquietudes entre pessoas escravizadas no período colonial. Silva (1997) esclarece que foi especificamente no dia 03 de setembro de 1798, a pedido de 14 pessoas escravizadas, os senhores permitiram que fosse organizada a irmandade da marujada de São Benedito de Bragança. Em gratidão à graça alcançada, os escravos saíram às ruas de Bragança dançando em frente às casas de seus senhores, fazendo evoluções coreográficas. Tal fato repetiu-se com novos agradecimentos nos anos seguintes dando origem à marujada, manifestação atrelada à esta de São Benedito, comportando o sagrado ao popular. Sendo permitida essa irmandade, os negros começaram a dançar de casa em casa d DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 91 história dessa festividade por um simbolismo que se reverte nos saberes e fazeres dessa tradição para seus integrantes. As crianças integrantes da marujada relatam que a marujada de Tracuateua é formada por muitos rituais os quais representam um saber e um fazer da festa. Ao Perguntar sobre o significado destes rituais, Nicole (09 anos) Rafael (11 anos) e Maria Guilhermina (12 anos) responderam que as danças e rezas são práticas que todos os marujos devem fazer, pois essas práticas estão relacionadas as suas promessas e dos demais devotos, visto que as crianças desenvolvem relações de um saber/fazer vivenciadas no seu cotidiano o que representa também atores sociais, nos seus mundos de vida e em suas e a infâncias (SARMENTO, 2000). O sentido da marujada é vista por seus integrantes como uma continuação de uma tradição, na qual filhos e netos dão continuidade para resistência dessa manifestação. Tal tradição é vivida por meio dos seus saberes e fazeres que estão presentes em seu contexto. Esses saberes compõem e são desenvolvidos por um movimento educativo organizados pelos seus sujeitos, pois Brandão (2006, p. 17) ao falar de educação leva-nos a perceber que ela consiste e existe pela existência da própria vida. Seu desenvolvimento está presente em qualquer espaço que haja a circulação de saberes e fazeres, visto que: [...] Ninguém escapa da educação. Em casa, na rua, na igreja ou na escola, de um modo ou de muitos todos nós envolvemos pedaços da vida com ela: para aprender, para ensinar, para aprender - e - ensinar. Para saber, para fazer, para ser ou para conviver, todos os dias misturamos a vida com a educação. (BRANDÃO, 2007, p. 03). [...] Ninguém escapa da educação. Em casa, na rua, na igreja ou na escola, de um modo ou de muitos todos nós envolvemos pedaços da vida com ela: para aprender, para ensinar, para aprender - e - ensinar. Para saber, para fazer, para ser ou para conviver, todos os dias misturamos a vida com a educação. (BRANDÃO, 2007, p. 03). Nessa perspectiva, destacaremos alguns desses principais saberes/fazer observados e praticados pelas crianças como uma prática educativa vivida e compartilhada pela percepção delas sobre tais rituais. 3 DA LEVANTAÇÃO À DERRUBAÇÃO: O SABER/FAZER DA MARUJADA PELAS NARRATIVAS DAS CRIANÇAS A festa da Marujada de Tracuateua/PA, em devoção a São Benedito e São Sebastião, condensa muitos dos elementos vivenciados pela cultura dos negros africanos, pois nessa festa se observa que o grupo vivencia aspectos como a fé a devoção que estão imbricados na a) A Levantação dos mastros: o início da festa O sentido desse ritual está no fato de que é a partir da hora que sobe o mastro, começa a festa, e os integrantes da marujada são abençoados por São Benedito e São Sebastião. Tal ritual acontece no dia 18 (dezoito) de janeiro, dia que antecede a primeira homenagem aos santos da festa. A maioria dos integrantes e pessoas que acompanham a marujada geralmente se faz presente nessa levantação do mastro. Na levantação do mastro começa a festa. [...] Os dois mastros é levantado, a gente tem que dançar, rezar. As pessoas batem palmas e as marujas dançam. Aí começa. (MARIA GUILHERMINA, 11 anos de idade – RODA DE CONVERSA – JANEIRO/2016) É Quando levanta o mastro, é a hora de começar a festa. É muito legal. Aí tem gente que dança no dia de São Benedito, no dia de São Sebastião [...]. Era bom se a festa tivesse mais dias. (RAFAELA, 12 anos- RODA DE CONVERSA – JANEIRO/2016). A fala dos intérpretes fica claro que a levantação dos mastros simboliza o início da festa por ser uma ocasião importante. É o momento em que todos os marujos e marujas são convidados e convocados a participarem, seja para erguê-lo ou dançar ao seu redor. Os mastros são enfeitados nas residências dos juízes, ou seja, marujos ou marujas promesseiros ficam encarregados e têm a responsabilidade de organizarem a ornamentação e a alimentação que será servida na festa, inclusive organizando a levantação dos mastros. As figuras a seguir mostram o exato momento em que os marujos estão levantando o mastro de São Sebastião. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 92 Figuras 3 e 4 – Ornamentação e Levantação do mastro de São Sebastião Fonte: Arquivo da Pesquisadora (2016) Nas palavras de Debora (11 anos de idade) esse momento da arrumação do mastro representa muita devoção, pois “esses enfeites do mastro são os marujos que dão, porque eles fazem promessa pra dar. Quem faz a promessa, tem que oferecer”. Fonte: Arquivo da Pesquisadora (2016) Fonte: Arquivo da Pesquisadora (2016) Nas palavras de Debora (11 anos de idade) esse momento da arrumação do mastro representa muita devoção, pois “esses enfeites do mastro são os marujos que dão, porque eles fazem promessa pra dar. Quem faz a promessa, tem que oferecer”. a) A Levantação dos mastros: o início da festa Nas palavras de Debora (11 anos de idade) esse momento da arrumação do mastro representa muita devoção, pois “esses enfeites do mastro são os marujos que dão, porque eles fazem promessa pra dar. Quem faz a promessa, tem que oferecer”. b) Ritual alimentar: c) A procissão: Após o almoço de São Sebastião, no segundo dia da festa, precisamente no dia 20 de janeiro, as expectativas dos participantes se voltam para a procissão. É nesse momento que os devotos manifestam sua devoção pelos santos São Benedito e São Sebastião, pois esse ritual da procissão marca a religiosidade e crença na interseção dos referidos santos, conforme o que segue. Figura 7 - Procissão de São Benedito e São Sebastião Fonte: Arquivo da Pesquisadora (Janeiro/2016) Figura 7 - Procissão de São Benedito e São Sebastião Fonte: Arquivo da Pesquisadora (Janeiro/2016) Saindo da igreja matriz a procissão inicia as dezesseis horas e trinta minutos, percorrendo as principais ruas da cidade. Este percurso dura aproximadamente duas horas, após isso, a procissão retorna para a igreja. As marujas vão na caminhada formando duas filas nas laterais das ruas e os marujos levam o andor revezando-se entre eles na tarefa de carregar e proteger o andor durante toda a procissão, conforme pode ser observado na foto a seguir. As crianças também acompanham a procissão, principalmente aquelas que são promesseiras. Elas não possuem um lugar especifico nesse ritual, ficam espalhadas pelo meio ou no colo dos adultos e normalmente estão acompanhas dos seus responsáveis. A procissão reúne toda a comunidade de devotos de São Benedito e São Sebastião. Ressaltamos que esse ritual representa um movimento de união, congregando pessoas das mais diferentes classes socioeconômicas, pois a caminhada é formada por marujos e marujas, religiosos, padres, autoridades, políticos e comunidade em geral. Ao chegar na igreja, o andor com as imagens é recebido pelo padre, que abençoa os marujos e os participantes da procissão. Ainda nessa prática, os marujos e marujas ao chegarem em frente à igreja retiram seus chapéus da cabeça e colocam em direção as imagens dos santos, como forma de agradecimentos e em gesto de reverência. Isso pode ser comprovado pelas intérpretes Catarina (08 anos de idade), Kiara (09 anos) e Júlio (09 anos de idade): Catarina: Eu danço faz tempo, porque eu pago promessa. (CATARINA, 08 anos de idade). Kiara: Eu também acompanho a procissão, porque minha mãe fez uma promessa pra mim. (KIARA, 09 anos de idade). Júlio: Eu pago promessa porque eu fiquei doente.ai todo ano eu acompanho a procissão. A minha mãe diz que é pra agradecer. Porque eu fiquei bom. (JÚLIO, 09 anos). b) Ritual alimentar: O ritual do almoço e do jantar assumem diversos significados, entre os quais, se destaca a comunhão dos santos e seus devotos. Oferecer o almoço e o jantar oficiais da festa é a principal atribuição e responsabilidade dos juízes da festa. As refeições são servidas na parte interior do salão, onde se encontra o refeitório dos marujos e marujas, conforme figuras a seguir. Figuras 5 e 6 - Ritual alimentar de São Sebastião e São Benedito Fonte: Arquivo da Pesquisadora (Janeiro/2016) Isso pode ser observado na fala das intérpretes Kiara (09 anos de idade) ao ressaltar a importância desse momento: “eu gosto também da hora do almoço, porque a gente come, tem até sobremesa. Todas as crianças comem” e Raiana (10 anos de idade) ao esclarecer que “Essa hora do almoço é sempre bom. Eles servem todo mundo, ninguém fica sem comer”. Percebe- se que nesse ritual há dedicação dos juízes em servir aos marujos destacando a importância para a realização da festa. Figuras 5 e 6 - Ritual alimentar de São Sebastião e São Benedito Fonte: Arquivo da Pesquisadora (Janeiro/2016) Figuras 5 e 6 - Ritual alimentar de São Sebastião e São Benedito Fonte: Arquivo da Pesquisadora (Janeiro/2016) Fonte: Arquivo da Pesquisadora (Janeiro/2016) Isso pode ser observado na fala das intérpretes Kiara (09 anos de idade) ao ressaltar a importância desse momento: “eu gosto também da hora do almoço, porque a gente come, tem até sobremesa. Todas as crianças comem” e Raiana (10 anos de idade) ao esclarecer que “Essa hora do almoço é sempre bom. Eles servem todo mundo, ninguém fica sem comer”. Percebe- se que nesse ritual há dedicação dos juízes em servir aos marujos destacando a importância para a realização da festa. DOSSIÊ AMAZÔNIA 93 Catarina: Eu danço faz tempo, porque eu pago promessa. (CATARINA, 08 anos de idade). Kiara: Eu também acompanho a procissão, porque minha mãe fez uma promessa pra mim. (KIARA, 09 anos de idade). Júlio: Eu pago promessa porque eu fiquei doente.ai todo ano eu acompanho a procissão. A minha mãe diz que é pra agradecer. Porque eu fiquei bom. (JÚLIO, 09 anos). d) Os rituais da dança O estatuto da associação da marujada de Tracuateua (2000) define como danças tradicionais sete rituais de dança, tais como: a roda, o retumbão, o chorado, a mazurca, a valsa, o xote e a contradança. Essas dançam também representam o saber/fazer da marujada, pois as danças carregam uma simbologia e um movimento da história dessa tradição. A roda marca o início e o fim dos rituais da dança e restabelece, portanto, a simbologia do mito de origem da marujada (SILVA, 1997). Informa o autor que a dança lembra a origem nos terreiros de macumba, pois se pede permissão, mesmo que simbolicamente, para iniciar a dança. Tal fato revive o rito de duzentos e onze anos, quando os negros pediam permissão aos seus senhores para dançar de casa em casa. O ritual da roda é caracterizado pela circularidade dos gestos e movimentos, a dança é executada com os pés descalços, onde as mulheres marujas fazem um círculo no salão e dançam ao som dos instrumentos. Ao centro da roda dançam abraçados o capitão, capitoa e vice-capitoa que reverenciam ao público que assiste a dança. Nesse gesto, as autoridades da marujada informam e pedem licença para começar sua apresentação. Através dessa prática, pede-se licença simbolicamente aos presentes, autoridades, para iniciar a dança tal como, segundo a tradição, há quase dois séculos os negros pediam autorização aos seus senhores para dançar de casa em casa (SILVA, 1997, p. 209). Marcada pela circularidade, na roda existe uma hierarquia que deve ser respeitada. Trata-se de uma coreografia formada por um círculo humano de marujas. Nesse ritual, as marujas são orientadas pela capitoa, que carrega nos braços um bastão3, e a vice-capitoa, as quais são superiores das demais marujas hierarquicamente. Elas conduzem e comandam as marujas mostrando os movimentos e a direção da dança pelo salão. Depois de alguns minutos de dança, as líderes saem da roda para ficarem no centro dela, conforme figuras a seguir. 3 Dois ramos de flores artificiais nas cores vermelha e azul que formam um só. Esse ramo simboliza a sua autoridade. Silva (1997) informa que esse bastão de flores faz alegoria ao chicote com que os senhores açoitavam seus escravos. Era o símbolo do poder dos que estavam sob sua autoridade. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 94 muitos choram, muitos aplaudem e outros ficam de joelhos, talvez pedindo uma graça ou agradecendo outras. Certamente é um momento de emoção inesquecível. muitos choram, muitos aplaudem e outros ficam de joelhos, talvez pedindo uma graça ou agradecendo outras. Certamente é um momento de emoção inesquecível. c) A procissão: Descrever esse ritual da procissão é, sobretudo, falar da emoção expressa nos rostos dos fiéis, pois no momento em que o andor chega na igreja e ao som dos sinos, muitos rezam, DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 95 O retumbão e o chorado: O primeiro se constitui na sequência dos rituais. É o segundo ritmo dança da marujada e representa uma situação de “margem vivida pelo homem”, pois o marujo, a uma condição de centro, isto é, ao contrário da roda, nele é o marujo quem inicia o ritual. Como ressalta a fala do intérprete Júlio (09 anos de idade) quanto sua preferência pelo ritual: “Eu gosto mais do retumbão. [...]. Ah! Quando eu escuto o retumbão, eu quero logo dançar! O retumbão é bonito, é legal”. Nessa dança é sempre o marujo que inicia. Depois a gente chama a maruja” (RODAS DE CONVERSA – JANEIRO/2016). É g j ( ) Na ordem das apresentações o chorado é o terceiro ritual, isto é, a terceira dança. É uma variação do retumbão, a diferença está no ritmo que é mais lento e sensual e, no número de pares que se apresentam. Nessa dança somente um casal se apresenta por vez. Assim, como no retumbão é o marujo que dá início ao ritual. Para Bordallo da Silva (1981) e Silva (1997), o retumbão e o chorado possui compasso musical e ritmo do lundu, modificado progressivamente da senzala ao salão aristocrático. Descrevem que o termo retumbão tem origem portuguesa, pelo fato de que era possível escutar o ritmo de locais distantes, onde o som “retumbava”. A orquestra para entoar a dança, pois não há canto, compõem-se de tambores grandes, pandeiros, cuíca (onça), rabeca, viola, cavaquinho e violino. Esses ritmos apresentam “elementos que lembram não apenas o lundu”, isto é, a dança em roda ou em círculos; “o bailado aos pares; os movimentos do corpo; o castanholar dos dedos, a marcação das palmas e o uso da viola” (SILVA, 1997, p. 219), conforme figuras abaixo. Figuras 10 e 11- Ritual de dança: o retumbão e o chorado Fonte: Arquivo da Pesquisadora (Janeiro/2016) Figuras 10 e 11- Ritual de dança: o retumbão e o chorado Figuras 10 e 11- Ritual de dança: o retumbão e o chorado Fonte: Arquivo da Pesquisadora (Janeiro/2016) Ao observar esses três primeiros rituais da marujada e ao considerar o que diz Silva (1997, p. 219), nota-se que eles apresentam em suas coreografias, elementos, passos e gestos que lembram o lundu, a dança em roda ou em círculos, embora na marujada esse bailado seja mais ameno, “talvez por ter sofrido algum tipo de resistência eclesiástica”. d) Os rituais da dança Figuras 8 e 9 - O ritual de dança: a roda de São Benedito e São Sebastião Figuras 8 e 9 - O ritual de dança: a roda de São Benedito e São Sebastião Fonte: Arquivo da Pesquisadora (Janeiro/2016) Após a capitoa e vice-capitoa se dirigirem ao centro da roda, elas se juntam ao capitão e ambos fazem reverência aos juízes e juízas da festa, os quais permanecem sentados no salão esperando que todas as marujas da roda façam o mesmo gesto. Fonte: Arquivo da Pesquisadora (Janeiro/2016) Após a capitoa e vice-capitoa se dirigirem ao centro da roda, elas se juntam ao capitão e ambos fazem reverência aos juízes e juízas da festa, os quais permanecem sentados no salão esperando que todas as marujas da roda façam o mesmo gesto. Após a capitoa e vice-capitoa se dirigirem ao centro da roda, elas se juntam ao capitão e ambos fazem reverência aos juízes e juízas da festa, os quais permanecem sentados no salão esperando que todas as marujas da roda façam o mesmo gesto. DOSSIÊ AMAZÔNIA A mazurca, a valsa e o xote: Esses “constituem como danças tardias, ou seja, foram incorporadas posteriormente à estrutura ritualística da marujada”. A mazurca e a valsa possui características europeias, especificamente do século XIX, mas ao fazer parte dos rituais da marujada assumiu características próprias como dança em roda e o uso dos pés descalços. (SILVA, 1997). e) A derrubação do mastro e a varrição da festa: e) A derrubação do mastro e a varrição da festa: não existe só na escola ou nas instituições de ensino, Não há uma forma única nem um único modelo de educação; a escola não é o único lugar onde ela acontece e talvez nem seja o melhor; o ensino escolar não é a sua única prática e o professor profissional não é o seu único praticante. (BRANDÃO, 2006, p. 04). Por isso, a narrativas das crianças sobre suas experiências e vivencias na marujada revela esse espaço como um lugar de aprendizagens de humanidades, porque as crianças também instigam e protagonizam as práticas em seus contextos. Não são apenas os adultos que intervêm junto das crianças, mas as crianças também intervêm junto dos adultos. As crianças não recebem apenas uma cultura constituída que lhes atribui um lugar e papéis sociais, mas operam transformações nessa cultura, seja sob a forma como a interpretam e integram, seja nos efeitos que nela produzem, a partir das suas próprias práticas (SARMENTO, 2000, p. 152). Dessa forma, partindo da diversidade de conhecimento construídos no cotidiano da marujada, identificou-se saberes e fazeres inerentes a festa vivenciados e partilhados do/pelo movimento entre marujos e marujas de diferentes gerações por meio da escuta, da observação e da oralidade. Nesse movimento da cultura, onde ela circunscreve as expressões da criança de forma geral, como “as ideias, os valores, e as emoções são produtos culturais manufaturados”, ou seja, são tecidas pelas mãos dos sujeitos, sendo que para a compreensão dessas expressões ou desses signos é necessário compreender o significado e chegar até “os conceitos específicos das relações entre eles” (GEERTZ, 2014, p. 36-37). Em outras, palavras podemos dizer que os saberes e fazeres representam processos educativos vinculados as diversas práticas culturais, sendo estas a representatividade das expressões humanas em seus mais variados contextos caracterizando, dessa forma, as manifestações culturais como espaços de aprendizagens. e) A derrubação do mastro e a varrição da festa: Brandão (2002) na sua análise entre cultura e educação, deixa claro que a educação 97 DOSSIÊ AMAZÔNIA não existe só na escola ou nas instituições de ensino, e) A derrubação do mastro e a varrição da festa: A derrubada dos mastros representa o final da festa, conforme relata a intérprete Débora (11 anos): “Quando chega a varrição a gente fica triste, porque já vai terminar a festa” e Maria Guilhermina (12 anos): NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 96 Na hora que derrubam os mastros a gente começa a danças ao redor deles. Depois a gente fica esperando os brinquedos, (...) porque eles são dados para as crianças. Os brinquedos dos santos. Mais todos ficam meio triste porque a festa tá acabando. Ai tem que esperar o outro ano. (RODA DE CONVERSA/JANEIRO/2016). A derrubação dos mastros, conforme falam as crianças, significa que àquele ciclo se encerra para organização de ciclo vindouro. Figura 12 - Derrubação do mastro Fonte: Arquivo da Pesquisadora (Janeiro/2016) Na noite da derrubada dos mastros, data do dia 21 de todos os anos, os marujos e Figura 12 - Derrubação do mastro Fonte: Arquivo da Pesquisadora (Janeiro/2016) Na noite da derrubada dos mastros, data do dia 21 de todos os anos, os marujos e marujas são convidados para o ritual da varrição todos devem varrer a festa, isto implica dizer que os marujos e marujas varrem, no sentido de repetir todos os rituais de dança. Nesse ritual da varrição qualquer pessoa pode participar, desde que ela esteja vestida adequadamente segundo as exigências da associação. No final da festa, as pessoas presentes aplaudem e as marujas, ao som dos instrumentos musicais, fazem uma roda ao redor dos mastros e dançam. Em seguida todos se dirigem ao salão da marujada para juntos varrerem a festa, conforme explicam Maísa e Rafaela (12 anos): “(...) depois da derrubação dos mastros a gente varre a festa. Nesse dia todos dançam. E a gente pode escolher qualquer roupa a de são Benedito ou a de São Sebastião”. Podemos analisar, por meio desses saberes e fazeres vivenciados pela criança que a educação está presente nesse contexto na festa da marujada de Tracuateua/PA. Uma educação que está pautada na diversidade do conhecimento, de solidariedades entre os sujeitos e em um processo de compartilhamento e coparticipação de saberes por seus membros. Em outras palavras a educação “é um triplo processo: um processo de humanização, de socialização, de subjetivação/singularização” (CHARLOT, 2013, p. 167). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 98 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 De modo geral, foi possível perceber que as crianças que participam dessa manifestação cultural aprendem e compartilham saberes. Essa aprendizagem é desenvolvida por meio da escuta, do fazer, do compartilhar com os outros e pela própria vivência no grupo. São aprendizagem que se desenvolvem a partir de processos educativos que decorrem da produção e da circulação dos saberes. 4 CONSIDERAÇÕES FINAIS Este artigo procurou analisar o saber/fazer na festa da marujada em Tracuateua/PA por meio das falas das crianças, que chamamos de narrativas, numa perspectiva cultural que envolve a circulação de saberes e, por conseguinte uma relação de aprendizagem, onde seus sujeitos relacionam-se entre si e sob práticas constituídas por um simbolismo religioso que revelam os saberes e sua relevância para a diversidade educacional amazônica. As concepções teóricas levantadas e construídas nesta pesquisa nos possibilitaram buscar novas compreensões sobre a construção científica. Essas concepções tornaram-se essenciais para a compreensão de que uma pesquisa não se faz só, mas ela é o resultado de uma construção coletiva para aquilo que se propõem apresentar. Desse modo, este trabalho foi desenvolvido com as crianças participantes da marujada de Tracuateua, que demonstraram as suas relações com os saberes que estão inscritos no contexto dessa festa. Para atingir nossos objetivos propostos nos apropriamos de referenciais teóricos que nos orientaram sobre o objeto de pesquisa e sobre os sujeitos que escolhi pesquisar, a fim de compreender respostas para muitas indagações, uma vez que fazer pesquisa com crianças requer compreender que elas são sujeitos protagonistas. Assim, foi possível perceber os diferentes saberes e fazeres que perpassam as festas na Amazônia pelo olhar da criança que se inserem e possuem suas percepções nesses contextos, pois elas adentram nesses ensinamentos desde muito cedo o que também torna possível a compreensão delas sobre determinados fazeres e saberes da marujada. A criança consegue compreender, da maneira dela, todos os processos organizacionais e simbólicos desta manifestação cultural. REFERÊNCIAS ALVES, Laura Maria Silva Araújo. A infância em construção: as fontes de investigação. In: Pesquisa e educação na Amazônia: reflexões epistemológicas e políticas/ Sônia Maria da Silva Araújo, Laura Maria Silva Araújo Alves, Sônia de Jesus Nunes Bertolo (Orgs.) – Belém: EDUEPA, 2014. AMORIN, Ana Karine Jansen. Um fogo que se deita no mar: um estudo sobra a marujada do município de Quatipuru/PA. 2008. Tese (Doutorado em Artes Cênicas) - Universidade Federal da Bahia, 2008. AMORIN, Ana Karine Jansen. Um fogo que se deita no mar: um estudo sobra a marujada do município de Quatipuru/PA. 2008. Tese (Doutorado em Artes Cênicas) - Universidade Federal da Bahia, 2008. BARDIN, Laurence. Análise de conteúdo. Lisboa: Edições Setenta, 1994. 226 p. BRANDÃO, Luis Carlos. O que é Educação? (Coleção Primeiros Passos – n 20). São Paulo: Brasiliense, 2007. BRANDÃO, Luis Carlos. Educação como Cultura. São Paulo: Cortez, 2002. CHARLOT, Bernard. Da relação com o saber: elementos para uma teoria. Porto Alegre: Artes Médicas Sul, 2013. COHN, Clarice. Antropologia da Criança / Clarice Conh – Rio de Janeiro: Jorge Zahar Ed., 2005 FERNANDES, José Guilherme dos Santos. Pés que Andam Pés que Dançam. Memória, identidade e religião cultural na esmolação e marujada de São Benedito. Coleção Saberes Amazônicos. Belém: EDUEPA. 2011. GEERTZ, Clifford. A interpretação das culturas. 1ª Ed. 13 Reimp. Rio de Janeiro. LTC, 2014. 323 p. IBGE. Instituto Brasileiro de Geografia e Estatística. Censo 2010. Disponível em: http://www.ibge.gov.br/. Acesso: 24 de jan. de 2016. LAVILLE, Christian.; DIONNE, Jean. A construção do saber. Belo Horizonte: UFMG, 1999. 340 p. LAVILLE, Christian.; DIONNE, Jean. A construção do saber. Belo Horizonte: UFMG, 1999. 340 p. KRAMER, Sonia. Currículo de Educação Infantil e a formação dos profissionais de creche e pré-escola: questões teóricas e polêmicas. In: Por uma política de formação do profissional pré-escola: questões teóricas e polêmicas. In: Por uma política de formação do profissional de Educação Infantil/MEC/SEF/COEDI - Brasília: MEC/SEF/DPE/COEDI, 1994. p.16-31. pré escola: questões teóricas e polêmicas. In: Por uma política de formação do profissional de Educação Infantil/MEC/SEF/COEDI - Brasília: MEC/SEF/DPE/COEDI, 1994. p.16-31. MINAYO, Maria Cecília. Pesquisa social: teoria, método e criatividade. 28ª edição. Petrópolis. RJ/2015 DOSSIÊ AMAZÔNIA 99 OLIVEIRA, Ivanilde Apoluceno de; MOTTA – NETO, João Colares da. A construção de categorias de análises na pesquisa em educação. In: MARCONDES, Maria Inês; OLIVEIRA, Ivanilde Apoluceno de; TEIXEIRA, Elizabete (org.). Abordagens teóricas e construções metodológicas na pesquisa em educação. Belém: EDUEPA, 2011. PINHEIRO, Márcio Duarte. Elaborador das imagens de mapas. Janeiro/2016 – VOLUME – – – PINHEIRO, Márcio Duarte. Elaborador das imagens de mapas. Janeiro/2016. REIS, Maria Helena de Aviz. Marujada de Tracuateua/PA: Um olhar sobre as manifestações culturais e religiosas na Festividade de São Benedito e São Sebastião. In: I CONGRESSO LUSÓFONO DE CIÊNCIAS DAS RELIGIÕES, 2015, Lisboa. Anais [...]Universidade Lusófona de Humanidades e Tecnologias, Lisboa – Portugal, 2015 SARMENTO, Manoel Jacinto. A infância como construção social. In: PINTO, Manuel e SARMENTO, Manuel Jacinto (coord.). As crianças: contexto e identidades. Portugal: Universidade do Minho (Centro de Estudos da Criança), 1997. SILVA, Armando Bordallo da. Contribuição ao estudo do folclore amazônico na zona Bragantina. Belém, 2 ed. 1981. Coleção Victorino Chermont de Miranda. 93 p. SILVA, Dedival Brandão. Os Tambores da Esperança: um estudo sobre cultura, religião, simbolismo e ritual na festa de São Benedito da cidade de Bragança. Falangola Editora. 1997. THOMPSON, John B. Ideologia e Cultura Moderna: teoria social crítica na era dos meios de comunicação de massa. Petrópolis, RJ: Vozes: 1995. 101 DOSSIÊ AMAZÔNIA VOLUME 202 1 M ARÇO ARÇO 2 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 102 A produção do conhecimento se constitui, cada vez mais, numa trama de relações complexas, exigindo um trabalho integrador, novas formas de interação comunicativa entre as diferentes áreas do saber (SILVA, 2010). Não podemos mais insistir numa concepção de conhecimento que se isole das dimensões gerais em que este está sendo constituído, ou então, que se mantenha com uma visão unitária do contexto social em que os acontecimentos são produzidos. p É necessário compreender que existem outros saberes, diferentes modos de explicar, conceber e construir a realidade. Saberes que são constituídos nas bases da cultura, da vivencia e de práticas tradicionais ainda presentes em muitas comunidades do nordeste paraense. Trata-se dos saberes míticos, locais e/ou socioambientais adquiridos por meio da convivência e da interação do homem com a natureza. E são imprescindíveis para o entendimento e o desenvolvimento dos modos de vida das comunidades que ainda se organizam a partir de práticas socioambientais a exemplo da pesca, a agricultura, o extrativismo e etc. Tais práticas são imprescindíveis dentro das relações econômicas e culturais para muitas comunidades tradicionais e desenvolvem uma diversidade de saberes em contato com a natureza, que influenciam seus modos de vida, a cultura e identidade dos moradores, como apontam estudiosos como ALMEIDA (2010); DIÉGUES (1994); SILVA (2010); FURTADO (1993). Porém, diante das mudanças imposta por paradigmas capitalistas provocando uma nova relação entre o ser humano e a natureza gerando transformações relacionadas ao consumo e aos modo de produção, muitas comunidades tradicionais ao se adaptar aos elementos ditos modernos e tecnológicos, deixam para traz suas raízes e seus saberes e as novas gerações silenciam os ensinamentos de seus ancestrais, suprimindo sua cultura pela adoção de valores racionais e hegemônicos. Esse pressuposto, conduziu-nos a refletir os saberes da tradição no contexto da comunidade Segredinho a partir das seguintes questões de pesquisa: que saberes da tradição as crianças da comunidade segredinho possuem advindos das relações estabelecidas com o Lago do Segredo? Quais as percepções que as crianças possuem a respeito dos saberes da tradição e do mito que envolve o índio encantado no Lago do Segredo? Estudos anteriores de Rocha (2011) apontaram a relação econômica e cultural que a comunidade Segredinho tem com a pesca artesanal e a relevância dos saberes da tradição adquiridos por meio desta atividade. 3 Alguns autores denominam de Saber ambiental (LEFF, 2005); conhecimento cotidiano (LOPES, 1999); conhecimento popular (BRANDÃO, 2007); saberes primevos (CHASSOT, 2000), saberes tradicionais (DIÉGUES, 1994) como sinônimos ou com premissas semelhantes. Utilizarei esse conceito com base na epistemologia do termo abordada por Maria da Conceição de Almeida, (2010). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Porém, nesse artigo, sentiu-se a necessidade de evidenciar tais saberes por meio das compreensões das crianças. A comunidade Segredinho geograficamente está localizada no nordeste paraense, há 24 km do município de Capanema-PA. A principal atividade econômica desenvolvida é a pesca artesanal que é realizada em um lago que fica próximo da comunidade, sendo este o motivo principal que desencadeou a formação das residências e posteriormente a constituição da comunidade. O lago do Segredo é considerado pelos moradores um lugar mítico, para eles existe a presença de um ser encantado nesse espaço, um índio, que sumiu a muitos anos atrás por ter escondido o segredo sobre a existência do lago. Sendo assim, os moradores conservam este mito alegando ser o índio encantado responsável pela conservação dos recursos naturais presentes no ambiente, reproduzindo suas práticas de acordo com esta crença e transmitindo entre as gerações por meio das histórias que envolvem mistério, imaginação e simbolismo. Acreditam na influência dos seres mágicos a manutenção e o cuidado com o ambiente natural e divulgam suas histórias por meio de suas narrativas. Além disso, o Lago é o ambiente onde é realizada a atividade da pesca artesanal, que é desenvolvida por homens e mulheres e por muito tempo algumas famílias sobreviveram apenas com os recursos extraídos de lá (ROCHA, 2011). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 103 Assim, este artigo objetiva refletir sobre os saberes da tradição que são apreendidos por meio da pesca artesanal e o mito que envolve o Lago do Segredo a partir da percepção das crianças da comunidade Segredinho/Capanema-PA. Trata-se de um recorte da pesquisa de doutorado que está sendo desenvolvida na comunidade Segredinho/Capanema-PA. Para isso, utilizou-se a pesquisa qualitativa por esta “levar em consideração as subjetividades dos sujeitos e seu contexto histórico-social, buscando responder as questões intrínseca a dimensão social dos sujeitos” (MINAYO, 2016, p.34). j ( p ) Para melhor compreensão das discussões e interpretações, optou-se pela escrita narrativa do texto que teve como colabores duas crianças (01 menino e 01 menina) que por meio de uma roda de conversa expressaram as narrativas que envolveram os saberes adquiridos por meio da pesca artesanal e o mito sobre o índio encantado no lago do Segredo. A vivência, os detalhes apresentados na narrativa e a relação com a atividade da pesca foram os critérios que os definiram enquanto colaboradores na pesquisa. Enquanto técnica auxiliar também foi utilizada a entrevista semiestruturada, por ser esta a que permite compreender o significado atribuído pelos sujeitos a eventos, situações, processos ou personagens que fazem parte de sua vida cotidiana (DEMO, 2001). Para preservar a identidade das crianças, utilizarei nomes dos deuses que representam o sol e a lua na mitologia tupi guarani - Jaci e Guaraci- considerando a importância que os elementos naturais tem para a pesca e para os povos tradicionais. As narrativas serão apresentadas por meio de episódios e tratados a luz da análise de conteúdo, evidenciando a importância dos saberes, a transmissão entre as gerações e a percepção das crianças na manutenção de sua cultura e identidade. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Pois a água tem uma importância fundamental na constituição econômica das populações, pois trata-se de uma relação estabelecida desde os primórdios da história. Mas a pesca, não é apenas um meio econômico ou uma simples atividade humana. Para Moraes (2005), trata-se de um conjunto de relações de domínios variados que envolve uma multiplicidade de saberes, de fazeres e de instrumentos utilizados. É dessa perspectiva que entendo, que na comunidade Segredinho, a pesca associa todos estes elementos e se mantém como propulsora da cultura e da identidade local, pois no exercício desta atividade, os moradores “estabelecem distintas formas de leitura de mundo” (ALMEIDA, 2017, p. 53). Uma destas formas diferenciadas de interpretar e explicar os fenômenos são os mitos, que possuem uma forte presença na comunidade Segredinho. Por meio deles os moradores interpretam a si próprios, o seu passado e a sociedade em que vivem e encontram soluções para o desconhecido. Aliando a isso, suas crenças religiosas e as relações que estabelecem entre si e entre os saberes que vão constituindo, recriando e transmitindo para as novas gerações. Segundo Eliade (1972, p. 109), “o mito não é absolutamente uma teoria abstrata ou uma fantasia artística, mas uma verdadeira codificação da religião primitiva e da sabedoria prática”. E por meio destes, revelam importantes elementos que condicionam e mantém as relações entre os seres sobrenaturais e os seres humanos. Além dos mitos, encontram-se muitos saberes que são elaborados e construídos por meio da pesca artesanal, por essa atividade favorecer uma aproximação constante e ampla com a natureza. São estes: O conhecimento de ventos, das águas, marés, fundos submarinos, correntes, hábitos de peixes, dentre uma série de processos que são “naturalizados” e formam redes cognitivas próprias para a interpretação, apropriação e representação destes processos que por vezes são considerados saberes (CARDOSO, 2001, p. 86). Na comunidade, a convivência com o lago tem possibilitado a construção de saberes sobre os tipos de peixes e seus hábitos alimentares, a adequação dos instrumentos de acordo com o tipo de peixe a ser capturado, o tempo e as estações mais favoráveis, a localização dos cardumes, dentre outras habilidades que operam de forma prática e diferenciada do conhecimento científico. Além disso, existe “diálogo e complementaridade entre o real e imaginário que configura seu modo de vida” (MORAES, 2005, p. 149). 1 PRÁTICAS SOCIOAMBIENTAIS E A CONSTRUÇÃO DOS SABERES DA TRADIÇÃO EM COMUNDADES DE PESCADORES ARTESANAIS Compreende-se que os saberes são adquiridos por meio das relações humanas com seus ambientes, dentro de um determinado contexto e são transmitidos de pais para filhos, através de um aprendizado vivo e contínuo. Esses elementos se configuram dentro da definição apontada por Silva (2010) quando diz que os saberes são gerados a partir da vivência e convivência, apreendidos por meio das ações cotidianas e que são responsáveis pela conservação cultural dos povos. Assim, a partir do universo conceitual de saberes, optou-se por trazer o conceito de saberes da tradição3. Trata-se dos saberes adquiridos por meio da convivência e da interação do ser humano com a natureza. “E que ao longo dos tempos as comunidades tradicionais têm desenvolvido e sistematizado o que lhes permitem responder a problemas de ordem material ligados à natureza e seus recursos e ainda ligados a ordem mítica e simbólica” (ALMEIDA, 2010, p.57). São saberes que se traduzem na leitura diferenciada dos fenômenos, na interpretação de situações que cotidianamente exigem respostas mais imediatas e que são construídos, sobretudo, por meio das práticas socioambientais muito presentes e de valor acentuado nas comunidades tradicionais. Principalmente aquelas que mantém suas práticas econômicas ancoradas em modos de vidas que são conduzidas e organizadas pela relação socioambiental, como a pesca artesanal, muito comum em grande parte da nossa região norte, pela localização próxima do litoral e de rios e lagos. 104 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 2 A PERCEPÇÃO DAS CRIANÇAS SOBRE OS SABERES DA TRADIÇÃO DA COMUNIDADE SEGREDINHO/CAPANEMA-PA Segundo Moraes (2005) a produção do conhecimento se dá em distintos lugares, de formas variadas e com sistemas próprios de concepção. A pesca é um universo que apresenta um espaço frutífero e altamente rico em conhecimentos específicos de leitura do mundo. Mas para isto, é necessário estabelecer diálogos entre as gerações, no processo constante de transmissão, feito pela aproximação, acompanhamento e observação da atividade, através da ação, na qual experimentam, contrastam, atualizam e possam adquirir novos saberes, que vão servir para confirmar ou modificar alguma crença, possibilitando um contínuo aprendizado. Elementos muito perceptíveis na comunidade Segredinho, em que as crianças estão sempre próximas aos pais/mães durante as pescarias ou acompanhando as conversas que envolvem as narrativas sobre o mito local. E isso, torna-se um aspecto importante de aprendizado e manutenção da cultura local, como foi possível identificar nas narrativas que serão descritas e analisadas a seguir: Episódio 1: Sabe moça, um dia eu fui pro lago com a mamãe, fomos umas 09 horas e voltamos umas 17:00 horas, ela pesca lá e me levou, eu sempre vou com ela, gosto muito de ir lá. E aí quando a gente tava pescando a canoa virou, a gente se alagou, minha mãe ficou com medo, pois tava fundo onde a gente tava, mas minha prima me ajudou e me puxou de volta pra dentro da canoa. A mamãe ficou com medo de eu morrer, mas eu me virava, eu sei nadar, não tenho medo não, não tenho medo das histórias que contam sobre o lago e nem do índio que mora lá, sei que ele só faz mal pra quem maltrata o lago, escangalha as árvores. Depois disso, resolvemos ir embora, pois já tinha um pouco de peixe e tava ficando tarde. Pergunta da pesquisadora: E vocês não tem medo do índio encantado no lago? Ela respondeu: “não, ele só assusta se mexer com ele”. A pesquisadora insistiu: como mexer com ele? A menina continuou: “tem que entrar caladinho, não perturbar ele, não fazer maldade com o lago, se não ele afunda a canoa da gente. A pesquisadora brincou: Será que não foi ele que alagou a canoa de vocês? Ela retrucou: “não, ele conhece a gente, sabe que a gente é da comunidade e só vai pescar”. (Jaci, 10 anos). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 105 diferenciada, que tem estrutura própria, singular e específica, portanto, assentados na complexidade e em múltiplas dimensões. Os saberes têm suas raízes na tradição e, durante o exercício constante da ação sobre determinados fatos e fenômenos que se dão principalmente pela relação de troca entre seus membros, essa tem sido a condição primeira de transmissão e manutenção dos conhecimentos nas comunidades tradicionais. Pois, a relação de troca e o convívio estabelecido entre os moradores – pescadores e pescadoras, adultos e crianças - objetiva perpetuar os ensinamentos e ressignificá-los diante da realidade que está posta. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 g q g ( p ) Esses saberes apreendidos por meio da observação, do acompanhamento e da realização constante da atividade da pesca tem favorecido o aprendizado das crianças, que envolvidas e em interação com a atividade vão constituindo seu corpus de saberes também sobre a anatomia dos peixes, os instrumentos de pesca, os hábitos alimentares dos peixes, os pontos de pesca do lago, as espécies e etc. Juntamente com o conhecimento sobre o mito que circunda o ambiente e as histórias que vivenciam e a experimentam envolvendo o índio encantado no lago. Segundo Moraes (2005, p. 119), “os seres das águas, das florestas, dos vales, seres encantados, são elementos que dão significados e que fazem da pesca uma confluência entre o real e o imaginário”. É nessa teia de relações que está configurado o universo que compõem a vida dos moradores da comunidade Segredinho. Que são mantidas e recriadas por meio das narrativas, contadas tanto por adultos quanto por crianças, pois elas desde cedo vão aprendendo a respeitar e a conviver com seres naturais e míticos, atribuindo uma importância fundamental para sua sobrevivência e a conservação dos elementos naturais presentes no lago. Para Alves (2008, p. 39), “os mitos formam a consciência social. Com base neles, desenvolve-se toda uma concepção teórica sobre o sentido da vida, do viver, do morrer, e da existência do mundo”. Por isso, a necessidade de respeitar, difundir e compartilhar estes saberes construídos pelas comunidades tradicionais, como uma estratégia de conhecimento NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Segundo Alves (2008), a narrativa é tão importante para a vida humana que é impossível imaginar um mundo sem ela, um mundo sem histórias, mitos, contos ou dramas. Esse universo que envolve imaginação, tradição e saberes contribui para conservação e manutenção dos mitos da comunidade que mesmo as crianças sempre tem uma história para contar sobre o lago. Nesse caso também, vimos que Jaci, descreve a partir de uma experiência que ela viveu, o que também é muito comum entre as crianças, pois eles acompanham seus pais e mães constantemente ao lago e vivem suas experiências. Para Larrosa (2010, p.34), a experiência é entendida como uma expedição em que se pode escutar o “inaudito” e em que se pode ler o não lido, isso é, um convite para romper com as regularidades de um sistema que já dá um mundo interpretado e pronto. Outro elemento que chama a atenção no episódio narrado por Jaci é com relação ao respeito e consideração que o índio tem com os moradores locais, quando ela diz que: “sei que ele só faz mal pra quem maltrata o lago, escangalha as árvores”. Esse entendimento demonstra que para eles é necessário parcimônia ao entrar e estar no lago. Trata-se do que Morais (2008; p.132) conceitua como “precaução”, ou seja, a crença de que o mito pode vir a ajudar ou prejudicar os pescadores caso não demonstrem respeito a essas crenças. O que colabora para a conservação e o uso equilibrado de seus recursos naturais por meio das práticas socioambientais. Podemos ratificar mais uma vez que o lago do Segredo é parte integrante da vida da comunidade, quer seja por meio de seus recursos naturais, quer seja por meio dos saberes que são constituídos nele, ou seja, os moradores têm nas águas do lago, além de uma fonte de alimentação que faz parte do caráter prático e utilitário, pois a utilizam na atividade da pesca artesanal e também como uma fonte de referência simbólica e mítica. E, portanto, caracterizam-se em uma forma diferenciada, mas não única nem inferior de ler o mundo e os fenômenos, além de configurarem uma maneira de compreender a vida e de compor a ciência e o conhecimento. Episódio 2: Eu moro aqui desde que nasci, eu tenho 12 anos, meu pai e minha mãe pescam, meu pai vai mais vezes, a mamãe só de vez em quando. 2 A PERCEPÇÃO DAS CRIANÇAS SOBRE OS SABERES DA TRADIÇÃO DA COMUNIDADE SEGREDINHO/CAPANEMA-PA É possível inferir deste episódio que Jaci, que é moradora da comunidade assim como sua família conhece sobre o mito do índio e demonstra respeito por esse saber mantido pelos moradores da comunidade e que embora ainda criança já desenvolveu seu entendimento e firmou suas posições sobre o mito. O que se configura no que Moraes (2008) fala, que os saberes construídos e metamorfoseados culturalmente são transmitidos para as novas gerações como um processo educativo que se vale de todos os elementos constituintes, sejam eles míticos e imaginários ou práticos e materiais. Quando ela afirma: “não tenho medo das histórias que contam sobre o lago”, confirma que esta tradição é contada e recontada pelos mais idosos. Ou seja, acentua a permanência dos saberes da tradição por meio das narrativas orais. O que se configura no que Moraes (2008) fala, que os saberes construídos e metamorfoseados culturalmente são transmitidos para as novas gerações como um processo educativo que se vale de todos os elementos constituintes, sejam eles míticos e imaginários ou práticos e materiais. Quando ela afirma: “não tenho medo das histórias que contam sobre o lago”, confirma que esta tradição é contada e recontada pelos mais idosos. Ou seja, acentua a permanência dos saberes da tradição por meio das narrativas orais. 106 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 107 As crianças da comunidade convivem diretamente com os adultos e compartilham de suas atividades. É comum ver ao lado dos pescadores e pescadoras as crianças, em uma espécie de parceria e colaboração na realização da pesca, o que potencializa a construção de aprendizagens que se dão por meio da observação, da imitação e do compartilhamento das experiências. Os saberes são adquiridos de forma espontânea, sem um compromisso prévio, um rigor ou uma obrigatoriedade, mas no exercício das práticas do cotidiano e na construção de sua cultura. De acordo com D’ambrósio (2001, p. 22): O cotidiano está impregnado dos saberes e fazeres próprios da cultura. A todo instante, os indivíduos estão comparando, classificando, quantificando, medindo, explicando, generalizando, inferindo e, de algum modo, avaliando, usando os instrumentos materiais e intelectuais que são próprios à sua cultura. Trata-se portanto, de um processo educativo que envolve interação, comunicação, diálogo e experimentação de tudo que é vivido, percebido e inventado para gerar o saber, que é diferenciado mas não menor ou inferior ao conhecimento cientifico, possui estratégia e organização especifica, são saberes distintos que podem ser complementares. Quando, em sua narrativa Guaraci diz: “Já sei que a anujá tem o rabo diferente do jacundá. Meu pai conhece todos eles, sabe a diferença deles”. Ele confirma a assertiva de que a experiência também produz conhecimento, que vai ter importância no seu dia a dia, vai ter sentido e significado nos seus hábitos alimentares, na economia local e na conservação da cultura e da pesca na comunidade. Não se trata de um aprendizado adquirido nos manuais escolares mas no cotidiano, na experiência constante com a pesca e com os peixes. O conhecimento adquirido por Guaraci sobre as espécies de peixes existentes no lago, a estrutura de cada um, seus contornos, hábitos alimentares e até as diferenças já apreendidas por meio da convivência, subsidia um saber que é prático. Pois foi construído por meio da observação e das transmissões construídas entre as gerações de forma solidária e coletiva. É o que denomina Lévi- Strauss (1989, p.98) de “a ciência do concreto”. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Às vezes eu vou com eles, mas a mamãe diz que é perigoso, porque o lago é fundo. Se eles deixassem eu ia toda vez, eu também gosto de pescar, ficar no lago escutando o barulho do vento, botar a linha e esperar o peixe. A pesquisadora pergunta: qual o peixe que vc mais gosta? Eu gosto do traíra e do jacundá, de todos (risos), as vezes a gente pode se confundir com os peixes, eles são bem parecidos, mas eu já sei dizer o nome certo deles, já sei porque fico vendo o papai separar e dá o nome. Já sei que a anujá tem o rabo diferente do jacundá. Meu pai conhece todos eles, sabe a diferença deles. No inverno, a gente não vai pro lago porque fica muito cheio e os peixes descem é mais difícil pegar e aí as coisas ficam mais ruim, a gente come mais é carne e frango. (Guaraci, 12 anos). No episódio acima, enfatiza-se a importância dos saberes da tradição apreendidos pelas crianças, mas aqui a conotação é dada aos saberes que são gerados e apreendidos por meio da atividade da pesca que é realizada no Lago do Segredo, mais uma vez, este ambiente congrega uma série de elementos formativos constituintes da composição social, cultural, econômica e mítica da comunidade. Na narrativa, está presente também a transmissão dos saberes por meio do acompanhamento e da vivência das práticas entre as diferentes gerações. Tal organização é muito comum e presente nas comunidades que tem na pesca sua atividade econômica principal. Para Farias (2006; p.47), “essa transmissão, feita de geração em geração, de forma oral ou prática, constitui-se no conjunto de testemunhos, conservados ou desaparecidos que materializam a vida de uma sociedade”. Assim, são incorporados e compreendidos os sentidos e fenômenos extraídos por meio da convivência e da observação constante de uma atividade. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 108 saberes, pois são estes que condicionam suas atitudes e os mantém integrados a um modo de vida pautado na preservação e no equilíbrio com o ambiente. saberes, pois são estes que condicionam suas atitudes e os mantém integrados a um modo de vida pautado na preservação e no equilíbrio com o ambiente. Brandão (2007, p.75) ressalta que, dentro de sua cultura, em sua sociedade, as pessoas aprendem de maneira mais ou menos intencional, através do envolvimento dentro do corpo, da mente e da afetividade, entre as incontáveis situações de relações e de trocas entre os homens, é parte do processo pessoal de endoculturação e é também parte da aventura humana de ‘torna- se pessoa’. Portanto, são inúmeras as possibilidades de saberes ligados a pesca e aos mitos que permeiam o contexto das comunidades tradicionais e colaboram para alicerçar a cultura e a identidade dos povos, principalmente no cenário amazônico em que nos encontramos. Jaci e Guaraci são apenas duas crianças que, apesar da pouca idade, já apresentam uma riqueza de conhecimentos que fazem diferença em seus modos de vida e na relação com o Lago do Segredo. O que evidencia que a pesca tem o significado e a importância na comunidade que vai além da necessidade econômica, mas configura-se no modo de vida da população através de suas tradições, crenças e simbologias, elementos presentes no seu universo sociocultural. DOSSIÊ AMAZÔNIA Percebi também por meio do episódio, que Guaraci está inserido dentro do contexto econômico que a família desenvolve e isso lhe possibilita está integrado de forma participativa e formativa nesses espaços, o que lhe favorece o entendimento da realidade que se movimenta, que é modificada pelas ações humanas mas que também contribui para a transformação das pessoas, por meio da relação com a natureza aprendem a respeitar o tempo, as estações, o ambiente natural e seus mistérios. Quando ele ressalta: “no inverno, a gente não vai pro lago porque fica muito cheio e os peixes descem é mais difícil pegar e aí as coisas ficam mais ruim”. Demonstra seu letramento diante do mundo, sua compreensão diante das dificuldades e de como os moradores precisam se adaptar as contingências da natureza. Ou seja, há uma apreensão de sua realidade, apreender no sentido de aprender proposto por Freire (1996, p. 69) “aprender para nós é construir, reconstruir, constatar para mudar, o que não se faz sem a abertura ao risco e a aventura do espírito”. Assertiva que é importante dentro do contexto sociocultural, pois congrega um sentido transformador tanto de si quanto de sua realidade. Embora, os moradores sofram as privações ocasionadas pelo tempo que a natureza necessita para se recompor, eles desenvolvem a consciência de que esse momento é temporário e que precisam realizar outras atividades econômicas para sobreviver, do contrário, sua sobrevivência estaria em risco pelo esgotamento dos recursos. Compreende-se assim, o desenvolvimento de uma responsabilidade coletiva pela conservação dos recursos naturais, na maioria das vezes, orientada e reproduzida por meio dos DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 109 Ressalto ainda, que os resultados discutidos neste artigo corroboram para divulgar e valorizar os modos de vida das comunidades de pescadores artesanais que por meio dos saberes da tradição criam e recriam seus modos de vida, constroem suas narrativas, estruturam as regras que norteiam o seu comportamento e dão significados aos acontecimentos cotidianos. Tudo isso, dentro de uma configuração que dialoga com os aspectos materiais da vida, ligados a sua sobrevivência e a sua imaterialidade, ou seja, a imaginação, o mito, a criatividade e o encantamento diante do desconhecido. 3 CONSIDERAÇÕES FINAIS Compreender os saberes da tradição da comunidade Segredinho a partir da percepção de duas crianças, moradores da comunidade, representou também dar visibilidade as práticas socioambientais que são realizadas nas comunidades tradicionais e as crenças, mitos e costumes que estas potencializam, principalmente no cenário amazônico pela sua sociobiodiversidade. O contexto da comunidade Segredinho é diferenciado não apenas pela preponderância dos elementos naturais presentes no Lago do Segredo mas sobretudo, pela exuberância de saberes que são apreendidos por meio da relação com ele. E a pesca é um dos mecanismos que viabiliza essas aprendizagens, não somente para os adultos mas também para as crianças que desde cedo vão para o Lago acompanhar seus pais e mães nas pescarias. Foi perceptível que Jaci e Guaraci conhecem os segredos da pesca e do mito que circundam o ambiente e cotidianamente desenvolvem saberes ligados a; composição, tamanho, tipo dos peixes, os instrumentos mais adaptados a cada peixe, a identificação das espécies, os pontos mais propícios para a pesca e etc. Saberes adquiridos por meio da transmissão e da observação dos mais experientes na atividade, que passa de geração a geração e de pai/mãe para filho e filha. Sobre o mito do índio encantado no lago, as crianças demonstraram respeito, consideração e apreço pelas histórias contadas pelos mais velhos e reproduzem estas atitudes através de suas próprias experiências. Isso se torna importante à medida em que consolida a cultura e a identidade desta comunidade, que por meio desta geração poderá manter suas tradições e conservar seus valores, através dos comportamentos que são modificados em virtude da crença no mito. Por meio do reconhecimento da existência do índio encantado no Lago as crianças desenvolvem atitudes de preservação ao meio ambiente, respeito ao período de reprodução dos peixes e só retiram do lago o suficiente para seu sustento, ou seja, o mito influencia na tomada de ação frente as suas necessidades econômicas. Evidenciando, um saber que se concretiza culturalmente, socialmente e economicamente por meio da organização e do consumo consciente. O reconhecimento da tradição local demonstrado pelas crianças é de suma importância pois convalida uma cultura que se constrói em diferentes percepções de compreender e interpretar o mundo, expandindo a consciência para outras formas de fazer ciência, que se projeta nas relações, na experiência e na diversidade, o que materializa a capacidade do ser humana de aprender com a natureza sem contudo, destruir seus recursos. REFERÊNCIAS ALMEIDA, Maria da Conceição de. Complexidade, saberes científicos, saberes da tradição. São Paulo: Editora Livraria da Física: 2010. ALMEIDA, Maria da Conceição de. Complexidade, saberes científicos, saberes da tradição. São Paulo: Editora Livraria da Física: 2010. ALMEIDA, Maria da Conceição de. Ciências da complexidade e educação: razão apaixonada e politização do pensamento. 2. ed. Curitiba: Appris, 2017. ALVES, L. M. S. A. A tradição oral na Amazônia: a mitopoética dos espaços nas narrativas, visagens e assombrações. In: ALVES, M. L. A. et al. (Org). Cultura e educação: reflexões para a prática docente. Belém: EDUFPA, 2008. p. 24-47. BRANDÃO, Carlos Rodrigues. O Que é educação. São Paulo: Brasiliense, 2007. 116 p. CHASSOT, Attico. Alfabetização cientifica: questões e desafios para a educação. Ijuí-RS: Editora Unijuí, 2000. CHASSOT, Attico. Alfabetização cientifica: questões e desafios para a educação. Ijuí-RS: Editora Unijuí, 2000. RANDÃO, Carlos Rodrigues. O Que é educação. São Paulo: Brasiliense, 2007. 116 p. CARDOSO, Maria Bárbara da Costa. Saberes ribeirinhos quilombolas e sua relação com a educação de jovens. São Paulo: Moderna, 2001. D`AMBROSIO, Ubiratám. Educação para uma sociedade em transição. São Paulo: Papirus, 2001. D`AMBROSIO, Ubiratám. Educação para uma sociedade em transição. São Paulo: Papirus, 2001. DEMO, Pedro. Pesquisa: o princípio cientifico e educativo. 8. ed. São Paulo: Cortez, 2001. DIÉGUES, Antônio Carlos. O mito moderno da natureza intocada. São Paulo, NUPAUB, Universidade de São Paulo, 1994. ELIADE, Mircea. Mito e realidade. São Paulo: Perspectivas, 1972. FARIAS, Carlos Aldemir. Alfabetos da alma: histórias da tradição na escola. Porto Alegre: Sulina, 2006. FURTADO, Lurdes. Pescadores do Rio Amazonas: Um estudo antropológico da pesca ribeirinha numa área amazônica. Belém-PA: Museu Paraense Emílio Goeldi,1993. Citação no texto: Furtado (1993) FREIRE, Paulo. Pedagogia da autonomia. São Paulo: Paz e Terra, 1996. LARROSA, Jorge. Pedagogia profana: danças, piruetas e mascaradas. 5. ed. Belo Horizonte: Autêntica, 2010. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 110 LEEF, Henrique. Saber ambiental: sustentabilidade, racionalidade, complexidade, poder. 4. ed. Rio de Janeiro: Vozes, 2005. LÉVI-STRAUSS, Claude. A ciência do concreto. In: O Pensamento Selvagem. Tradução de Tânia Pellegrini, Campinas, SP: Papirus, 1989. LOPES, Alice R. Cassimiro. Conhecimento escolar: ciência e cotidiano. Rio de Janeiro: EDUERJ, 1999. MINAYO, Maria Cecília de Souza (org). Pesquisa Social: teoria, método e criatividade. Petrópolis, RJ: Vozes, 2016. MORAES. Sérgio Cardoso de. Saberes da Pesca: Uma arqueologia da ciência da tradição. 2005. Tese (Doutorado em educação) - Universidade Federal do Rio Grande do Norte. Centro de Ciências Sociais Aplicada: Natal, 2005. MORAES, Sergio Cardoso de. Fragmentos de saberes tradicionais. In: ALVES, M. L. A. et al. (Org.). Cultura e educação: reflexões para a prática docente. Belém: Edufpa, 2008. p. 109-132. ROCHA, Nádia Suei Araújo da. A pesca feminina na comunidade Segredinho: município de Capanema-PA. 2011. Dissertação (Mestrado em Gestão de Recursos Naturais) – Programa de Pós-Graduação em Gestão de Recursos Naturais e Desenvolvimento Local, Universidade Federal do Pará, Belém, 2011. SILVA, Márcia Regina Farias da. Ciência, natureza e sociedade: diálogo entre saberes. São Paulo: Livraria da Física, 2010. 111 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA – VOLUME – – SABERES NA AMAZÔNIA SABERES NA AMAZÔNIA M ARÇO REVISTA DO PROGRAMA DE PÓS GRADUAÇÃO EM LINGUAGENS E M ARÇO NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 112 identificarmos como os sujeitos constroem saberes, que são aperfeiçoados por seus corpos durante os longos anos de experiência empírica. identificarmos como os sujeitos constroem saberes, que são aperfeiçoados por seus corpos durante os longos anos de experiência empírica. Os saberes e práticas locais, além de explicitarem os modos de vida e visões de mundo, também constituem domínios e instrumentos técnicos necessários para diversos grupos sociais em zonas litorâneas no Brasil conseguirem suas fontes de rendas, incluso os que reproduzem suas atividades no território da Resex Marinha Caeté-Taperaçu. Nesse sentido, esses grupos estabelecem uma relação de dependência com os territórios que constroem, se apropriam e dominam. Além da captura do caranguejo-uçá (Ucides cordatus), pescam turu, siri e peixes. Também participam de atividades agroextrativistas como a pequena agricultura. Este último fator caracteriza o que autores como Alex de Mello (1993) e Lourdes Furtado (1993) denominam: pescadores polivalentes. Ressalta-se que o grupo social acompanhado, os tiradores de caranguejos da comunidade do Pontinha do Bacuriteua, município de Bragança, Nordeste do Estado do Pará, reproduz lógicas e práticas vinculadas ao Capitalismo, como a monetarização, a quantidade de caranguejos coletados (produção) vinculada aos ganhos auferidos, em fricção com o modo de vida baseado nos saberes tradicionais locais. Essas práticas se dão no manguezal localizado em Bragança-PA, descrito por Ipojucan Campos (2012, p. 150) enquanto “[...] fonte de sobrevivência econômica para milhares de pessoas, ele significa lugar de “livre” atividade financeira que, inevitavelmente, reflui na cidade de Bragança”. A interpretação do pesquisador é que existem, no manguezal, dinâmicas socioculturais, quando o assunto “localiza-se nas estratégias de sobrevivência” (p. 155). Desta feita, é necessário interpretar o manguezal e sua estrutura como componente do humano e também do não-humano, caracterizando o que o economista equatoriano Alberto Acosta (2016) chama de “bem viver”, no caso a partir da perspectiva das populações indígenas do Equador e da Bolívia. É um lugar de constituição de territórios e territorialidades (LITTLE, 2004; HAESBAERT, 2007), “marcas de poder” e símbolos projetados pelos homens que diariamente estão dentro do ecossistema. O uso deste ambiente como fonte de sobrevivência e reprodução social é bastante antigo, pois antes da chegada dos europeus à Amazônia, os povos nativos já utilizavam os recursos do manguezal (SOFFIATI, 2004). 3 Intermediários que compram dos tiradores e vendem ao consumidor e ou a outros marreteiros. DOSSIÊ AMAZÔNIA Esse segundo se subdivide nos subtópicos: “Técnicas e tecnologias e o regime de marés” ou seja, como são construídas tecnologias e as técnicas usadas a partir de um conhecimento empírico, no caso específico, o regime de marés; “A composição do solo e a flora do manguezal”, discorre sobre como os conhecimentos dos tiradores sobre a estrutura física do bioma e como isso é importante ao êxito nas empreitadas diárias; e “A coleta do caranguejo: saberes e práticas”, mostra-se e identifica-se como tiradores de crustáceo usam os conhecimentos locais em suas práticas empíricas diárias. O desenvolvimento do artigo divide-se em dois tópicos. No primeiro, “Quem são os tiradores de caranguejo da Pontinha do Bacuriteua?”, a intenção é apresentar os tiradores de caranguejo; o segundo denomina-se “Saberes e práticas socioambientais dos tiradores de caranguejo”: onde analisamos os principais saberes e práticas dos tiradores sobre aspectos ambientais e o ciclo biológico do crustáceo. Neste, mostra-se como tais fatores repercutem na atividade e na escolha dos territórios. Esse segundo se subdivide nos subtópicos: “Técnicas e tecnologias e o regime de marés” ou seja, como são construídas tecnologias e as técnicas usadas a partir de um conhecimento empírico, no caso específico, o regime de marés; “A composição do solo e a flora do manguezal”, discorre sobre como os conhecimentos dos tiradores sobre a estrutura física do bioma e como isso é importante ao êxito nas empreitadas diárias; e “A coleta do caranguejo: saberes e práticas”, mostra-se e identifica-se como tiradores de crustáceo usam os conhecimentos locais em suas práticas empíricas diárias. 4 Trata-se de encantados, assombrações, fantasmas e fenômenos inexplicáveis que se manifestam em mangues, rios ou florestas, tais como a Mãe-d’água e a Curupira, por exemplo. DOSSIÊ AMAZÔNIA 113 fora do ecossistema; as questões simbólicas como a existência de “visagens”4 no local de trabalho; as técnicas e tecnologias utilizadas à pesca; o tempo de entrada e de saída à labuta e a alimentação dos trabalhadores. Com a proposta em dar continuidade as observações e conclusões dos cientistas sociais, a pesquisa, em foco, intenciona compreender como um grupo social específico, tiradores de caranguejo da Pontinha do Bacuriteua, desenvolvem suas práticas socioambientais a partir dos saberes locais, utilizados à pesca do caranguejo-uçá na Amazônia, especificamente em Bragança. Para isto, os procedimentos metodológicos decorreram do contato dos pesquisadores com habitantes da Pontinha do Bacuriteua. Nesta localidade, parte significativa da população vive da coleta e comercialização do caranguejo-uçá, estando o Bacuriteua entre as comunidades de maior produção pesqueira na região (DOMINGUES, 2008). Os pesquisadores mantiveram diálogos com os interlocutores, através, primeiramente de entrevistas informais, e posteriormente com entrevistas gravadas, feitas, também, digitação das falas dos atores. Metodologicamente, para a compreensão desse modo de vida, foi necessária uma visão mais interdisciplinar sobre pesca artesanal e comunidades, englobando áreas de conhecimento como antropologia, sociologia e história. Nossa intenção foi o de dialogar com diferentes saberes para evitar que os limites de uma única abordagem impeçam o entendimento dos fenômenos analisados, pois trata-se de homens que há décadas mantém interações com o ambiente natural e que, sobre este, desenvolveram e partilham muitos saberes, sem os quais não seria possível a coleta do caranguejo. Durante os anos de 2010 a 2017 os autores deste estudo tiveram contatos com pessoas que vivem na e da holocenose em foco. Os dados aqui discutidos advêm das pesquisas para a dissertação de mestrado de ambos. Somando as duas pesquisas, foram realizadas entrevistas semiestruturadas com trinta coletores de caranguejo, além de observação não participante das atividades dos tiradores de caranguejo. A pesquisa consistiu em observação direta e entrevistas semiestruturadas com trabalhadores do manguezal da comunidade Pontinha do Bacuriteua. O desenvolvimento do artigo divide-se em dois tópicos. No primeiro, “Quem são os tiradores de caranguejo da Pontinha do Bacuriteua?”, a intenção é apresentar os tiradores de caranguejo; o segundo denomina-se “Saberes e práticas socioambientais dos tiradores de caranguejo”: onde analisamos os principais saberes e práticas dos tiradores sobre aspectos ambientais e o ciclo biológico do crustáceo. Neste, mostra-se como tais fatores repercutem na atividade e na escolha dos territórios. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 O manguezal como local necessário à sobrevivência desses grupos sociais e como território de suas identidades, modo de vida e pertencimento, dos que residem na zona costeira de Bragança foi identificado nas pesquisas de Alves (2015a, 2017b); Oliveira (2015); Oliveira (2013); Oliveira & Maneschy (2014); Oliveira (2018) e Campos (2013). Os autores citados observaram os movimentos dos homens em tal ecossistema e as rotas de comercialização do caranguejo-uçá na PA-458 (Estrada que liga Bragança à praia de Ajuruteua e utilizada pelos trabalhadores para entrar no manguezal). A Estrada é utilizada, também, ao escoamento da produção de parte do pescado desembarcado na área costeira de Bragança, quais sejam: nas Vilas do Bonifácio, do Tamatateua, do Castelo, do Bacuriteua e nos portos de paradas, em pontes que se localizam na estrada. A constatação do ecossistema como lugar essencial às atividades de populações que residem às proximidades das áreas de manguezais por parte da sociologia e de outras ciências humanas tem conduzido à produção de pesquisas e divulgação de seminários, palestras, artigos, ensaios, dissertações e teses que identificam e mostram o modo de vida desses grupos humanos. Os estudos de Maneschy (1993a); (2003b); Souza (2013); Oliveira (2015), Oliveira (2018) e Alves (2017) centram-se em aspectos socioeconômicos, tais como as transações entre tiradores e os marreteiros3 e nas identidades locais. Os estudos desses autores também mostram as atividades realizadas por mulheres e crianças no manguezal; as relações de gêneros dentro e DOSSIÊ AMAZÔNIA NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 114 As populações tradicionais locais têm seu modo de vida diretamente vinculado às atividades da pesca, mais especificamente à artesanal, e aos territórios que eles se apropriam. Antônio Carlos Diegues (2005) evidencia que a categoria população tradicional engloba uma diversidade de populações, entre as quais se destacam na Amazônia: os povos indígenas, os caboclos e os ribeirinhos. Esses grupos dominam saberes empíricos sobre rios, igarapés e manguezais e dos seres vivos encontrados nos territórios aonde desenvolvem suas atividades. Suas vivências são associadas às áreas rurais, com grande dependência do território que as cercam, tendo como premissas para os seus usos os ciclos (biológicos, climáticos, astronômicos, entre outros) para a produção e reprodução de seus modos de vida. E uma das principais atividades reproduzidas na Pontinha do Bacuriteua é a pesca artesanal aqui entendida a partir de Diegues (2005), e refere-se a pescadores que se concentram nas regiões litorâneas, rios e lagos, possuindo um modo de vida baseado na pesca, mas exercendo outras atividades como: o extrativismo vegetal, o artesanato e a pequena agricultura. Especificamente, no Nordeste Paraense, destacam-se a coleta de mexilhão, turu, cipós, plantas medicinais, lenha, madeira para carvão, entre outros (FURTADO et al., 2006) como atividades entre os pescadores artesanais, o que ocorre na Pontinha do Bacuriteua, principalmente como função das mulheres. Os tiradores de caranguejo acompanhados podem ser inseridos na categoria de pescadores artesanais, pois exercem suas atividades com base em conhecimentos empíricos sobre o ambiente em que atuam. Observando pela lente de Diegues (2005, p. 6, 7), pescadores artesanais têm: [...] um modo de vida baseado principalmente na pesca, ainda que exerçam outras atividades econômicas complementares, como o extrativismo vegetal, o artesanato e a pequena agricultura. Embora, sob alguns aspectos, possa ser considerada uma categoria ocupacional, os pescadores, particularmente os chamados artesanais, apresentam um modo de vida peculiar, sobretudo aqueles que vivem das atividades pesqueiras marítimas. Frequentemente, alguns grupos como os jangadeiros e praieiros identificam-se primordialmente como pescadores. A unidade de produção é, em geral, a familiar, incluindo na tripulação conhecidos e parentes mais longínquos. Apesar de grande parte deles viver em comunidades litorâneas rurais, alguns moram em bairros urbanos ou peri-urbanos, construindo aí uma solidariedade baseada na atividade pesqueira [...]. Entretanto, no lugar desta pesquisa, os trabalhadores não se denominam pescadores e, sim, tiradores e/ou coletores. 1 QUEM SÃO OS TIRADORES DE CARANGUEJO DA PONTINHA DO BACURITEUA? Nesse tópico, evidenciaremos as principais características apresentadas pelo grupo acompanhado. As categorias principais trabalhadas no tópico são populações tradicionais e saberes locais. Essas escolhas se dão pela literatura científica disponível nas ciências sociais, a partir de pesquisas que observaram grupos similares, e que foram utilizadas nas dissertações feitas pelos autores. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 A observação de Oliveira e Henrique (2018) identifica que os mariscadores, pelas diferenças de suas atividades, incluso o recurso explorado, técnicas, instrumentos, produção e comercialização, não se consideram pescadores. É recorrente, inclusive, fazerem distinção entre suas atividades e a dos pescadores de rios, lagos e marés. O labutador do manguezal na percepção dos entrevistados exerce função diferente, pois tem outra especialidade, ou seja, outra forma de trabalho. A lida com o mangue é muito dificultosa em relação ao trabalho nas águas, visto que exige uma série de habilidades corporais tais como: o afã com as ferradas de insetos, os cuidados nas caminhadas e a esforços físicos nas caminhadas diárias (ALVES, 2020). Porém, observando do ponto de vista científico, não há equívocos em denominar os trabalhadores dos manguezais de pescadores artesanais, pois os mangues dependem das águas das marés para existir, são ecossistemas que se localizam entre águas de rios, marés e o solo, razão porque seu “terreno” é lamoso. Das formas de coleta mais utilizadas pelos tiradores acompanhados, destacam-se o braceamento e o gancho. Oliveira (2013) as descreve: A primeira é a mais corrente. O tirador introduz seu braço na galeria do caranguejo até alcançá-lo e trazê-lo à superfície. Embora possa parecer “fácil”, simples coleta, ele praticamente tem que se deitar no solo de maneira a que seu braço possa penetrar mais DOSSIÊ AMAZÔNIA 115 fundo na toca. Para protegerem as mãos e o braço, é comum utilizarem espécies de luvas feitas de tecido resistente, agregando proteção especial de tecido para os dedos. Já o gancho é uma prática mais recente e sua difusão decorre da busca de maior produtividade e da maior concorrência na exploração do recurso. Consiste da utilização de uma barra de madeira, geralmente o cabo de uma vassoura, com um pedaço de ferro em formato de gancho na extremidade, que é introduzido nas galerias dos caranguejos que estão mais profundos, principalmente em áreas de sedimentação lamosa, popularmente conhecida como “tijuco” (OLIVEIRA, 2013, p. 61). Outra característica importante para a reprodução da pesca artesanal e dos saberes tradicionais é a transmissão geracional, via oralidade, geralmente de pai para filho, e que darão o suporte necessário para o desempenho na atividade, sobrevivência no mangue, sustento da família e reprodução social do grupo, ou seja, são saberes necessários sobre os ecossistemas para o desenvolvimento das atividades extrativistas (OLIVEIRA, 2013; OLIVEIRA; MANESCHY,2018; OLIVEIRA, 2018). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Outros autores (CAMPOS, 2013; MANESCHY, 1993) também ressaltam a transmissão geracional da profissão como fator decisivo para a reprodução social dessas atividades e modos de vida. Em Oliveira (2013), os dados apontam que todos os pais dos entrevistados trabalharam em atividades ligadas à pesca artesanal e à agricultura (geralmente lavoura) foram, na maioria das vezes, iniciados pelo pai no manguezal. Eis um relato típico: “Toda a vez que ele [o pai] ia [ao manguezal] eu ia com ele” (Z., 30 anos, entrevistado em 08.08.2011). E nas coletas de dados realizadas por Oliveira (2013), junto aos tiradores de caranguejo atuantes nas áreas abrangidas pela Resex, os discursos sempre evidenciam o grande saber acerca dos fenômenos naturais e seu uso nas atividades extrativistas pelos pescadores artesanais. Decerto, o saber empírico aprofundado das relações entre animais, plantas, marés, do ecossistema e território, é fundamental para a reprodução social das populações e de suas atividades extrativistas. Outros resultados: os tiradores de caranguejo da Pontinha do Bacuriteua acompanhados se caracterizam por início na atividade extrativista do caranguejo ainda durante a adolescência; apresentam alta evasão escolar; se autorrepresentam como especialistas na atividade, embora também atuem em atividades complementares e têm dificuldade em serem reconhecidos como categoria profissional; enfrentam problemas de saúde geralmente com alguma relação com as condições de seu trabalho; seu regime semanal de trabalho ocorre de terça a sábado, com folga geralmente no domingo e segunda-feira. Desse modo, temos uma breve caracterização de questões colocadas no cotidiano desses homens e que nos ajudam na compreensão, a seguir, de como são construídos, quais sentidos e como são realizadas as práticas socioambientais em articulação com os saberes locais. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Os tiradores de caranguejos constroem suas cosmovisões pela relação empírica com natureza e sociedade, seus modos de vida, formas de organização social e para a prática das atividades extrativistas e sentidos para as relações sociais estabelecidas entre si e com os outros, o que aqui denominamos de saber tradicional local. Para a compreensão desse saber torna-se fundamental a territorialidade, no sentido do controle de espaço, dos recursos disponíveis e para a defesa dos territórios destinados às atividades extrativistas (BEGOSSI, 2004). Assim, para a autora, os pescadores artesanais criam “marcos” e “marcas” delimitando pontos de pesca, referências no mar e na terra, importantes nos processos relacionados ao lugar pesqueiro e à tomada de decisão de localização, formas de escolha e apropriação. Há a construção de uma racionalidade própria dos grupos, que nesse processo de ocupação os dotam de sentidos culturais, construindo o reconhecimento e sua apropriação social, a partir das práticas identitárias e culturais do grupo social (SILVA, 2007; FURTADO, 1997). No caso de manguezais onde atuam os tiradores da Pontinha do Bacuriteua, a partir da não existência de territórios delimitados de forma fixa em áreas de coleta do caranguejo, como afirmado por Oliveira (2013) e Oliveira; Maneschy (2014), teremos territorialidades não fixas e os saberes dos extrativistas, eivados em suas percepções do ambiente; do caranguejo e dos peixes, irão nortear suas escolhas durante o desenvolvimento da atividade extrativista. Essa não fixidez vincula-se a caracterização dos mangues, enquanto, categoria de base comum de recursos ou territórios comuns, nos quais a apropriação dos recursos ocorre em comum com determinados grupos, não significando livre acesso (MCKEAN; OSTROM, 2001). Essas últimas autoras consideram a propriedade comum uma forma de propriedade privada, não no sentido de ser dividida em parcelas com donos definidos, mas sim apropriada por direito por um grupo, grupos e/ou comunidades bem definidas, podendo ter um caráter exclusivista e de exclusão de outros grupos5. Apesar dessa não fixidez, no contexto dos grupos sociais evidenciados, para que os territórios sejam apropriados há a necessidade do uso das territorialidades. Elas dizem respeito aos processos pelos quais o grupo ou grupos sociais envolvidos institucionalizam a apropriação e o controle, constituindo o reconhecimento social do território. 5 A principal descoberta de Oliveira (2013) e Oliveira & Maneschy (2014) é que os tiradores de caranguejo da Pontinha do Bacuriteua possuem uma dupla percepção sobre a instituição de territórios no manguezal. A primeira é de que o manguezal é uma área de livre acesso e de recursos comuns, sem propriedade privada, sem a existência de territórios delimitados e de acesso exclusivo de um determinado grupo e excludente de outros, ou seja, os tiradores não instituem territórios permanentes, pois “o manguezal é de todo mundo”. Essa percepção se manifesta antes da definição sobre qual área será explorada no dia. A segunda percepção é de que no momento da chegada e ocupação de uma determinada área pelos tiradores, elas se tornam territórios temporários, ou seja, a partir do momento em que os trabalhadores iniciam suas atividades naquela área apropriada temporariamente os territórios aparecem, com acesso apresentando algumas restrições, observação de regras no seu uso, disputas e conflitos. O direito temporário a esse território dura o tempo em que efetivamente o grupo está explorando os recursos naturais da área, sendo geralmente diário ou durante algumas horas. Nesse sentido, o manguezal “é de quem chegar primeiro”. Por fim, há fluidez entre as duas percepções, pois elas se alternam constantemente, dependendo da situação dos tiradores em determinado momento. 6 O trabalho de Silva (2011) interpreta as tradições e modernidade relacionando o conhecimento ecológico local, o manejo na pesca e os conflitos pesqueiros em comunidades no rio Negro, Brasil. 2 SABERES E PRÁTICAS SOCIOAMBIENTAIS DOS TIRADORES DE CARANGUEJO O objetivo deste tópico é analisar os principais saberes e práticas dos tiradores sobre aspectos ambientais e o ciclo biológico do caranguejo, mostrando como tais fatores repercutem na atividade e na escolha dos territórios. As categorias principais trabalhadas são saber tradicional local e práticas socioambientais. O trabalho com essas categorias se deu em virtude da literatura científica disponível nas ciências sociais, a partir de pesquisas que observaram grupos similares, que foram utilizadas nas dissertações feitas pelos autores e das observações feitas no acompanhamento das atividades dos tiradores de caranguejo da Pontinha do Bacuriteua. Nossa intenção é de articular essas categorias entre si e com os dados empíricos, evidenciando os saberes e práticas reproduzidas acerca dos saberes e práticas socioambientais. 116 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 6 O trabalho de Silva (2011) interpreta as tradições e modernidade relacionando o conhecimento ecológico local, o manejo na pesca e os conflitos pesqueiros em comunidades no rio Negro, Brasil. 2.1 TÉCNICAS E TECNOLOGIAS E O REGIME DE MARÉS As tecnologias e técnicas são construídas e usadas a partir de um conhecimento empírico, no caso específico destacamos o regime de marés. A escolha da técnica depende de alguns fatores, tais como: intenção de aumento da produção, a condição do solo e a profundidade em que está o caranguejo nas galerias. Não foram observadas diferenças entre tiradores jovens e adultos quanto às práticas de coleta. Porém, essa evolução técnica - o uso do gancho - não é acompanhada de melhores condições para o desenvolvimento das atividades, o que acarreta falta de roupas mais adequadas para adentrar no manguezal, equipamentos e instrumentos que proporcionem condições para o desenvolvimento da atividade sem impactar o ecossistema (MANESCHY, 1993). O clima na região caracteriza-se, tendencialmente, por um período bastante chuvoso, que ocorre de dezembro a maio, e um período mais seco, com muito sol e calor, que acontece de junho até novembro (GLASER; CABRAL; RIBEIRO, 2005). Diegues (2005) reafirma a importância da estacionalidade como elemento para a organização social e a identidade das populações tradicionais que vivem da pesca artesanal. A vida destas e suas atividades econômicas não podem ser desvinculadas do seu saber e dependência das duas estações: das águas e da seca. g Glaser, Cabral e Ribeiro (2005) relacionaram a importância da estacionalidade com as atividades econômicas desenvolvidas nas comunidades onde vivem os tiradores. Eles diversificam as atividades buscando meios complementares de garantir a renda familiar, principalmente no período de maior precipitação, também pela influência do período reprodutivo do caranguejo, sendo que a proximidade das comunidades com os recursos naturais no seu entorno facilita essa diversificação. Já no período mais seco e com maior abundância do caranguejo a extração se intensifica. A maioria dos entrevistados indicou que a presença de chuva, principalmente aquelas torrenciais e associadas às marés, tem relação direta com a baixa produtividade nesse período. Um entrevistado sobre essa relação assim se manifestou: “Quando chove grosso demora mais pra vazar a maré” (M., entrevistado em 08/08/2011). A chuva prolonga a submersão das áreas do manguezal, reduzindo as possibilidades de coleta do caranguejo. Resta ao tirador aguardar a diminuição da chuva e a vazante da maré. “No inverno, quando chove muito, não presta, a gente trabalha e não pega nada. Na chuva fina ainda pega, na chuva grossa não pega” (M., entrevistado em 08/08/2011). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 As territorialidades são as regras sociais e os padrões pelos quais grupos sociais se apropriam de seus espaços de vida e trabalho, reivindicando-os como seus e mobilizando-se em sua defesa contra grupos rivais (SILVA, 2011; ABREU, 2010; BEGOSSI, 2004).6 O uso dos termos no plural indica a heterogeneidade desses processos dentro de um grupo e/ou entre grupos diferentes, que diferem nas perspectivas sobre territórios e práticas inerentes ao seu uso (OLIVEIRA, 2013; OLIVEIRA; MANESCHY, 2014). ) A partir dos territórios e territorialidades, os saberes tradicionais locais são usados e influem nas tecnologias e técnicas construídas e utilizadas nas atividades extrativistas. 6 O trabalho de Silva (2011) interpreta as tradições e modernidade relacionando o conhecimento ecológico local, o manejo na pesca e os conflitos pesqueiros em comunidades no rio Negro, Brasil. DOSSIÊ AMAZÔNIA 117 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 118 em visualizar as “áreas esbandalhadas”7 e/ou em “descanso”, devido à submersão das áreas pela chuva, dificultando o reconhecimento dos territórios recém-utilizados; e, por fim, aumento da característica polivalente do grupo. em visualizar as “áreas esbandalhadas”7 e/ou em “descanso”, devido à submersão das áreas pela chuva, dificultando o reconhecimento dos territórios recém-utilizados; e, por fim, aumento da característica polivalente do grupo. Já na estação seca há mais facilidade no trabalho. O caranguejo tem mais dificuldade para penetrar mais fundo no solo, que não está tão “mole”. O discurso de facilidade na coleta também evidencia que a “safra” do caranguejo ocorre em outubro, quando é menor o índice pluviométrico. Nos trechos abaixo sobressai a pouca profundidade dos caranguejos nas galerias. “Agora no verão fica raso, em agosto, setembro, outubro. A lama engrossa e ele [o caranguejo] não cava. (S. C., entrevistado em 13/07/2011) ”. Os principais saberes levados em consideração nas práticas socioambientais no período mais seco são: os territórios com sedimentação lamosa estão mais “secos” e com menor influência das marés e os caranguejos, também, apresentam dificuldade em se “enterrar” nas galerias, ficam mais rasos e facilitam sua coleta; há facilidade em visualizar as “áreas esbandalhadas” e/ou em “descanso”, propiciando o reconhecimento dos territórios recém- utilizados; e, por fim, a quantidade de galerias e o tamanho da sua circunferência são primordiais na escolha dos territórios, pois estão relacionadas com uma possível grande quantidade de caranguejos disponíveis e pela valorização dos machos graúdos, discernível pelo maior tamanho das galerias. Portanto, como afirma Oliveira (2013), a estacionalidade tem implicações, junto às marés e ao ciclo biológico do caranguejo, na produtividade da coleta do caranguejo. O regime de marés também funciona como regulador das atividades dos tiradores. Schmiegelow (2004, p.119) apresenta a conceituação científica de maré como um fenômeno regular no meio marinho caracterizado pelas oscilações nos níveis das águas, resultado da influência gravitacional exercida pelo sol e, sobretudo, pela lua, originando elevações e reduções de níveis das águas do mar. Geralmente, o seu movimento ocorre de doze em doze horas, duas vezes ao dia, constituindo duas enchentes da maré, conhecidas como preamar, e duas vazantes da maré, denominadas baixa-mar. O autor também discorre sobre as marés de quadratura, que ocorrem nas fases da lua minguante e da lua crescente e são responsáveis pelas menores amplitudes de maré. 7 As áreas esbandalhadas são pontos dentro do manguezal que foram recentemente explorados, onde os tiradores coletaram caranguejo, e que se apresentam “pisoteadas”, com marcas visíveis de que outros trabalhadores “mexeram” naquele local, portanto encontram-se esbandalhadas e sem muitos caranguejos graúdos (OLIVEIRA, 2013; OLIVEIRA; MANESCHY, 2014). 2.1 TÉCNICAS E TECNOLOGIAS E O REGIME DE MARÉS Os tiradores afirmam que quando chove no manguezal as áreas de tijuco são “lavadas” e ficam mais “moles”, por se constituírem de sedimento lamoso, facilitando o caranguejo em se “enterrar” no solo, dificultando a coleta via braceamento e, em alguns lugares, mesmo com o “gancho”. O deslocamento dos tiradores nessa condição é difícil, principalmente, para carregar os caranguejos capturados de volta para a embarcação, tarefa que se torna extenuante. Também é necessário ressaltar que o período de chuva coincide com um dos ciclos biológicos do caranguejo indicados pelos tiradores como de baixa produtividade: o período de reprodução (OLIVEIRA, 2013). O grupo faz articulações entre as condições de estacionalidade com a escolha dos territórios para a atividade, com os seguintes aspectos: tendência de escolher áreas com a presença de mais areia, devido à dificuldade que os caranguejos têm em se “enterrar” nas galerias, ficando mais rasos e facilitando sua coleta; tendência de escolher territórios mais próximos das margens dos furos e igarapés, também denominadas pelos tiradores de caranguejo de cabeceiras, evitando o deslocamento para áreas mais afastadas e que possuem maior presença de sedimentação lamosa, para que não percorra grandes distâncias. Há, também, dificuldade NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 119 Para os tiradores de caranguejo, as marés influenciam diretamente no seu trabalho, pois nas marés de maior amplitude é maior a tendência de submersão do solo do manguezal pelas águas, o que dificulta a coleta de caranguejo. Por conta disto, os trabalhadores aguardam a vazante da maré para executar suas atividades. Nas de menor amplitude, as de quadratura, quando o solo do manguezal tem a tendência menor de ser “invadido” pelas águas dos rios e do mar, as coletas são realizadas com maior facilidade, o que possibilita o aumento da produtividade. p Observou-se que os tiradores da Pontinha do Bacuriteua utilizam os termos lançante e maré de quarto para se referir às marés de sizígia e quadratura, respectivamente. Os tiradores da Pontinha do Bacuriteua afirmam que a etapa de marés lançantes tem início a partir de um ou dois dias após as luas de quarto minguante e quarto crescente e se estendem até um ou dois dias após as luas nova e cheia, então recomeça o período de marés de amplitude decrescente, as de quarto. Durante as lançantes, a cada dia a maré aumenta de amplitude, até chegar aos dias de maiores marés, que alguns tiradores se referem como “cabeças de lanço”. O manguezal fica submerso dificultando o trabalho, quando eles não conseguem visualizar bem as galerias, consequentemente seu diâmetro e, também, perceber se esses territórios foram recém-usados (“esbandalhados”): “Maré lançante, crescendo, fica mais complicada [a coleta]” (S. L., entrevistado em 13/07/2011). Como já afirmado por Maneschy (1993), a dificuldade de coletar caranguejos durante as cheias da maré repercute na diminuição da produção. Os tiradores da Pontinha do Bacuriteua percebem as diferenças diárias no comportamento da maré, pois observam que a enchente atrasa em uma hora a cada dia. Esse movimento influencia na quantidade de horas que ficam no manguezal, haja vista que eles têm de aproveitar as horas de vazante durante o dia claro para poderem trabalhar. Desse modo, se a enchente começar a ocorrer mais próxima do meio dia, resta-lhes pouco tempo para que ela vaze antes que anoiteça, obrigando os tiradores a sair mais cedo do mangue, diminuindo sua produtividade. Seus turnos de trabalho diários, portanto, variam conforme esses fluxos das marés. A gente sabe quando [a maré vai encher]. Ela vai modificando, 1 hora, 2 [horas] de diferença. Cada dia tarda uma hora [para encher]. (C., entrevistado em 08/08/2011)”. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Já as marés de sizígia, que ocorrem nas fases da lua nova e lua cheia, são responsáveis pelas maiores amplitudes de maré. p p p Outra definição de maré é feita por Baptista Neto, Ponzi; Sichel (2004) que agrega às influências gravitacionais da lua e do sol, as forças ligadas ao movimento da Terra. As marés são formadas pela ação combinada de forças de atração gravitacional entre a Terra, Lua e por forças centrífugas geradas pelos movimentos de rotação em torno do centro de massa do sistema Sol-Terra-Lua que se localiza no interior da Terra, a uma distância de um quarto do raio terrestre, as forças estão em equilíbrio impedindo que a lua seja atraída para a superfície da Terra (BAPTISTA NETO; PONZI; SICHEL, p.190). Assim como Schmiegelow (2004), Baptista Neto, Ponzi; Sichel (2004) colocam que durante um dia qualquer ponto da terra passa duas vezes pela enchente e vazante da maré, sendo que o sol é responsável pelas variações das marés de sizígia e de quadratura. Os autores também mostram as influências das marés nas zonas costeiras, caso das áreas exploradas pelos tiradores da Pontinha do Bacuriteua, que invadem os estuários e baías, submergem os manguezais, e são também responsáveis pela manutenção da diversidade biológica dessas áreas, transportando sedimentos e modelando a linha da costa. DOSSIÊ AMAZÔNIA O cara é acostumado na maré e sabe quando enche. É uma hora mais por dia. Fica deitado, pescando no barco [esperando a maré vazar] (M., entrevistado em 08/08/2011). A fala a seguir indica a variação no horário de trabalho em função do horário da vazante: “Conforme a maré saí do porto de lá 8 horas, fica esperando a maré escoar e entra (no mangal), sai 3, 4 horas (da tarde). (S. L., entrevistado em 13/07/2011)”. Na adaptação ao horário da maré, eles também procuram atender aos horários do mercado. Daí o fato de estarem cedo pela manhã posicionados próximo ao local escolhido, aguardando na margem do rio ou igarapé o momento em que podem adentrar e dar início ao trabalho. “Tem que esperar vazar a maré. Tem horas que 10 horas a maré vaza, tem dia que meio dia e a gente vai até 5 hora trabalhando. O cara tem que esperar ela vazar” (M., entrevistado em 08/08/2011). Contudo, nos dias em que a maré enche mais cedo, final da madrugada e início da manhã, mais cedo será sua vazante, deixando mais tempo disponível para o trabalho, garantindo uma captura maior; nessa situação os trabalhadores retornam do manguezal no final da tarde e início da noite. “Sai cedo [o tirador do manguezal] quando enche tarde. Quando chega [de manhã] e ela tá cheia espera vazar” (Z., entrevistado em 08/08/2011). As marés de quarto, de menor amplitude, na percepção dos tiradores não “lavam” o manguezal, ou seja, não submergem o solo, propiciando aos tiradores o desenvolvimento de suas atividades sem a premência do tempo, como ocorre durante as lançantes. “A maré de NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 120 quarto, porque ela não lava [inunda] o mangal, eles [caranguejos] fica mais raso” (F., entrevistado em 18/07/2011). quarto, porque ela não lava [inunda] o mangal, eles [caranguejos] fica mais raso” (F., entrevistado em 18/07/2011). Essa não submersão do manguezal torna o solo menos encharcado, “mais duro” e, como afirmado abaixo, com o caranguejo tendo dificuldade em cavar suas galerias. A primeira citação relaciona a maré lunar e a estação do ano como fatores que influenciam no seu trabalho e sua preferência pela maré de quarto: “É na maré de quarto. No inverno fica ruim que a lama fica mole e ele (caranguejo) cava” (S. C., entrevistado em 13/07/2011). 2.2 A COMPOSIÇÃO DO SOLO E A FLORA DO MANGUEZAL Em relação ao ambiente, os tiradores da Pontinha do Bacuriteua necessitam de saberes acerca da composição do solo e da flora para a escolha das áreas de trabalho. Essa escolha influencia na produtividade e no tipo de caranguejo que é coletado. Com isso, existe um mapeamento mental das áreas segundo essas características e, dependendo das circunstâncias do momento, será feita a escolha e esses elementos serão considerados. O solo do manguezal é dividido pelos tiradores em “areal” e “tijuco”. Nas áreas com predominância de areia, as marés que submergem o manguezal transportam esse elemento para dentro do mangue, por isso sua presença é mais constante nas cabeceiras dos furos e igarapés e em áreas próximas à praia de Ajuruteua. No “tijuco” há predominância de sedimentação lamosa. Esses solos ficam em regiões mais centrais no manguezal, tendo como referência sua distância em relação aos rios e igarapés. O “tijuco” é subdividido em “tijuco mole” e “tijuco duro”. Este último é caracterizado pela presença de pneumatóforos8, conhecidos pelos tiradores por siribal. São áreas com menor possibilidade de atolamento devido à retenção de sedimentos pelos pneumatóforos, tornando o solo mais “duro”; já o tijuco mole ocorre em áreas sem pneumatóforos, cuja tendência é, então, a][ de atolamento do tirador em seu deslocamento no manguezal. A composição do solo é importante na percepção dos tiradores acerca das possibilidades de sucesso na coleta, pois nas áreas com maior concentração de areia caranguejos têm maior dificuldade de penetração no solo, o que facilita a coleta no braço. Já nas áreas com maior concentração de tijuco, o caranguejo consegue penetrar mais facilmente e mais profundamente nas galerias, o que dificulta a coleta no braço. Essas características do solo vão influir na técnica de coleta, por exemplo, os tiradores usam o gancho preponderantemente no “tijuco mole”, pois os caranguejos estão mais fundos pela facilidade em cavar suas galerias. É o que afirma o tirador quando compara os dois tipos de elementos. A gente já sabe, num ponto e outro [revezam os locais de coleta], sempre na parte de mais areia, fica mais fácil, parte de tijuco [sedimentação lamosa] fica mais difícil, fica mais fundo [o caranguejo], o caranguejo fica mais ou menos dois metros no fundo (S. L., entrevistado em 13/07/2011). Porém, outros tiradores afirmaram que, geralmente, as cabeceiras estão “esbandalhadas”, o que ocorre quando o lugar foi recentemente explorado por tiradores, pela maior facilidade de chegar nelas. DOSSIÊ AMAZÔNIA Em suma, no discurso dos tiradores é sempre evidenciado o saber acerca dos fenômenos naturais. É comum a expressão “a gente sabe”, colocada para demonstrar a experiência adquirida na atividade e a vivência com o ambiente natural e seus fenômenos. A organização e o desenvolvimento do trabalho diário dependem desses saberes. Outros aspectos do ambiente também vão influir, a partir dos saberes, na atividade de tiração de caranguejo, como a composição do solo e a flora do manguezal. 8 Os pneumatóforos são os sistemas radiculares que permitem a fixação das árvores de Avicennia germinans e também atuam como raízes respiratórias (FONSECA, 1995). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 121 entre um e dois quilômetros para efetuar as coletas: “Vê esbandalhado [o local da coleta] nem pensar (M., entrevistado em 08/08/2011)”. entre um e dois quilômetros para efetuar as coletas: “Vê esbandalhado [o local da coleta] nem pensar (M., entrevistado em 08/08/2011)”. Também deve levar em conta que os rios e igarapés são os principais meios de acesso ao manguezal. Nas áreas mais próximas desses cursos de águas há tendência de escassez de recursos, tanto pela facilidade no acesso, quanto pela interferência do regime de marés nesses locais. Assim, é mais difícil encontrar caranguejos machos e graúdos, havendo a necessidade de adentrar o manguezal e buscar as áreas de tijuco, como mostra o trecho abaixo: “Mais dentro do mangal, tem mangal que a gente anda mais longe. Na beira do rio tá mais esbandalhado e mais dentro é mais raso o caranguejo (C., entrevistado em 08/08/2011)”. Portanto, a avaliação das áreas de trabalho leva em conta, também, a composição do solo. Dependendo da necessidade dos tiradores, do regime das marés, do ciclo biológico do caranguejo e da estacionalidade será realizada a escolha de qual área é mais propícia naquele momento. Em relação ao solo, há sinais deixados por outros tiradores que são percebidos e indicam a utilização ou não daquele trecho do manguezal. Domingues (2008) e Reis (2007) afirmam que os tiradores observam os sinais existentes nesse trecho e quando detectam que está “mexido” ou “esbandalhado” geralmente decidem por não o utilizar e saem em busca de outros espaços sem sinal de uso recente. Outra prática importante é o descanso das áreas de coleta. Após o uso de uma área, os tiradores dão um intervalo de tempo, uma espécie de pousio, para o retorno. Domingues (2008) conceitua essa prática de descanso como uma forma de manejo e em sua pesquisa percebeu que o intervalo de tempo varia bastante. Reis (2007) afirma que os tiradores não retornam no dia seguinte ao local do dia anterior, mas em dias alternados. Nas entrevistas no Bacuriteua, o intervalo de tempo para o descanso das áreas, de acordo com os tiradores, varia de semanas a até meses. O descanso tem o intuito de permitir que os caranguejos repovoem a área e/ou aflorem das galerias, tornando-os mais “rasos”. Os tiradores afirmam que todo dia exploram uma área diferente: “Não, a gente vamos em outro lugar, cada dia em um lugar. 2.2 A COMPOSIÇÃO DO SOLO E A FLORA DO MANGUEZAL Há relatos de que os tiradores têm que adentrar o manguezal 2.3 A COLETA DO CARANGUEJO: SABERES E PRÁTICAS Neste tópico discutiremos como os tiradores de caranguejo da Pontinha do Bacuriteua percebem os seguintes aspectos do caranguejo-uçá: ciclo biológico, sexo e os tipos coletados. Neste tópico discutiremos como os tiradores de caranguejo da Pontinha do Bacuriteua percebem os seguintes aspectos do caranguejo-uçá: ciclo biológico, sexo e os tipos coletados. Vale recorrer novamente a Domingues (2008, p.34-35) que discute sobre o ciclo biológico anual do caranguejo a partir da percepção dos tiradores da península bragantina Vale recorrer novamente a Domingues (2008, p.34-35) que discute sobre o ciclo biológico anual do caranguejo, a partir da percepção dos tiradores da península bragantina, ressaltando três fases principais: a “andada”; a troca da carapaça, também conhecida como “troca da camisa”; e a “safra”. Durante os meses de janeiro a abril ocorre a andada, também conhecida como “souatá”, que é o período reprodutivo da espécie, quando os caranguejos estão fora das galerias, “andando” pelo solo do manguezal. Esse fenômeno ocorre em determinadas fases da lua durante esses meses. Há diferenças, pois nos três primeiros meses há o predomínio dos machos fora das galerias e, no mês de abril, há o predomínio da fêmea (condurua) no solo. Os tiradores referem- se a essa andada das fêmeas como o “souatá das conduruas”. A partir de junho e mais intensamente em julho se inicia a troca da carapaça ou troca da “camisa” do caranguejo, quando ele “fica de leite”, impróprio para o consumo e suas galerias são “tapadas” pela própria espécie. A troca vai até final de agosto, ou início de setembro. De outubro em diante inicia o período de “safra” e, progressivamente, de engorda do caranguejo, quando a produtividade dos tiradores é maior e diretamente proporcional à oferta de caranguejos. Em resumo, temos a seguinte configuração do ciclo biológico do caranguejo na percepção dos tiradores. Maneschy (1993, p.43), referindo-se aos tiradores do Município de São Caetano de Odivelas, resumiu de maneira muito semelhante ao saber dos tiradores sobre o ciclo biológico do caranguejo e sua produtividade. De acordo com a autora, no período reprodutivo do caranguejo há uma intensificação da coleta do crustáceo, que se torna “fácil” para ser coletado na visão dos tiradores, pois os caranguejos estão fora das galerias e mais expostos. dominância de bosques de mangueiro (Rhizophora mangle) (DOMINGUES, 2008, p.33). Os tiradores da Pontinha do Bacuriteua fazem a mesma classificação sobre as árvores baseada no conhecimento ecológico local citado por Domingues (2008) e eles também estabelecem a relação entre o raizal (Mangueiro) e o caranguejo cabelo branco, que se trata do caranguejo-uçá (Ucides Cordatus). Há, na percepção deles, preferência dos caranguejos pelas folhas dessa árvore, como afirmado abaixo: “Tem na areia caranguejo bonito, gordo, cabelo branco e tem o da lama. Muito caranguejo no raizal (S. C., entrevistado em 13/07/2011)”. A composição do solo, relacionada com as árvores e com o tipo de caranguejo requerido pelo mercado se apresenta como mais um fator do ecossistema cuja percepção dos tiradores de caranguejo da Pontinha do Bacuriteua levam em conta no momento da escolha das áreas de coleta e a instituição dos territórios temporários. Por fim, além de todos esses saberes construídos a partir de uma “leitura” realizada pelos tiradores acerca do ecossistema, também temos construções sociais sobre o ciclo biológico do caranguejo que afetam sua coleta. DOSSIÊ AMAZÔNIA Passa um mês, dois meses pra voltar [a uma área já utilizada]. Se for de novo lá, o bicho tá fundo” (C., entrevistado em 08/08/2011)”. De acordo com os tiradores, quanto menor o espaço de tempo entre uma coleta e outra na mesma área menor a produtividade e o tamanho dos caranguejos, por isso um tempo maior é recomendado. “Em uma semana, o cara vai hoje e na outra semana vai de novo, mas não pega muito não (Z., entrevistado em 08/08/2011)”. Além da facilidade dos caranguejos para cavar as galerias que se localizam no tijuco mole, há mais um fator percebido pelos tiradores que favorece a concentração de caranguejos machos, graúdos: sua alimentação das folhas do Mangueiro (Rhizophora Mangle). Quando os tiradores fazem referência ao mangueiro destacam suas raízes aéreas, denominando-as de “raizal”. Domingues (2008), sobre o saber dos tiradores acerca da flora do manguezal, afirma: Os coletores demonstraram possuir um conhecimento sobre as populações de caranguejos e os bosques de mangue. Segundo eles há manguezais que são berçários, pois há grande quantidade de caranguejos pequenos e fêmeas. Mas existem bosques em que o caranguejo é mais raso ou mais fundo (a relação raso ou fundo corresponde a profundidade da toca), há também locais onde o caranguejo é graúdo. Também os tiradores associam as características da população de caranguejo às peculiaridades do sedimento e da espécie arbórea dominante no bosque. Os manguezais denominados areais (nomenclatura utilizada para o sedimento com maior presença de areia), normalmente possuem caranguejos menores e flora dominada por siribeiras (Avicennia germinans) e menos frequentemente tinteiras (Laguncularia racemosa). Em contrapartida, há locais onde predomina o tijuco (nomenclatura utilizada para o sedimento com maior presença de silte e argila), onde o caranguejo é maior e há NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 122 dominância de bosques de mangueiro (Rhizophora mangle) (DOMINGUES, 2008, p.33). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 123 manguezal; no dizer de um trabalhador entrevistado por Souto (2007, p.74), o caranguejo "fica tudo doido aqui no mangue". manguezal; no dizer de um trabalhador entrevistado por Souto (2007, p.74), o caranguejo "fica tudo doido aqui no mangue". De acordo com os tiradores, a andada ocorre nos primeiros quatro meses do ano, sendo dividida em primeira andada que ocorre de janeiro a março quando macho e fêmea estão se reproduzindo e acontecendo no mês de abril a desova das fêmeas no manguezal, também conhecido por andadas das conduruas. “Final da andada fica buchuda. Março, abril é a derradeira andada, a da condurua, só da condurua. Janeiro é a primeira andada, eles ficam namorando. (S. C., entrevistado em 13/07/2011)”. Os tiradores relataram que há muita dificuldade durante esse período, principalmente durante a semana em que o IBAMA proíbe a coleta do caranguejo. Seus relatos compreendem que as políticas públicas e sociais voltadas especificamente para o período do defeso não levam em conta o contexto em que vivem. Por exemplo, a cesta básica que foi distribuída em um determinado ano pelo IBAMA foi considerada de má qualidade e com produtos que não fazem parte da dieta local. As decisões políticas são vistas como tomadas de cima para baixo: Na andada fica difícil, porque a gente não vai (Z., entrevistado em 08/08/2011). Proíbe, fica sem pegar, os homem [técnicos do IBAMA] tão na estrada pra cima e pra baixo. A gente dá um jeito na boia e vai passando. Fica uma semana sem pegar. Uma vez deixaram uma cesta básica com uma farinha branca. Passava um dia no fogo e a farinha pulava na panela [reclamação sobre os produtos da cesta] (G., entrevistado em 18/07/2011). De janeiro a março é fixado o defeso, quando são proibidos de coletar o caranguejo. Há fiscalização por parte dos técnicos do IBAMA e do ICMBio para que se evite a captura da espécie, como citado na fala anterior. É durante esse período que buscam atividades complementares com mais frequência, destacando-se a pesca de linha e a coleta de sururu, dentre outras. Os tiradores afirmam que ficam de cinco a oito dias sem poder trabalhar. No ano de 2012 foram espalhadas na área da RESEX matérias de divulgação do período de defeso, buscando sensibilizar a população e os tiradores em relação ao período de reprodução do caranguejo-uçá. 2.3 A COLETA DO CARANGUEJO: SABERES E PRÁTICAS Essa condição colabora para que muitas pessoas das comunidades próximas do mangue adentrem, incluindo mulheres e crianças pequenas, para apanhar os caranguejos, sendo que no caso das crianças, geralmente, é visto como um período propício para a transmissão dos saberes sobre a prática da coleta. Essa ideia de que o caranguejo se torna presa fácil nesse período é compartilhada também por Souto (2007, p.77) que, em estudo realizado no município de Acupe, no Estado da Bahia, coloca essa prática, na visão do IBAMA, como grande ameaça aos estoques de caranguejos-uçás existentes na região. Outra percepção dos tiradores sobre o comportamento do caranguejo durante a andada é a suposta “loucura” dos bichos, que ficam “perdidos” no NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Além disso, como já afirmado, há a intensificação das atividades complementares, principalmente a pesca de linha para a subsistência; há a dependência em relação aos comerciantes que “fiam” seus produtos para as famílias. Muitas vezes os patrões são donos ou ligados a esses comércios, havendo uma relação de subordinação dos tiradores em relação a esses sujeitos. “Fica difícil, a gente tira fiado no patrão no comércio. Quando trabalha a gente paga” (Z., entrevistado em 08/08/2011). Maneschy (1993) criticou a fixação de um período de defeso sem levar em consideração “as estruturas econômicas” e a realidade em que estão inseridos os tiradores, incluindo o mercado que possui demandas cada vez maiores de produção do caranguejo. A autora também destacou a importância do conhecimento científico, citando as Ciências Biológicas, para a delimitação desses períodos de defeso, juntamente com os saberes dos tiradores de caranguejo. p j g j Já a segunda etapa do ciclo biológico do caranguejo percebida pelos tiradores é a troca de carapaça ou troca da “camisa”, cientificamente conhecida por ecdise. Souto (2008), após pesquisar os pescadores artesanais do município de Acupe, na Bahia, verificou o saber acerca da ecdise; os pescadores relataram que essa troca na carapaça não se dá de maneira simultânea para todos os caranguejos, mas que determinado contingente a realiza e outro não, só vindo a fazê-lo mais tarde, o que permite que a coleta continue em menores quantidades. Souto (2007, p.75) também verificou o saber dos tiradores sobre essa “não sincronia” dos caranguejos na troca da carapaça. p ç De acordo com Souto (2007), esse período do ciclo biológico do caranguejo implica diretamente na cadeia produtiva. Domingues (2008, p.40) também analisa como a troca da “camisa” afeta o trabalho dos tiradores da Vila do Tamatateua, localizada no município de Bragança, onde esses trabalhadores têm que procurar alternativas visando complementar a renda mensal familiar e encontram muitas dificuldades nessa busca. O autor diz que “essas restrições, por sua vez, acabam por estimular a redução dos esforços na dedicação a esta atividade, forçando-os a dedicar-se às outras atividades produtivas”, corroborando para o grande número de tiradores que também complementam sua renda com outras atividades. Alguns tiradores denominam esse período de troca da carapaça como “falha” devido à menor disponibilidade de caranguejos destinados ao consumo humano. Durante esse período os caranguejos “ficam de leite”, expressão usada pelos tiradores, e impróprio para o consumo humano a seu ver. 9 Quando os tiradores afirmam que os caranguejos estão tapados significa que a abertura da galeria onde o bicho fica para proceder a troca da carapaça está bloqueada. DOSSIÊ AMAZÔNIA A comercialização é proibida nesses dias, daí a importância de sensibilizar também os consumidores e os agentes nos diferentes elos da cadeia de comercialização do crustáceo. Nas conversas com os tiradores, o entendimento deles sobre o defeso é de que as decisões são tomadas sem consultá-los e que já ocorreram erros nas datas do defeso. Alguns afirmam que não há a necessidade do defeso, pois os caranguejos se reproduzem em grande quantidade e rapidamente e que existem estratégias de conservação, sendo coletados somente machos e graúdos. “Condurua não, só graúdo. Porque é proibido, elas que “filha” eles [responsáveis pela reprodução da espécie]. Se a gente pega, daqui a uns tempos não tem mais caranguejo pra gente pegar” (G., entrevistado em 18/07/2011). Outra fala atribui problemas de descumprimento de normas por parte de pessoas de fora. Há, também, referências quanto à captura de fêmeas em alguma proporção: “A RESEX facilitou, ninguém vai, mas outro de fora não respeita defeso e não fica bem, fica no máximo 8 dias sem pegar, vai trabalhando e reservando um pouquinho [de dinheiro para se manter no período da proibição]” (S. L., entrevistado em 13/07/2011). Em virtude do defeso do caranguejo, os tiradores da Pontinha do Bacuriteua utilizam estratégias para alimentar as famílias durante esse período. Dizem que quando são divulgadas as datas do defeso, na semana anterior há um aumento na produtividade, quando se intensifica a coleta de caranguejos para aumentar a renda e possibilitar a compra de alimentos durante o período parado, fato afirmado na fala anterior. É necessário prestar atenção a essa estratégia do aumento na produtividade, pois se de fato há esse aumento para compensar a parada, ele pode, então, neutralizar em parte os efeitos do defeso, que visa justamente proteger a espécie. 124 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 125 difícil, mas quando trocam a casca eles ficam raso (C., entrevistado em 08/08/2011)”. Portanto, é um período de grande dinâmica no que tange à atividade extrativista, devido à dependência do tirador em relação aos caranguejos que ainda não iniciaram a troca, ou que já a fizeram, e os que estão passando por esse processo. Passado o período de troca da “camisa”, inicia-se a “safra” do caranguejo, momento em que há grande quantidade de caranguejos disponíveis para a coleta e quando inicia a “engorda” e o crustáceo está mais “graúdo”, com casco acinzentado e com a carne de gosto “doce”, de acordo com os tiradores. É o período em que aumenta a produtividade e diminui a demanda pelo produto. De um lado, porque já passou o mês de férias e de pico de consumo e, de outro lado, em grande parte dos municípios do nordeste paraense ocorrem os círios municipais, cuja culinária nem sempre engloba o caranguejo, fazendo com que seja o período do ano em que é pago o menor valor pela cambada. Porém, diferentemente do período da andada, quando os caranguejos se tornam presas fáceis, na fase da safra o comportamento do caranguejo se modifica, tornando-se mais “arisco” e dificultando a sua coleta. Souto (2007) enumera diversas expressões utilizadas pelos tiradores de Acupe, na Bahia, para sinalizar a “esperteza” dos bichos nesse período, tais como: “veiacos”, "astúcia" e "tarimba". Quando se considera a percepção dos tiradores sobre o ciclo biológico do caranguejo há relações diretas entre a produtividade dos trabalhadores e o preço do caranguejo. Durante a andada o preço da cambada é baixo e há a proibição por parte do Estado. Na safra essa relação se inverte, pois há maior oferta de caranguejo e menor demanda por parte do mercado e, consequentemente, o preço da cambada é menor. “Agora [julho] no máximo 10 cambadas, 8, 9, 7. Em outubro pega 15, 20. Nesse tempo de agora [julho] a gente tá vendendo de R$ 8,00. A partir de outubro tira mais, mas diminui o dinheiro, a gente vende de R$ 3,00, de R$ 4,00 (Q., entrevistado em 18/07/2011)”. Apesar da possível defasagem no texto, o importante é evidenciar as diferenças de preço em relação ao ciclo biológico que interfere na quantidade e tamanho disponível para coleta. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Um dos tiradores entrevistados por Souto (2007, p.75) fez o seguinte relato: “Agora vai começar uma fase ruim, ruim! Vai levar no mínimo uns trinta dias aí para o cara sofrer. Porque ele (o caranguejo) tá panhando leite. Quando panha leite, não presta. Ele fica molinho, molinho". Essa restrição no consumo do caranguejo que “fica de leite”, segundo Souto (2007, p.75), dá-se em consequência do alto teor de carbonatos nas vísceras e carne do caranguejo, acarretando um sabor desagradável para o paladar humano e provocando “diarréia e dores abdominais e alterações no sistema nervoso, como letargia e entorpecimento”. Os caranguejos nessa fase não são coletados pelos tiradores de caranguejo na Pontinha do Bacuriteua, segundo o que se apurou aqui. A característica identificada pelos tiradores de que o caranguejo está trocando a “camisa” é a “tapagem”9 da galeria feita pelo próprio crustáceo, que se aloja e inicia o processo de mudança da carapaça. Os tiradores quando percebem que as galerias estão “tapadas” saem em busca de galerias abertas, sendo uma das características verificadas no momento da escolha dos territórios para a coleta. A não sincronia dessa etapa faz com que os tiradores continuem indo ao manguezal, porém cai a quantidade de caranguejos coletados. E efetuam maior mobilidade dentro do manguezal, buscando as tocas abertas. “Tem muito tapado e muito destampado. Fica muito NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 126 terem maior aceitação no mercado e maior preço, sendo que “mesmo as fêmeas, quando coletadas, passam por uma seleção de tamanho” (SOUTO, 2007). terem maior aceitação no mercado e maior preço, sendo que “mesmo as fêmeas, quando coletadas, passam por uma seleção de tamanho” (SOUTO, 2007). Domingues (2008, p.34), a partir dos relatos dos tiradores, identifica as principais diferenças de sexo dos caranguejos elencadas pelos sujeitos em sua pesquisa: o tamanho da carapaça das conduruas é menor que dos machos; “a presença de pêlos em maior ou menor quantidade na região inferior das patas; o perfil mais ou menos delgado da unha (primeiro segmento das patas)”; e o “imbigo” (abdômen), localizado na parte inferior da carapaça, maior nas fêmeas para abrigar os ovos após o acasalamento. Os tiradores também utilizam caraterísticas na entrada das galerias feitas pelos caranguejos para diferenciar o sexo. Maneschy (1993, p.29) relata que os trabalhadores distinguem as galerias da condurua pelas marcas das patas em torno dos buracos; as “fêmeas deixam sulcos mais finos e profundos” e quando os tiradores se enganam na coleta e apanham uma fêmea imediatamente a soltam, garantindo, na visão destes, a reprodução da espécie, conforme os relatos colhidos pela autora em São Caetano de Odivelas. Domingues (2008) também trata sobre esse assunto dizendo que os machos fazem as galerias com maior circunferência na abertura e deixam rastros mais espessos, devido a seu maior tamanho em relação à condurua. Portanto, em relação à escolha dos territórios para desempenho da atividade extrativista, os tiradores tendem a buscar locais onde exista muitas galerias e com grande circunferência, que configuram a presença de caranguejos machos e graúdos. Em relação aos tipos de caranguejos coletados pelos tiradores da Pontinha do Bacuriteua dois se destacam: o caranguejo cabelo branco e o caranguejo cabelo vermelho. DOSSIÊ AMAZÔNIA Portanto, os saberes acerca das marés, ciclo biológico do crustáceo, a identificação do tamanho, sexo e as condições dos diferentes espaços próprios dos manguezais são aspectos fundamentais para as estratégias e a organização da atividade extrativista, influenciando em preço, demanda e qualidade do produto, entre outros (DOMINGUES, 2008). Torna-se mais claro, também, o quanto se constituem elementos que interferem na relação entre esses trabalhadores e os patrões, principalmente, no que tange aos preços praticados na compra das cambadas pelos últimos. Outro aspecto que influencia na comercialização é o sexo e o tamanho dos caranguejos coletados. De acordo com Domingues (2008, p.35), o mercado é quem regula a seleção dos caranguejos a serem coletados. É estabelecido o preço a ser pago a partir do tamanho do caranguejo, sendo proporcional, ou seja, quanto maior o tamanho maior o preço pago. A compra de fêmeas é restringida. Acerca dessa proporcionalidade do valor pago com o tamanho do crustáceo, Reis (2007, p.217) afirma que “os caranguejos pequenos não são recusados pelos comerciantes, mas recebem um preço menor, em relação às cambadas de graúdos”. Os tiradores também recebem o respeito e valorização dos patrões quando são coletadas cambadas com caranguejos graúdos, tendo o objetivo claro de estimular a competição entre tiradores e promover a coleta de caranguejos maiores, mais aceitos no mercado. Souto (2007) relaciona as preferências nas coletas com o tamanho, sexo e com a estação climática. Os tiradores do município de Acupe buscam coletar machos e graúdos principalmente no verão, mas no inverno, devido a facilidade que os caranguejos têm em escavar as galerias devido ao solo do manguezal se apresentar mais “mole”, há maior esforço no trabalho e, com isso, coletam algumas fêmeas. Porém, o autor ressalta que o aspecto mais importante nessa escolha do espécime a ser coletado é o seu tamanho, mais do que o sexo, por 3 CONSIDERAÇÕES FINAIS Os principais resultados foram de que os tiradores de caranguejo da Pontinha do Bacuriteua acompanhados se caracterizam por início na atividade extrativista do caranguejo ainda durante a adolescência; alta evasão escolar; pela transmissão geracional da atividade; pela grande experiência na atividade extrativista; estão no ramo há mais de 35 anos; se autorrepresentam como especialistas na atividade, embora também atuem em atividades complementares e têm dificuldade em serem reconhecidos como categoria profissional; enfrentam problemas de saúde geralmente com alguma relação com as condições de seu trabalho; seu regime semanal de trabalho ocorre de terça a sábado, com folga geralmente no domingo e segunda-feira; quanto à organização do trabalho, as formas de coleta mais utilizadas pelos tiradores são o braço e o gancho. Sobre as implicações e influências dos ciclos biológicos do caranguejo e outras características do ambiente na organização da sua atividade extrativista e produtividade, levando em consideração os conhecimentos tradicionais locais que os tiradores reproduzem, os resultados indicaram que a estacionalidade, juntamente com fatores ligados às marés e ao ciclo biológico do caranguejo, incidem na percepção dos tiradores de caranguejo quanto a facilidade ou dificuldade em coletar o caranguejo, relacionando-se diretamente com a produtividade; o regime de marés é conhecido pelos tiradores de caranguejo da Pontinha do Bacuriteua e necessário para o desempenho de suas atividades, repercutindo diretamente no seu tempo diário de trabalho, nas técnicas e tecnologias utilizadas e, consequentemente, na sua produtividade; a composição do solo relacionada com as árvores presentes no manguezal se apresenta como mais um fator do ecossistema que os tiradores de caranguejo da Pontinha do Bacuriteua levam em conta no momento da escolha das áreas de coleta e a instituição dos territórios temporários. Resumindo, para a compreensão da atividade extrativista do caranguejo, suas demandas mercadológicas e os preços praticados entre tiradores e patrões, o conhecimento do ciclo biológico, o sexo e os tipos de caranguejo são fundamentais no que tange à organização social do grupo e sua reprodução, pois dependendo desse estágio biológico as relações entre esses grupos, tiradores e patrões, se estabelecem de maneira diferente, afetando a organização do trabalho e os ganhos de renda dos tiradores. Além disso, os conhecimentos sobre o sexo e o tipo de caranguejo e as pressões do mercado terão influência na escolha dos territórios para a prática de suas atividades. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Eles apontam as seguintes características do caranguejo com cabelo branco: casco com a cor azulada; localizado em áreas com predominância de tijuco mole; unha (pata) com pêlos brancos, daí a denominação de cabelo branco; tem maior concentração de gordura e, portanto, são maiores que o caranguejo com cabelo vermelho; tem o sabor mais adocicado; concentram- se no raizal; predominam nas áreas do Rio Taperaçu e do Furo da Ostra, ambas na península bragantina; são considerados mais perecíveis ou “mais fracos”, sendo destinados a Bragança e a mercados mais próximos. Em relação ao de cabelo vermelho, os tiradores da Pontinha do Bacuriteua destacam as seguintes características: casco com a cor entre o vermelho e o preto; localizado em áreas de transição entre terra firme e manguezal; tem a unha (pata) com pêlos vermelhos ou pretos, daí a denominação cabelo vermelho; tem menor concentração de gordura e, portanto, menores que o caranguejo com cabelo branco; são conhecidos como “Puruê”; concentram-se no siribal (pneumatóforos); são predominantes nas áreas do Furo do Taici e Rio Caeté, ambas localizadas próximas da Pontinha do Bacuriteua; considerados menos perecíveis que o de cabelo branco, ou seja, “ele não morre muito”, sendo destinados à Bragança e, também, a mercados mais distantes, por sua baixa perecibilidade. Durante a pesquisa de campo, conversamos com três patrões na Feira principal do município de Bragança e todos concordaram que o cabelo branco é o tipo que mais vendem e todos foram unânimes em afirmar que há dificuldade em comercializar o cabelo vermelho em Bragança, sendo destinados a outros mercados, inclusive o de Belém. Os tiradores afirmam que coletam os dois tipos. Porém, o tamanho maior do caranguejo cabelo branco em relação ao vermelho estimula explorarem mais as áreas onde esse caranguejo tende a prevalecer. Portanto, para a compreensão da atividade extrativista, suas demandas mercadológicas e os preços praticados entre tiradores e patrões, o saber do ciclo biológico, o sexo e os tipos de caranguejo são fundamentais no que tange à organização social do grupo e sua reprodução. 127 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 3 CONSIDERAÇÕES FINAIS No contexto da pesquisa foram identificadas nas falas dos tiradores de caranguejos: frases, metalinguagens, prosopopeias e outros referenciais que apontam muitos elementos como aprendizagem para os trabalhos no manguezal, pois, de acordo com o grupo: “não se entra de qualquer jeito”, são necessárias roupas apropriadas; andanças adaptadas; tocar-se na lama; utilizar o gancho de forma cautelosa para não matar o crustáceo; destreza para agarrar o caranguejo e amarrá-lo ou colocá-lo no saco; saber as horas da vazante e da enchente da maré; observar os solos onde possivelmente há mais crustáceo; o ciclo biológico do caranguejo, etc. Esses são saberes utilizados nas práticas socioambientais que requerem dos trabalhadores aprendizados ao longo do contato com o meio ambiente pelo contato com de pessoas mais experientes na atividade, que ensinaram aos aprendizes técnicas e uso de tecnologias. O manguezal apresenta-se aos sujeitos pesquisados como lugar das atividades e das reproduções de suas cosmovisões e modos de vida. Esses grupos apropriam-se do território via práticas, regras sociais, modos de manutenção e transmissão de direitos, a partir da construção social e perspectivas, incorporando-o ao seu contexto. Portanto, os esforços do artigo concentraram-se em apresentar como os saberes e práticas socioambientais são utilizados pelos tiradores da pontinha do Bacuriteua em suas atividades diárias à coleta de caranguejos. Esses conhecimentos foram transmitidos de gerações passadas e são utilizados ao manejo no ecossistema costeiro bragantino, o que constituem importante fonte para pensar em ações e/ou NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 128 políticas públicas no território da Resex Marinha caeté-Taperaçu que contextualizem a atividade e evidenciem demandas importantes para esse grupo social. políticas públicas no território da Resex Marinha caeté-Taperaçu que contextualizem a atividade e evidenciem demandas importantes para esse grupo social. FURTADO, L. G. 1993. “Reservas pesqueiras” uma alternativa de subsistência e de preservação ambiental: reflexões a partir de uma proposta dos pescadores do médio REFERÊNCIAS ACOSTA, Alberto. O viver bem: uma oportunidade para imaginar outros/ Alberto Acosta: tradução de Tadeu Breda. – São Paulo: Autonomia Literária. Elefante, 2016. ABREU, G. C. de. 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NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 130 OLIVEIRA, Marcelo do Vale. “É de quem chegar primeiro”: Territorialidades entre tiradores de caranguejos da Vila do Bacuriteua, Bragança/Pa. XV ENCONTRO DE CIÊNCIAS SOCIAIS DO NORTE E NORDESTE e PRÉ-ALAS BRASIL., 2012, Teresina. Anais [...]. Teresina-PI, 2012. OLIVEIRA, Marcelo do Vale. “É de quem chegar primeiro”: Territorialidades entre tiradores de caranguejos da Vila do Bacuriteua, Bragança/Pa. XV ENCONTRO DE CIÊNCIAS SOCIAIS DO NORTE E NORDESTE e PRÉ-ALAS BRASIL., 2012, Teresina. Anais [...]. Teresina-PI, 2012. OLIVEIRA, Marcelo do Vale. Trabalho e territorialidade no extrativismo de caranguejos em Pontinha de Bacuriteua, Bragança-Pará. Dissertação (Mestrado em Biologia Ambiental) – Universidade Federal do Pará (UFPA), Bragança, 2013. OLIVEIRA, Marcus Vinicius. A estrada para o “progresso”: política, cultura e natureza em Bragança, Pará (1970-1996). Dissertação (Mestrado em História Social) – Universidade Federal do Pará (UFPA), Belém, 2015. ALVES, Alexandre de Brito. Estrada Bragança-Ajuruteua e sobrevivência no manguezal (1975-1991). São Carlos: Pedro & João Editores, 2020. OLIVEIRA, Marcus Vinicius; HENRIQUE, Márcio Couto. No meio do caminho havia um mangue: impactos socioambientais da estrada Bragança-Ajuruteua, Pará. Revista Manguinhos, v.25, n.2, abr.-jun. 2018. Disponível em: http:www.revistahcsm.coc.fiocruz.br. Acesso: 9 set. 2020. SCHAEFFER-NOVELLI, Y. Manguezal: ecossistema entre terra e mar. São Paulo: Carribean Ecological Reserach, 1995. SILVA, C. N. A percepção territorial-ambiental em zonas de pesca. Belém: Bol. Mus. Para. Emílio Goeldi, Ciências Humanas, v. 2, n. 3, set./dez. 2007, 25-32p. SILVA, A. L. da. Entre tradições e modernidade: conhecimento ecológico local, conflitos de pesca e manejo pesqueira no rio Negro, Brasil. Belém: Boletim do Museu Paraense Emílio Goeldi, Ciências Humanas, v. 6, n. 1, jan./ abr. 2011, 141-163p. SCHMIEGELOW, J. M. M. O planeta Azul: Uma Introdução às ciências marinhas. Rio de Janeiro: Interciência, 2004, 202p. SOFFIATI, Arthur. Da mão que captura o caranguejo à globalização que captura o manguezal. In: II ENCONTRO DA ASSOCIAÇÃO NACIONAL DE PÓS-GRADUAÇÃO E PESQUISA EM AMBIENTE E SOCIEDADE., 2004, Indaiatuba. Anais [...]. Indaiatuba, 2004. SOUZA, Camilla. 2013. Relações de gênero em Bacuriteua (PA): imaginário do homoerotismo masculino entre coletores de caranguejo. Dissertação (Mestrado em Linguagens e Saberes na Amazônia) – Universidade Federal do Pará (UFPA), Bragança, 2013. RAMALHO, Cristiano Wellington Noberto. O sentir dos sentidos dos pescadores artesanais. Revista de Antropologia, SÃO PAULO, USP, 2011, v. 54 nº 1. Disponível em http: www.revistas.usp.br/ra/about. Acesso: 28 set. 2020. 131 DOSSIÊ AMAZÔNIA – - SABERES NA AMAZÔNIA INTRODUÇÃO Os conhecimentos tradicionais são o resultado de uma coevolução entre as sociedades e seus ambientes naturais. Esta evolução simultânea permitiu a conservação de um equilíbrio no qual o manejo dos recursos, bióticos e abióticos, está diretamente relacionado com mitos, lendas, regras, valores e saberes. Este entendimento de que a diversidade cultural caminha lado a lado com a diversidade ecológica e que uma depende da outra é crescente. Entretanto, o etnocentrismo predominante na construção do conhecimento científico, não favorece o reconhecimento dos sistemas de saberes organizados de outras culturas (DIEGUES, 2000). No Brasil, o Decreto Federal 6040 instituiu, em 27 de fevereiro de 2007, a Política Nacional de Desenvolvimento Sustentável dos Povos e Comunidades Tradicionais (PNPCT), definindo-os, em seu artigo 30, parágrafo 1, como: grupos culturalmente diferenciados e que se reconhecem como tais, que possuem formas próprias de organização social, que ocupam e usam territórios e recursos naturais como condição para sua reprodução cultural, social, religiosa, ancestral e econômica, utilizando conhecimentos, inovações e práticas gerados e transmitidos pela tradição. (BRASIL, 2007, p. 1). Estes, também denominados saberes tradicionais, conhecimento ecológico local ou etnoconhecimentos, são adquiridos através de observações extensivas dos elementos naturais e transmitidos por via oral, de geração a geração (BERKES et al., 2000; HUNTINGTON, 2000). O resgate desses saberes empíricos, visando incluí-los nas estratégias de desenvolvimento socioambiental se dá a partir da construção de pontes entre as disciplinas tradicionais da academia e as diversas culturas. Uma destas pontes, a etnobiologia, investiga as relações do homem com os seres vivos e com o mundo natural (MARQUES, 1991). Dentro desta, a etnozoologia é definida como um estudo transdisciplinar dos pensamentos e percepções (mitos e crenças), dos sentimentos (representações afetivas) e dos comportamentos (ações) que constituem as relações entre populações humanas e os animais que as cercam (MARQUES, 2002). Portanto, a etnozoologia traduz-se como o estudo da ciência zoológica do “outro”, por ser construída a partir do referencial teórico acadêmico, levando em consideração as perspectivas culturais de cada grupo humano (CAMPOS, 1994). Meu interesse pelos conhecimentos tradicionais foi desenvolvido à medida que eu submergia, enquanto Bióloga em busca da diversidade zoológica, em diferentes contextos socioambientais. Despertou nos primeiros contatos com agricultores do entorno do Parque Nacional Serra de Itabaiana (Sergipe). CAMPUS BRAGANÇA VOLUME NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 132 Data de submissão: 13.10.2020 Data de aprovação: 01.12.2020 1 DIVERSIDADE ZOOLÓGICA E ETNOCONHECIMENTOS: A PERSPECTIVA DA ETNOZOOLOGIA Os estudos da etnozoologia incluem os sistemas empregados na classificação dos elementos presentes nos ecossistemas. Os quais, segundo Ellen (1986), refletem o modelo organizacional social de uma comunidade. Denominadas de etnotaxonomias, estas classificações relacionam os aspectos naturais mais notáveis aos humanos (cor, tamanho, forma do corpo, odor), bem como os aspectos de significância cultural (BROWN, 1986). Kellert (1983) afirma que humanos percebem os animais nas formas de (1) intenção, (2) saber e/ou (3) simbólica. Dentre estas crenças, saberes e fazeres, a significância das diferentes espécies animais nos diversos conhecimentos tradicionais pode ser estudado, como nos instigam Vargas-Clavijo e Costa-Neto (2010), através da compreensão das atitudes humanas para com os demais animais. Em geral, os etnozoólogos se impressionam com a coerência dos saberes e práticas que as populações tradicionais possuem e demonstram, em graus diversos, sobre as espécies de animais com as quais convivem e interagem (POSEY, 1982; SCHEPS, 1993). Esses estudos contribuem, tanto para a elaboração de estratégias de manejo e conservação da fauna, quanto dos diversos simbolismos e representações, historicamente construídos nas diferentes culturas, sobre os animais. Posey (1986, p. 16) recomendou aos etnobiólogos que não nos prendamos somente a dados estatisticamente relevantes, pois para ele a contradição e a anomalia são os princípios culturais básicos a serem investigados em maior extensão. Nisto é que a Etnobiologia difere da Biologia, esta última trata os fenômenos socioculturais como dados estatisticamente irrelevantes, enquanto a Etnobiologia deve considerar a contradição e a anomalia como elementos fundamentais ao desenvolvimento dos métodos de pesquisa. Assim, a continuidade entre conhecimentos tradicionais e conhecimentos científicos foi construída, nos estudos aqui compilados, utilizando múltiplos métodos e técnicas, na perspectiva quali-quantitativa. Esta triangulação é defendida por Ruediger e Riccio (2004) como uma estratégia para amenizar os possíveis vieses das pesquisas que envolvem as percepções humanas do mundo natural. Para compreender as percepções de pescadores artesanais, em diferentes espaços/contextos da costa amazônica paraense, diferentes técnicas foram empregadas no registro e análises das histórias orais. A história oral centra-se na memória humana e sua capacidade de rememorar o passado enquanto testemunha do vivido. Podemos entender a memória como a presença do passado, como uma construção psíquica e intelectual de fragmentos representativos desse mesmo passado, nunca em sua totalidade, mas parciais em decorrência dos estímulos para a sua seleção. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 133 a contribuição dos etnoconhecimentos, de pescadores artesanais polivalentes na Amazônia costeira paraense, sobre animais marcantemente presentes nos locais de moradia e/ou pesca, para o entendimento da diversidade faunística. Bem como para a reflexão de questões ambientais e de manejo dos componentes animais. INTRODUÇÃO Intensificou durante as expedições às várzeas do aluvião entre os rios Solimões e Japurá (Amazonas), onde foi implantada a Reserva de Desenvolvimento Sustentável Mamirauá, quando tive muitos encontros com ribeirinhos pescadores e indígenas. Aprofundou-se nos manguezais da Amazônia costeira paraense (Reserva Extrativista Marinha (RESEX-MAR) Caeté-Taperaçu, RESEX-MAR Tracuateua e arredores) onde convivi com pescadores artesanais, extrativistas e agricultores. O olhar para as formas de vida e a escuta dos saberes desses grupos humanos transformaram minha atitude no mundo e a abordagem das minhas pesquisas. Passei a me dedicar ao entendimento das teias que envolvem homens e animais, a partir da perspectiva ética-êmica da etnozoologia. Este artigo foi construído a partir de dados secundários de trabalhos desenvolvidos, de forma assíncrona, entre 2015 e 2018, com foco em grupos zoológicos diferentes em cada local de estudo (tartarugas-marinhas e aves). Os resultados foram aqui apreciados, visando ressaltar NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 134 de dados, quanto mais abertas são as perguntas, maior a liberdade do sujeito para responder de acordo com sua lógica e conceitos. Também se seguiu a Hermenêutica Objetiva, de Övermann (1979), na qual se leva o sujeito estudado a expressar-se de forma reconstrutiva, buscando na particularidade do próprio sujeito uma compreensão esclarecida e crítica da realidade social. E ainda os pressupostos do método de Descrição Densa. de Geertz (1973), o qual vai além da técnica etnográfica, pois o que interessa não é apenas a interpretação ou explicação de fatos isolados, mas dos conjuntos que se constroem levando em consideração as diversas estruturas conceituais e significativas que moldam as ações humanas. de dados, quanto mais abertas são as perguntas, maior a liberdade do sujeito para responder de acordo com sua lógica e conceitos. Também se seguiu a Hermenêutica Objetiva, de Övermann (1979), na qual se leva o sujeito estudado a expressar-se de forma reconstrutiva, buscando na particularidade do próprio sujeito uma compreensão esclarecida e crítica da realidade social. E ainda os pressupostos do método de Descrição Densa. de Geertz (1973), o qual vai além da técnica etnográfica, pois o que interessa não é apenas a interpretação ou explicação de fatos isolados, mas dos conjuntos que se constroem levando em consideração as diversas estruturas conceituais e significativas que moldam as ações humanas. Seguindo princípios éticos da pesquisa com seres humanos, inicialmente foram apresentados, às comunidades estudadas, por meio do termo de consentimento livre e esclarecido (TCLE), os objetivos, finalidades e métodos das pesquisas. Bem como solicitado o consentimento para os registros imagéticos e áudios. Os dados secundários aqui interpretados (AMORAS, 2017; ALVÃO, 2016; BARBOZA et al., 2018; BARBOZA et al., 2019) foram obtidos com base na aproximação participativa, ou seja, no acompanhamento e auxílio na realização de práticas cotidianas, com a preocupação de observar o ambiente e participar dos eventos sociais que ali se desenrolavam. Nesta etapa, era possível identificar os papeis de cada sujeito no grupo, e assim planejar a aplicação das demais técnicas, individualmente ou em Grupos Focais2. As memórias, acontecimentos e atividades relacionadas aos animais foram gravados em áudio (quando possível e consentido) ou registradas em formulários e diários de bordo. Os registros em áudio foram, posteriormente, transcritos com apoio do programa Listen N Write Free 1.17.0.2. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Os textos resultantes foram editados manualmente, quando necessário. Nestes textos, foi aplicada a análise de conteúdo3 de acordo com Bardin (2011). As técnicas de campo empregadas foram: (1) observação participante (CHIZZOTTI, 1995; QUEIRÓZ et al., 2011); (2) entrevistas baseadas em uma lista de tópicos previamente escolhidos, com enfoque “emicista-eticista” como proposto por Costa-Neto (2003) para a pesquisa etnozoológica; (3) lista livre de etnoespécies foram construídas, durante as entrevistas, à medida que cada domínio cultural foi trabalhado. Foram considerados como elementos culturalmente mais importantes, aqueles que apareceram em muitas listas e em uma ordem de importância semelhante, de acordo com Albuquerque e Lucena (2004); e ainda, quando pertinente, foram aplicadas as (4) técnicas projetivas com uso de fotografias de animais, da categoria taxonômica abordada por cada pesquisa, para confirmar as espécies às quais os agentes da pesquisa (pesquisador e pesquisados) se referiram nos momentos anteriores, sanando possíveis discrepâncias4. 3 Um conjunto de técnicas de análise das comunicações, que utiliza procedimentos sistemáticos e objetivos de descrição do conteúdo das mensagens. Levando em conta a frequência dos temas extraídos do conjunto dos textos/fala, as representações dos conteúdos ou de suas expressões foram codificadas por recorte, agregação e enumeração de unidades de registro (palavras ou temas). As unidades de registro foram analisadas através das frequências e por análise temática da enunciação. 4 De acordo com Frank (1939), as técnicas projetivas oferecem acesso ao mundo dos sentidos, significados, padrões e sentimentos, revelando aquilo que o sujeito não consegue expressar. Segundo Pinto (2014), a associação de palavras com estímulos visuais é útil para projetar as sensações e atitudes. 2 Seguindo a definição de Powell e Single (1996): um conjunto de pessoas selecionadas e reunidas por pesquisadores para discutir e comentar um tema, a partir de suas experiências pessoais. 2 Seguindo a definição de Powell e Single (1996): um conjunto de pessoas selecionadas e reunidas por pesquisadores para discutir e comentar um tema, a partir de suas experiências pessoais. 3 Um conjunto de técnicas de análise das comunicações, que utiliza procedimentos sistemáticos e objetivos de descrição do conteúdo das mensagens. Levando em conta a frequência dos temas extraídos do conjunto dos textos/fala, as representações dos conteúdos ou de suas expressões foram codificadas por recorte, agregação e enumeração de unidades de registro (palavras ou temas). As unidades de registro foram analisadas através das frequências e por análise temática da enunciação. 4 De acordo com Frank (1939), as técnicas projetivas oferecem acesso ao mundo dos sentidos, significados, padrões e sentimentos, revelando aquilo que o sujeito não consegue expressar. Segundo Pinto (2014), a associação de palavras com estímulos visuais é útil para projetar as sensações e atitudes. 1 DIVERSIDADE ZOOLÓGICA E ETNOCONHECIMENTOS: A PERSPECTIVA DA ETNOZOOLOGIA Não é somente a lembrança de certo indivíduo, mas de um indivíduo inserido em um contexto familiar ou social, por exemplo, de tal forma que suas lembranças são permeadas por inferências coletivas, moralizantes ou não (MATOS; SENNA, 2011). As técnicas foram utilizadas seguindo a Metodologia Geradora de Dados, proposta por Posey (1986), que aponta importantes sugestões para um diálogo de campo em que os referenciais do “outro” sejam respeitados e, sobretudo, para que se compreendam os conceitos a partir da própria cosmologia e cosmogonia do grupo pesquisado. Nesta metodologia de coleta DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 135 alta diversidade de ecossistemas, alguns mais contrastantes (mata, cerrado, campinas, manguezal, costeiros) e outros de transição, por vezes muito sutis, na composição biótica da contínua área de floresta (AB’SABER, 2002). Parte dela está em contato com o Oceano Atlântico proporcionando uma extensa região amazônica costeira. A costa amazônica compreende 35% dos, aproximadamente, 8.500 km da costa brasileira. As especificidades desse litoral, entre o rio Oiapoque no Amapá (5°N, 51°W) e a baia de São Marcos no Maranhão (2°S, 44°W), incluem precipitação anual elevada (até 3.300mm), temperaturas também elevadas (>20 °C) com baixa variação anual, extensa plataforma continental (cerca de 330km), regime de macromarés (com valores máximos chegando a 6m no Pará, 8m no Maranhão, e 12m no Amapá), descarga de dezenas de estuários e do maior rio do mundo (Amazonas) e elevado escoamento de sedimentos (nutrientes e matéria orgânica) (PEREIRA et al., 2009). Estas condições proporcionam o desenvolvimento de grande biodiversidade, bem como de uma ampla gama de modos de apropriação social, econômica e cultural dos ambientes costeiros. A principal atividade produtiva dos moradores da costa amazônica é a pesca artesanal, definida por Diegues (2005) como “pequena pesca, cuja produção em parte é consumida pela família e em parte é comercializada.” As técnicas secularmente empregadas pelos indígenas serviram de base para a pesca nessa região, a qual é reconhecidamente uma atividade tradicional pela antiguidade de sua prática na Amazônia, pelos métodos e técnicas de captura, formas de acondicionamento e preparação do produto (FURTADO, 2006). Segundo Furtado (1990), a pesca artesanal no Pará, abrange as pescarias flúvio-lacustres e costeiras ou marítimas (até 10 milhas do litoral paraense), e apresenta duas formas de organização social. Na forma polivalente, os pescadores se dedicam a várias atividades durante o ano, como roçados, extrativismo, coleta, criação de gado e pesca. Estes pescam para consumo próprio durante o ano e, sazonalmente, para comercialização. O elemento familiar predomina nas unidades de produção. Enquanto os pescadores monovalentes se dedicam às atividades de pesca durante todo o ano, tendo as parcerias como unidades de produção. As comunidades de pescadores polivalentes onde os estudos foram realizados situam- se na mesorregião nordeste paraense, na costa atlântica da Amazônia brasileira. Sendo quatro na/ou entorno da Reserva Extrativista Marinha (RESEX-MAR) Caeté-Taperaçu, uma na RESEX-MAR Tracuateua e uma na área onde se propõe a expansão das áreas de RESEX-MAR no município Salinópolis. 2 PESCADORES ARTESANAIS DA AMAZÔNIA COSTEIRA PARAENSE: AS COMUNIDADES ESTUDADAS A Amazônia é um bioma que se estende por uma grande porção da América do Sul, abrangendo partes de nove países. Com extensão de 8 milhões de km², trata-se de uma área com 4 De acordo com Frank (1939), as técnicas projetivas oferecem acesso ao mundo dos sentidos, significados, padrões e sentimentos, revelando aquilo que o sujeito não consegue expressar. Segundo Pinto (2014), a associação de palavras com estímulos visuais é útil para projetar as sensações e atitudes. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 136 empresas de processamento. Os pescadores dessas comunidades trabalham na costa da microrregião bragantina do Salgado, utilizando as ilhas como “rancho”5. empresas de processamento. Os pescadores dessas comunidades trabalham na costa da microrregião bragantina do Salgado, utilizando as ilhas como “rancho”5. A comunidade Quatipuru-Mirim habita uma ilha que dista cerca de 40 km da sede do município de Tracuateua, onde pescam (peixe e camarão) para auto-sustento e comercialização durante o ano todo. É uma das três que se localizam dentro dos limites da RESEX-MAR Tracuateua, da qual são usuárias mais 55 comunidades localizadas no entorno da área. A qual situa-se contígua à oeste da RESEX-MAR Caeté-Taperaçu. A comunidade de Santa Rosa localiza-se na área onde o Instituto Chico Mendes de Conservação da Biodiversidade/Ministério do Meio Ambiente propôs, em 2017, a implantação de uma nova RESEX-MAR no município de Salinópolis. Situada à margem direita do rio Urindeua, que corre do sul para o norte do município e deságua no Oceano Atlântico, esta comunidade possui energia elétrica, três estabelecimentos comerciais, uma igreja católica, um campo de futebol, uma escola municipal de educação básica, um posto de saúde e associação de moradores. O rio Urindeua, navegável apenas por pequenas embarcações, é de grande importância econômica para os moradores da Santa Rosa. As principais atividades das cerca de 35 famílias, são a agricultura familiar, a pesca e a coleta de caranguejo. Os etnoconhecimentos de pescadores sobre tartarugas marinhas foram registrados nas comunidades Vila dos Pescadores e Vila Bonifácio (Bragança) (BARBOZA et al., 2018; BARBOZA et al., 2019). Sobre Maçaricos (Charadriiformes) na comunidade Santa Rosa (Salinópolis) (AMORAS, 2017). Sobre aves residentes e visitantes das Ilhas da microrregião bragantina - Canelas, Baiaco, Pilão e Quatipuru Mirim - foram escutados pescadores das comunidades Castelo e Taperaçu-Porto, e residentes na Cidade (Bragança); e ainda comunidade Quatipuru-Mirim (Tracuateua) (ALVÃO, 2016). g p q ( ) g p g g j 7 Quelônio de água doce, conhecido no Brasil como “cágado”, na Amazônia, em geral, como “tartaruga da Amazônia”. 5 Denominação dada pelos pescadores a locais de apoio durante os longos períodos de pesca. ç p p p g p p 6 O grupo quelônios (Ordem Chelonia ou Testudinata) agrupa tartarugas, cágados e jabutis. DOSSIÊ AMAZÔNIA Reservas Extrativistas são territórios de uso comum, regulamentadas com vistas à conservação dos recursos naturais, das culturas locais e meios de vida dos povos tradicionais. A regulação do uso dessas áreas é feita por cogestão, ou seja, gestão compartilhada entre a administração pública e os povos tradicionais, com princípios participativos. Para a implementação deste modelo de gestão, são criadas Associações de Usuários, como representantes do povo tradicional. A Vila dos Pescadores estabeleceu-se na linha de praia, da península de Ajuruteua, no município de Bragança, ainda na segunda década do século XX. A forte erosão produzida pela variação no nível do mar apontada por Souza-Filho (2011), levou o governo local a construir uma nova vila com equipamentos urbanos (posto de saúde e escola), e induzir a transferência das famílias de pescadores artesanais para este novo local, situado próximo a um canal de maré. Entretanto, a formação da nova vila, denominada Bonifácio, em 1995, não extinguiu completamente a antiga Vila dos Pescadores. As vilas situam-se dentro dos limites da RESEX- MAR Caeté-Taperaçu. Também situada na RESEX-MAR Caeté-Taperaçu, as comunidades Castelo (dentro) e Taperaçu-Porto (entorno), se destacam por possuírem portos que permitem o atracamento de barcos de médio porte, importantes para a economia local, pois fornecem pescado para as DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 137 Os ninhos, geralmente localizam-se distante dos ranchos de pescadores e das casas, no caso de locais com residentes. Os pescadores classificam as aves quanto aos locais dos ninhos, dividindo-as em: aves que nidificam no chão (maçaricos, gaivotas) e as que nidificam sobre os galhos de árvores do manguezal (guará, galça, taquiri, maguari, entre outros). “Ah fica lá em cima, lá em cima, eles fazem o linho lá em cima, lá em cima da siribeira do manguero...” Segundo os pescadores, várias mudanças nas áreas de nidificação relacionadas à geomorfologia das ilhas e perseguição de aves pelas pessoas estão ocasionando a diminuição de aves e de ovos nas ilhas de pesca. Os predadores dos ovos de aves mais citados pelos pescadores foram os cachorros, seguidos pelos gaviões, cobras e humanos. “Não ser uma praia muito ‘habituada’ num tem muita população, acho que devido também não ter energia elétrica, se tivesse, acho que só a iluminação espantava eles.” Também na etnozoologia de pescadores no rio Urideua (na altura da comunidade Santa Rosa), é amplo o conhecimento sobre maçaricos. Observam, ao longo do ano, maior abundância dessas aves no período seco (denominado verão na região), ou seja, nos meses de julho a dezembro. Variações sazonais na diversidade também são percebidas. Há relatos de que somente o Maçariquinho (Actitis macularius) é presente durante todo o ano. Nas narrativas dos pescadores, estas aves limícolas8 são avistadas diariamente alimentando-se, principalmente, do caranguejo-maracoanim, em praias e enseadas. Também em áreas de descanso, nos manguezais circundantes. Os principais predadores são o gavião- caburé e as cobras. Embora não seja o nosso objetivo aqui, confrontar os etnoconhecimentos sobre os animais com a zoologia científica, podemos afirmar que esta correlação é positiva. Ainda que construída e transmitida de formas diferentes da Ciência, como afirmam Santos-Fita e Costa- Neto (2007), as etnozoologia locais nos fornecem o recorte de maior significância para os humanos que vivem em contato direto com os elementos naturais. Este recorte agrega primor na tomada de decisões sobre ações que afetem os modos de vida das comunidades e povos tradicionais, na medida em que respeita o direito desses grupos sociais de serem ouvidos. Este direito foi preconizado na Convenção 169, da Organização Internacional do Trabalho (OIT), aprovada pela Organização das Nações Unidas em 1989. g g 9 Taxonomia ou classificação biológica é uma ciência biológica que busca agrupar e categorizar as espécies de seres vivos (extintas e viventes). DOSSIÊ AMAZÔNIA Esse Tratado Internacional sobre os direitos humanos de povos indígenas e tradicionais está em vigor no Brasil desde 2004 através do Decreto 5051 (BRASIL, 2004). 3 ETNOZOOLOGIA: COMO OS ANIMAIS SÃO PERCEBIDOS PELOS PESCADORES A presença de tartarugas marinhas nas praias e águas continentais da microrregião bragantina foi descrita pela quase totalidade dos pescadores da Vila do Bonifácio e Vila dos Pescadores (península de Ajuruteua) que participaram da pesquisa. As interações entre esses pescadores e as tartarugas marinhas ocorrem durante suas práticas pesqueiras (rede, curral e espinhel), e ainda em seus trajetos entre o mar e as comunidades. “[...] as redes de arrastão no fundo espanta as tartarugas, o que não malha nessas redes corre pra frente.” Embora não percebam a temporalidade do processo reprodutivo das tartarugas marinhas, descrevem desovas da tartaruga-cinza e da aruanã (Chelonia mydas e Lepidochelys olivacea). “Tartaruga faz um rebaixo, arrudeio, para não cercarem os ovos, faz forçado marca.” A adaptação aos ambientes costeiros é percebida pelos sabedores locais que distinguem os quelônios6 marinhos de outros grupos de ambientes dulcícolas e terrestres. “Aruanã tem a parte traseira cheia; Chimirra7 tem a parte traseira seca.” Esta aguçada percepção de pescadores quanto à diversidade morfológica, fisiológica e comportamental também foi registrada para aves costeiras. Sendo o montante de informações sobre cada espécie bastante heterogêneo entre os pescadores artesanais que arrancham nas ilhas, o que indica diferentes graus de significância cultural de cada uma delas para este grupo social. Os locais (ilhas Quatipurú-Mirim, Maciel, Baiaco, Canelas, Otelina, Carrapato e Ilha do Cachorro) e períodos de nidificação (entre os meses de maio a agosto) de diversas espécies foram claramente descritos. 8 Aves da Ordem Charadriiformes, geralmente associadas a zonas úmidas costeiras, como estuários e lagoas. 9 Taxonomia ou classificação biológica é uma ciência biológica que busca agrupar e categorizar as espécies de seres vivos (extintas e viventes). Aves da Ordem Charadriiformes, geralmente associadas a zonas úmidas costeiras, como estuários e lagoas NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Segundo os pescadores das Ilhas, quarenta e cinco etnoespécies que residem ou visitam as ilhas da microrregião bragantina, sendo as mais citadas guará, galça, taquiri, gaivota e maçarico. Nas narrativas identificamos citações de variações nos caracteres de coloração, tamanho, tipo de bico e vocalização. Dezessete etnoespécies de maçaricos foram citadas para as margens do rio Urindeua. As mais frequentes foram Maçaricão, Vira-pedra e Maçariquinho-da-beira-de-igarapé. Os pescadores locais utilizam para a distinção entre as etnoespécies as categorias locais classe, pinta e qualidades. “Eu faço porque tem as qualidades né, tem o grande, tem o menor e tem os que são pintadinho e outros que são diferente um do outro.” Duas correntes teóricas foram propostas, inicialmente, para explicar os critérios utilizados nas classificações tradicionais (folk). A intelectualista ou cognitivista, defendida por Berlin et al. (1973), sugere que as pessoas classificam os organismos para satisfazer a necessidade inata de organizar o mundo à sua volta ou meramente por curiosidade. Enquanto a corrente utilitarista, proposta por Hunn (1977), afirma que as pessoas classificam os componentes do mundo natural que possuem valor para elas. Posteriormente, Atran (1998) propõe um modelo único de classificação, contemplando as duas correntes. Considera que os organismos são classificados em nível genérico com base em características culturais universais inatas. E que o nível específico está condicionado ao valor local dos seres naturais para as pessoas. p 11 A Lei nº 5.197, de 3 de janeiro de 1967, dispõe sobre a proteção à fauna e dá outras providências. 10 Animais da Classe Crustacea, infra-classe Cirripedia. 10 Animais da Classe Crustacea, infra-classe Cirripedia. 10 Animais da Classe Crustacea, infra-classe Cirripedia. 11 A Lei nº 5.197, de 3 de janeiro de 1967, dispõe sobre a proteção à fauna e dá outras providências. ETNOTAXONOMIA: COMO OS PESCADORES CLASSIFICAM OS ANIMAIS Os pescadores listaram 26 etnoespécies de tartaruga-marinha, dentre as quais foi possível confirmar a presença das cinco espécies ocorrentes no litoral brasileiro (Ceará a Santa Catarina) pela ciência e nas políticas ambientais: tartaruga-amarela (Caretta caretta), tartaruga- cor-de-vinho (Eretmochelys imbricata), tartaruga-cinza (Chelonia mydas), aruanã (Lepidochelys olivacea) e tartaruga-de-couro (Dermochelys coriacea). Os caracteres diagnósticos neste sistema de classificação são morfológicos e comportamentais, tal qual a taxonomia9 zoológica clássica. Estes mesmos aspectos são utilizados na etnotaxonomia de aves. A diversidade de aves é localmente organizada em um complexo sistema de classificação, baseado em atributos de formas, modos de vida e funcionalidade no ambiente. As espécies afins sendo reunidas em grupos, denominados por eles de “tipo” ou “marca” de aves, caracterizando os etnogêneros locais. 138 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 5 INTERAÇÕES ENTRE PESCADORES E ANIMAIS O uso dos elementos da natureza pelos povos tradicionais evoluiu, a partir das necessidades para a sobrevivência, até o acúmulo de saberes através da observação e experimentação de fenômenos e características destes elementos. Os saberes acumulados favorecem e mantêm o conhecimento sobre os territórios, sendo necessário que se ressalte a importância da transmissão desses saberes às novas gerações. Assim, a biodiversidade deixa de ser apenas um conceito biológico, em relação a diversidade genética dos indivíduos, e dos ecossistemas, tornando-se o resultado de práticas milenares dessas comunidades. Este entendimento conduziu as chamadas etnociências ao interesse pela diversidade biológica, que também está ameaçada pela mundialização de modelos culturais dominantes (DIEGUES, 2000). No estudo das tartarugas-marinhas registramos o aproveitamento de animais capturados acidentalmente para consumo de carne, bem como a coleta de ovos dos ninhos são descritos como hábitos alimentares esporádicos. Além do uso alimentar, os pescadores de Ajuruteua usam a banha e o casco da tartaruga para tratamento de dores em geral, asma e processos inflamatórios. E ainda, as cracas10 incrustadas no casco da tartaruga para “doenças dos olhos”. Além do uso zooterápico, os moradores das Vilas Bonifácio e dos Pescadores também usam partes das tartarugas marinhas para confecção de zooartesanato, o que indica uma percepção estético-afetiva desses animais. Quanto às interações humano-maçaricos, os pescadores admitiram consumir o maçarico como iguaria alimentar, mas afirmam que esse hábito foi abandonado por saberem que a caça de animais silvestres é ilegal11. Declaram reconhecimento da importância desses animais para o equilíbrio ambiental local e demonstram admiração contemplativa. Alguns relatam, com desaprovação, terem presenciado atitudes humanas de desprezo (balar, atirar e matar) para com os maçaricos. Segundo Alves e Souto (2010), os conhecimentos acerca dos animais, transmitidos de geração em geração por tradição oral, são bem integrados com outros aspectos culturais. Sendo DOSSIÊ AMAZÔNIA 139 resultado de inúmeras civilizações e tradições que se desenvolveram ao longo da história, os conhecimentos etnozoológicos são parte importante do patrimônio cultural humano. resultado de inúmeras civilizações e tradições que se desenvolveram ao longo da história, os conhecimentos etnozoológicos são parte importante do patrimônio cultural humano. 6 CONSIDERAÇÕES FINAIS A integração entre estudos etnozoológicos e levantamentos faunísticos é necessária quando o objetivo maior é a conservação da fauna em áreas habitadas por populações humanas. A etnozoologia dos pescadores da Amazônia costeira paraense sobre tartarugas- marinhas e aves-costeiras revela que esses trabalhadores tradicionais produzem e transmitem conhecimentos, não apenas sobre aqueles grupos animais que utilizam como fonte de proteínas e de renda, mas sobre todos os animais que compõem o sistema onde vivem. Os conhecimentos etnozoológicos, consistentes com a zoologia e a taxonomia zoológica de tartarugas-marinhas e aves-costeiras, constituem um conjunto de informações teórico- práticas que nos ajudam a conhecer a diversidade animal local, e deve ser considerado em planejamentos de ações sobre conservação e uso destes territórios. As interações entre humanos e tartarugas-marinhas e humanos e aves aqui relatadas constituem-se em práticas sustentáveis, uma vez que não interferem no equilíbrio ambiental de forma significativa. E ainda nota-se nas narrativas dos pescadores que suas práticas tradicionais evoluem de acordo com mudanças na sociedade. Tais como a promulgação de leis ambientais, que passam a ser consideradas por eles em suas atitudes junto aos elementos naturais. ATRAN, Scott. Folk biology and anthropology of science: cognitive universals and cultural particulars. Behavioral and Brain Sciences. v. 21, p. 547-609. 1998. REFERÊNCIAS AB'SABER, Aziz N. 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R.R; SOUTO, W.M.S.S; MOURÃO, J.S. (org.). A Etnozoologia no Brasil. v. IV. Recife: NUPEEA. 2010. 143 DOSSIÊ AMAZÔNIA UNIVERSIDADE FEDERAL DO PARÁ – CAMP US BRA GAN ÇA VOLUME IX – REVISTA DO PROGRAMA DE PÓS GRADUAÇÃO EM LINGUAGENS E SABERES NA AMAZÔNIA – NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 144 há vinte anos e mãe há cinco. Tracuateua se tornou um município recentemente, tendo apenas vinte e quatro anos de emancipação do município de Bragança/PA, que fica a aproximadamente 200 km da capital Belém/PA e possui cerca de 30 mil habitantes, segundo o último censo demográfico (IBGE, 2019). há vinte anos e mãe há cinco. Tracuateua se tornou um município recentemente, tendo apenas vinte e quatro anos de emancipação do município de Bragança/PA, que fica a aproximadamente 200 km da capital Belém/PA e possui cerca de 30 mil habitantes, segundo o último censo demográfico (IBGE, 2019). Minhas perguntas surgiram após um contato com uma benzedeira, que inicialmente aconteceu sem qualquer objetivo acadêmico. Em 2015, após a chegada de meu primeiro filho, tudo mudou, a casa mudou, os amigos mudaram, os programas em famílias mudaram e tanta transformação foi por conta de um novo ser que passou a integrar a família. O leitor poderia indagar: qual o sentido de eu narrar aqui a chegada de uma criança na família com a pesquisa que aqui estou apresentando? Neste sentido, em uma determinada manhã, já com três meses de vida, meu filho começou a ter febre alta logo cedo, às 6h da manhã, acompanhada de calafrios e choro. Pensei que poderia ser uma das viroses típicas do inverno amazônico; assim, como é de praxe, levei meu filho ao pediatra. O pediatra receitou antitérmico e vitaminas. Porém, após a febre ceder, apareceu outro sintoma – diarreia –, com fezes um pouco “esverdeadas”, o que chamou a atenção da avó. Embora a cor das fezes chamasse atenção, o cheiro não era característico de algum tipo de infecção alimentar. Mesmo assim, fiquei atenta ao sintoma manifestado. Já seguíamos para o terceiro dia e meu filho não apresentava melhoras. Mesmo quando os sintomas abrandavam, eles retornavam pontualmente às 6h, 12h, 18h e 00h. E foi essa pontualidade sintomática que fez com que a segunda mãe de meu filho concordasse comigo e com a avó em sair para pedir ajuda a uma benzedeira. Fomos então à casa da Sra. T., benzedeira. – NÚMERO 01 DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 145 As orações e as rezas procuram buscar a necessidade de instrumentos, através de objetos que se utilizam durante as benzeções e dependem de cada benzedor. Os instrumentos incluem: plantas, água, alho, roupas, fotos, terço. Mas normalmente eles em sua maioria utilizam ramos ou galhos para o benzimento. Tendo em vista este contexto, pretendo analisar nesta pesquisa o ofício de benzer como produção de conhecimento, etnografando práticas de benzeção do município de Tracuateua/PA a partir desta outra forma de saber e de suas práticas de cura. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Sugeri a ida especificamente até a casa dela porque já a conhecia há muito tempo e já tive sua ajuda outras vezes, pois desde minha infância convivo com benzedeiras, parteiras e experientes; inclusive, na minha casa, todos nós, exceto minha irmã caçula, nascemos com o auxílio de parteira, carinhosamente chamada de “vovó Mercedes”, que também era benzedeira. No final da tarde fomos à casa da dona T.. Meu filho ainda chorava e estava com muita febre. Ao chegarmos lá, pedi para falar com dona T., que estava cuidando da neta. Ela nos deu cadeiras para nos acomodar e eu contei todo o ocorrido. Ela então pediu para tirar toda a roupa dele, entrou na casa, retornou com um alho na mão e começou a rezar e benzer o meu filho com o alho. Ela passava o alho no corpo dele e ao mesmo tempo fazia orações. Neste momento, fazia um vento muito forte. Lucas, meu filho, chorava enquanto ela fazia o sinal da cruz nele e o abençoou. Após benzê-lo, ela disse que ele ficaria bom logo que voltasse para casa, que o problema havia sido quebranto e ele havia sido admirado por uma mulher, e ela estava grávida. Voltamos para casa, Lucas chegou suado, sem febre. Esperamos chegar 18 horas para ver se a febre voltava, mas não voltou. Meia noite também a febre não apareceu, assim como os outros sintomas. Assim, felizmente, meu filho logo estava bem. Neste momento, este estudo buscará se clarificar, mesmo com a complexidade do método etnográfico, complexo no sentido de sua relação essencial e interdisciplinar com diversas áreas (antropológica, filosófica, biológica etc.), sendo capaz de construir algo de forma que cada uma das áreas citadas possa renovar ou enriquecer suas formas de apreensão da realidade acerca do assunto estudado. Estou considerando que as benzedeiras e os benzedores em seu ofício mantêm uma relação fenomenológica com o mundo. Neste sentido, faço um paralelo com Tim Ingold (2015) ao tratar sobre os elementos não humanos, que estão também no mundo interagindo com os humanos, a exemplo dos materiais que são feitos os objetos e produzem uma materialidade. As plantas também são matérias, ainda que não produzam objetos, fazendo parte do arsenal que compõem a prática da benzeção, apreendida cotidianamente a partir de experiências deixadas por outras gerações, e ganha uma habilidade mais específica no presente. 1 FUNDAMENTAÇÃO TEÓRICA A cidade de Tracuateua pertence à mesorregião do Nordeste do Pará e à microrregião Bragantina, também conhecida como zona do Salgado. O município possui aproximadamente 30.959 habitantes, conforme estimativa do IBGE e densidade populacional de quase 29,39 hab/km². (IBGE, [2019]). Segundo informações da Secretaria de Estado de Turismo (2014), Tracuateua fica a aproximadamente 188 km da capital Belém e a 15 km do município de Bragança. No estudo em questão, dei prioridade para os locais onde se localizavam benzedores conhecidos pela comunidade, a saber, os povoados de Santa Maria, Tracuá e a Comunidade Quilombola do Jurussaca (Fig. 1). Figura 1 – Mapa do Município de Tracuateua, com destaque para as comunidades pesquisadas Figura 1 – Mapa do Município de Tracuateua, com destaque para as comunidades pesquisadas Figura 1 – Mapa do Município de Tracuateua, com destaque para as comunidades pesquisadas Fonte: elaborado pela Autora. A comunidade de Santa Maria encontra-se situada na zona rural de Tracuateua. Nesta comunidade vivem aproximadamente 40 famílias, a economia vem da agricultura familiar (produção de grãos, milho, feijão, mandioca), da pesca de subsistência e da criação de animais Fonte: elaborado pela Autora. A comunidade de Santa Maria encontra-se situada na zona rural de Tracuateua. Nesta comunidade vivem aproximadamente 40 famílias, a economia vem da agricultura familiar (produção de grãos, milho, feijão, mandioca), da pesca de subsistência e da criação de animais A comunidade de Santa Maria encontra-se situada na zona rural de Tracuateua. Nesta comunidade vivem aproximadamente 40 famílias, a economia vem da agricultura familiar (produção de grãos, milho, feijão, mandioca), da pesca de subsistência e da criação de animais NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 146 de pequeno porte (galinha, patos, porcos e gado). Possuem 1 (uma) escola de ensino fundamental, 2 (duas) igrejas (católica e evangélica) e 1 uma Unidade Básica de Saúde – UBS. Nesta comunidade, temos a benzedeira Dona T. de pequeno porte (galinha, patos, porcos e gado). Possuem 1 (uma) escola de ensino fundamental, 2 (duas) igrejas (católica e evangélica) e 1 uma Unidade Básica de Saúde – UBS. Nesta comunidade, temos a benzedeira Dona T. Bem próximo da sede do município a cerca de 4 km, temos a comunidade de Cajueiro Grande/Tracuá, embora a proximidade da sede seja tamanha, isso não faz diferença alguma na vida dos que residem na mesma. 1 FUNDAMENTAÇÃO TEÓRICA A comunidade conta apenas com uma escola de ensino fundamental menor, e não possui nenhum serviço básico (posto de saúde, água encanada, coleta de lixo etc.), lá vivem cerca de 20 famílias, que vivem basicamente da agricultura e roças de mandioca. Nesta comunidade temos um benzedor, o Seu S.T. Por fim, a comunidade Quilombola de Jurussaca abriga atualmente 45 famílias, descendentes de escravos. Segundo depoimento de moradores mais antigos da localidade, os fundadores da comunidade fugiram das fazendas nos arredores de Bragança (REIS; VIEIRA, 2016). Possuem como atividade econômica principal a agricultura familiar de subsistência, criação de pequenos animais e extrativismo. Na comunidade, é muito comum ainda encontrar moradores que detêm conhecimentos sobre plantas medicinais encontradas nas matas nativas, e também moradores que se interessam e conhecem sobre plantas cultivadas em quintais, o que é reforçado pelo benzedor estudado, o Sr. J.C ç p Conforme Reis e Vieira (2016, p. 4), que estudaram o ritual de beberagem na comunidade de Jurussaca, a história do município “está inserida no circuito que ‘abriga’ comunidades afrodescendentes e indígenas’”. Seu surgimento está diretamente relacionado à construção da estrada de ferro Belém-Bragança, em 1908, que promoveu o processo de povoamento pela vinda de trabalhadores da região nordeste (REIS; VIEIRA, 2016). Conta-se que o nome Tracuateua veio da referência dos trabalhadores da estrada de ferro, que reclamavam da quantidade de formigas pretas (tracuás) no local (SETUR/PA, 2014). Historicamente, as mulheres têm se mostrado profissionais com êxito na prática da medicina, seja na alimentação, no asseio, na cura de doenças e também na saúde espiritual daqueles que estão por perto. Não por acaso, pode-se dizer que esta relação entre o feminino na história e os cuidados com o próximo advém do conceito da maternidade e da interdependência humana com a mãe/mulher, tendo se estendido para além da comunidade familiar, fazendo assim com que surgissem as benzedeiras, rezadeiras e conselheiras (MACIEL; GUARIM NETO, 2006). Ainda no que diz respeito à prática de rituais realizadas no passado, pode-se estabelecer uma diferença importante quanto a participação do homem e da mulher, o que se mantém até hoje se comparamos com as práticas de benzimento feitas pelos benzedores e benzedeiras de Tracuateua. Esses aspectos apresentados acima configuravam um modelo das relações entre homens e mulheres (MOTTA-MAUÉS, VILLACORTTA, 2000). DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 147 No mesmo sentido, Boaventura de Souza Santos (2004), em sua teoria crítica pós- moderna, abre espaço para a luta emancipatória e o desafio representado pela articulação entre os saberes científicos, bem como entre outros saberes. Essa discussão epistemológica torna-se fundamental neste estudo, pois nos deparamos frequentemente com o saber hegemônico científico tentando suprimir os demais saberes. Diante das argumentações de Boaventura, temos a análise das relações entre a prática médica oficial e aos tratamentos populares que apresentam essa divisão: a medicina com o empírico, a farmacopeia, o racional e o orgânico, entre outros; e do lado das práticas populares, encontraríamos o simbólico, o ritual, o mágico, o psicológico, o sagrado e o social. Não há como ignorar ou deixarmos de relacionar a prática dos benzedores aos pressupostos de Ingold quando faz suas indagações que destacam que desde o simples hábito de tomar um chá para algum tipo de enfermidade receitado por nossos avós ou alguém mais velho, quando continuamos a propagar essas receitas aos nossos filhos, damos prosseguimento a um ritual de conhecimento que sem dúvida contribui a várias gerações. Foram muitos relatos até chegar aos benzedores, uma vez que há inúmeras pessoas que foram curadas de algumas enfermidades. Isso não é algo recente, as práticas de benzimento fazem parte do cotidiano das pessoas, dos costumes tradicionais e da cultura religiosa também, merecendo ser reconhecidos de algum modo como detentores de saberes e de um trabalho único. O reconhecimento dos benzedores e de seus saberes populares tradicionais, como o do saber científico, é imprescindível para corroborar a existência de uma epistemologia dos benzedores. 1 FUNDAMENTAÇÃO TEÓRICA É importante considerar a diferença que existe entre essa classificação simbólica de homens e mulheres e a sua classificação social, o que implica numa autêntica inversão de papeis ou posições. Enquanto na primeira classificação o homem ocupa uma posição estável e a mulher transita entre domínios, na segunda, a ele é permitido circular e atuar, livre e efetivamente em qualquer domínio de qualquer área do sistema social, ao passo que a mulher são impostos limites rígidos, restringindo-se drasticamente as oportunidades para seu desempenho na sociedade (MOTTA-MAUÉS; VILLACORTTA, 2000). Vale esclarecer, ainda, o que entendo sobre doenças naturais e não naturais. A doença que acomete o ser humano fisicamente é classificada como natural, o que é explicado por Maués (1990), e as doenças não naturais seriam aquelas causadas além do caráter físico, já chegando ao nível espiritual do ser humano. No entanto, os benzedores de Tracuateua apresentam uma nomenclatura diferente das encontradas em seu trabalho de campo; segundo eles, há doenças que são para médicos e outras para benzedores. TNOGRAFANDO AS BENZEDEIRAS E OS BENZEDORES DE TRACUATEUA Nesse sentido, Reis e Vieira (2016) corroboram que “seja qual for o discurso ou o meio, a representação do fato é uma referência e assim, temos que nos aproximar dela” Essa aproximação do sagrado é perceptível nos rituais de prática dos benzedores e benzedeiras, reforçando dessa maneira vários tipos de reflexões acerca das práticas de cura. Conforme Nascimento (2010) e Quintana (1999), na literatura, há pesquisas que evidenciam a constante interlocução dos benzedores com o além, ou seja, com a ordem divina, sagrada. Estabelecer o diálogo com benzedores foi essencial para construção de todo o método de pesquisa. O calendário para coleta de dados e visitas in loco foi dividido conforme a disposição dos benzedores e as pessoas atendidas, a cada visita foram fotografadas e feitas anotações em diário de campo; além disso, o termo de consentimento foi apresentado a todos os participantes. Solicita-se que os sujeitos (benzedores e pessoas atendidas por eles), relatem detalhadamente como acontece todo o processo de benzimento, desde as manifestações de doenças, até a cura após o benzimento. ç p A metodologia e a postura epistemológica sugerida neste estudo científico permite analisar epistemologicamente o ofício dos benzedores, delineando e tecendo um estudo que respeite e corrobore com aquilo que o outro compreende e acredita como certo, a partir do seu próprio saber, o qual está baseado numa cultura e na particularidade de cada pessoa, bem como, descrevendo o ato de benzimento, a sobreposição do saber científico sobre outros saberes, identificando as razões para a desvalorização das práticas de benzimento e meios para sugerir políticas públicas que valorizem esses saberes populares. Em conversas com pessoas idosas da região, encontramos os benzedores, que chamaremos de Dona T, Sr. J.C e Sr. S.T. Entre os mais conhecidos atualmente estão dona T., que é somente benzedeira, e seu S. T., experiente e benzedor. Ambos residem em áreas rurais do município. Dona T. possui 65 anos e seu S.T., 75 anos. Ambos são católicos. Seu J.C, 59 anos, é benzedor e reside na comunidade quilombola de Jurussaca. Segundo Maués (1994, p. 76-77), os especialistas locais que tratam de doenças não naturais são o benzedor e o experiente, sendo que ambos possuem métodos específicos no tratamento das doenças. O esclarecimento de Maués é fundamental, pois apresenta os conceitos e as diferenças de algumas palavras utilizadas neste estudo, tais como experientes e benzedores. TNOGRAFANDO AS BENZEDEIRAS E OS BENZEDORES DE TRACUATEUA Antes de iniciar o relato de experiência com os Benzedores e Benzedeiras do município de Tracuateua, é imprescindível apresentar as descrições dos métodos utilizados para esta pesquisa e para a coleta do material etnográfico. Conforme Malinowski (1978, p. 22-23): Na etnografia, o autor é ao mesmo tempo, o seu próprio cronista e historiador; suas fontes de informação são, indubitavelmente, bastante acessíveis, mas também extremamente enganosas e complexas, não estão incorporadas a documentos materiais fixos, mas sim ao comportamento e memória dos seres humanos. Na etnografia é frequentemente imensa a distância entre a apresentação final dos resultados da pesquisa e o material bruto de informações coletadas pelo pesquisador. Sob essa diretriz, inicialmente, Ingold (2015, p. 19) esclarece que: O objetivo da etnografia é descrever as vidas de outras pessoas além de nós mesmos, com uma precisão e sensibilidade afiada por uma observação detalhada e por uma prolongada experiência em primeira mão. Em Ingold (2015, p. 385), o conceito apresentado sobre o que é a etnografia significa escrever e falar sobre pessoas, destacando-se a ação de se escrever e sobre quem se irá escrever, o que permite o entendimento acerca do seu próprio conhecimento. No entanto, não é qualquer relato sobre um povo que torna a técnica uma etnografia; ao contrário, é necessário um olhar mais aguçado, artístico. As descrições etnográficas se tornam de certo modo mais uma produção única do que apenas um método científico, sem deixar de ser apresentada com coerência, precisão e realidade (INGOLD, 2015). p Quando se fala que as práticas socioculturais e religiosas de grupos étnicos no Brasil apresentam características próprias de uma história das mentalidades, como afirma Chartier (1991), significa dizer que essas práticas são distintas e coletivas, mas são representadas coletivamente e elevadas a uma só denominação. Nesse sentido, corrobora que os símbolos são NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 148 apresentações do sagrado, sendo uma prática simbólica que resume o ethos de uma comunidade (REIS; VIEIRA, 2016, p. 8). Nesse sentido, Reis e Vieira (2016) corroboram que “seja qual for o discurso ou o meio, a representação do fato é uma referência e assim, temos que nos aproximar dela”. apresentações do sagrado, sendo uma prática simbólica que resume o ethos de uma comunidade (REIS; VIEIRA, 2016, p. 8). DOSSIÊ AMAZÔNIA 149 intercessora entre os comandos do homem e do reconhecido localmente como sagrado, que é capaz de agir sobre tais aparecimentos. intercessora entre os comandos do homem e do reconhecido localmente como sagrado, que é capaz de agir sobre tais aparecimentos. Quando se fala do ritual terapêutico, podemos citar desde o início da benzeção, onde a pessoa é colocada em um assento (uma cadeira comum), num local onde haja luz, ou em direção da mesma, e assim são feitas as benzeções, com as orações auxiliadas com folhas ou ramos de algumas plantas, ou mesmo com a utilização das mesmas embebidas em álcool etílico. Ao ser indagados sobre umas características que defina quando a pessoa está aquebrantada, os benzedores relatam que há uma peculiaridade entre os sintomas; todo instante, os olhos permanecem com lágrimas. p g Os benzedores de Tracuateua possuem uma rotina como de qualquer outra pessoa; entre o trabalho da casa e da roça, seguem realizando os benzimentos. Dona T. realiza os trabalhos domésticos e cumpre outras rotinas como a maioria das pessoas, trabalha em casa, cuida dos netos e realiza os benzimentos sempre que solicitada. Ela também produz alguns produtos para tratamentos de tosse, gripe e inflamações, agregado ao dom de benzer realiza cursos sobre plantas medicinais ofertados pela Pastoral da Igreja Católica. Entre esses produtos estão xaropes, garrafadas, chás e extratos. Seu S.T levanta às 5h da manhã todos os dias e sua rotina consiste em cuidar de seu pequeno sítio; isso compreende a limpeza do lugar, o cuidar da roça e das pequenas criações. Os benzimentos acontecem no decorrer do dia, sempre que ele é procurado. Seu S.T também sugere o uso de plantas e xaropes para o tratamento quando é necessário. O senhor J.C assim como dona T e seu S.T também cuida de seu pequeno sítio, cria animais de pequeno e grande porte, e possui horários reservados para seus trabalhos que consiste em benzimento, rezas, amarrações, orações etc. Todos os benzedores afirmam que sempre que alguém procura ajuda, eles já sabem o que a pessoa tem, e porque estão lá. E que sempre são avisados “por eles” com antecedência. Durante a benzeção, é possível perceber que os benzedores se concentram, silenciosos rezam, algumas vezes em voz audível, outras não, e utilizam palavras milagrosas, que não são reveladas. TNOGRAFANDO AS BENZEDEIRAS E OS BENZEDORES DE TRACUATEUA O experiente lança mão de chás, “leite de paus” (seiva de plantas), “emplastros, banhos, defumações, pomadas, fricções, vomitórios, excrementos de animais, saliva humana e outros” (MAUÉS, 1990, p. 204), bem como remédios de farmácia. Além disso, é fundamental o experiente “dar a sua finesa”, isto é, indicar para o paciente o resguardo e as proibições no período de tratamento para que a substância não se torne venenosa no paciente. As referências sobre os benzedores na região são inúmeras. Dona T. é uma das benzedeiras mais conhecidas na região de campos. Muitas pessoas a procuram, de crianças a idosos, mas principalmente mulheres grávidas. S.T. também é muito procurado, mas seu maior público são crianças. J.C. atende a todos, sem distinção de gênero e faixa etária. Ambos rezam durante as benzeduras. Rezam orações católicas conhecidas como: Pai nosso, Ave Maria, Santa Maria e Creio em Deus Pai, há também uma oração que permanece em segredo, as fazem silenciosamente, quase inaudíveis. Conforme os benzedores são nestes momentos que ocorrem à cura e algo é sentido apenas por eles. Segundo Dona T, normalmente os benzedores são procurados para curar as doenças que os médicos não conseguem resolver, mas que para os benzedores são reconhecidas por sintomas pontuais: febre alta, acompanhada de calafrios e choramingo, diarreia, fezes “esverdeadas” e sobressaltos (pequenos sustos), que é característico do que é chamado de quebranto (informação oral, 2018). Isso segue a existência da prática do benzimento, que é uma arte DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA O que se pode perceber é que o benzimento, para essas pessoas simples detentoras de um conhecimento literalmente poderoso, é o ato de tornar benzido ou sagrado algo a ponto de curar. É o ato de cruzamento, ou seja, simbolicamente fazer o sinal da cruz diante de alguém, algo ou local e realizar uma espécie de operação do objeto ou sujeito a ser benzido e curado. Embora seja reconhecido, o saber popular dos benzedores não é levado em consideração pela medicina convencional. A maioria dos pacientes dos benzedores enfrentam resistência quando relatam aos profissionais médicos que fazem tratamento paralelo junto aos benzedores. O benzedor da comunidade do Cajueiro Grande não é tão detalhista quanto Dona T. Numa segunda-feira, em minhas observações em campo, ele recebeu um garotinho em sua casa, a mãe já havia lhe procurado fazia alguns dias. Ela apareceu bem cedo, antes das 7h da manhã, mas seu S.T. já estava acordado há bastante tempo. Ele foi até o quintal, pegou uma folha bem vistosa de peão-roxo (Jatropha gossypiifolia), e trouxe na mão. Ouviu atentamente o relato da mãe e logo em seguida sentou a criança meio chorosa em uma cadeira. Ele rezou baixinho, fez orações, pegou a folha de peão-roxo e passou pela cabeça e corpo do garotinho. Após benzer a criança, seu S.T. disse à mãe que o garoto era “gaiato”, muito falador e chamava muita atenção dos outros, por isso era fácil de “pegar” quebranto. Ao terminar de benzer a criança, a folha de pião-roxo estava completamente murcha e a criança mais saudável do que nunca. Aqueles que possuem informações mais especializadas sobre o cultivo e a utilização das plantas, de ervas e suas formas de cura e para tratamento e que podiam colocar em prática os benzimentos ou as simpatias eram conhecidas ou identificadas como benzedores e benzedeiras. Atualmente, apesar das inúmeras dificuldades por quais passam, possuem de certo modo um NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 150 prestígio junto às suas comunidades, sendo respeitados pelos seus serviços à saúde do povo de determinado lugar. Para a produção de chás é necessário a identificação do problema por qual a pessoa está acometida. DOSSIÊ AMAZÔNIA Em alguns casos de problemas gastrointestinais (dores de barriga ou cólicas abdominais), são utilizadas folhas secas ou verdes, que são fervidas em pequenas quantidades com água e depois de coadas são definidas para utilização em horários determinados. Figura 2 – Benzimento utilizando “Pião - roxo” Fonte – Acervo da autora. Os xaropes na maioria das vezes são utilizados com as partes das plantas, açúcar ou mel, Figura 2 – Benzimento utilizando “Pião - roxo” Figura 2 – Benzimento utilizando “Pião - roxo” Fonte – Acervo da autora. Fonte – Acervo da autora. Os xaropes na maioria das vezes são utilizados com as partes das plantas, açúcar ou mel, isto somente quando o paciente pode utilizá-los. Normalmente as folhas são cozidas junto com mel ou açúcar e depois armazenadas em pequenos potes ou frascos de vidros para consumo e tratamento. Os xaropes na maioria das vezes são utilizados com as partes das plantas, açúcar ou mel, isto somente quando o paciente pode utilizá-los. Normalmente as folhas são cozidas junto com mel ou açúcar e depois armazenadas em pequenos potes ou frascos de vidros para consumo e tratamento. Os xaropes na maioria das vezes são utilizados com as partes das plantas, açúcar ou mel, isto somente quando o paciente pode utilizá-los. Normalmente as folhas são cozidas junto com mel ou açúcar e depois armazenadas em pequenos potes ou frascos de vidros para consumo e tratamento. As garrafadas são feitas com partes das plantas, cascas, entre outras ervas. Ficam armazenadas em garrafas até adquirirem o ponto de consumo, sendo que existem garrafadas para cada doença específica, seja gastrite, inflamações femininas, sendo estas as mais conhecidas, por estar relacionadas à saúde feminina (VALE, 2018, p. 3). Dentre as similaridades observadas entre os benzedores e benzedeiras participantes deste estudo, podemos ainda destacar: a) o saber advindo de seus antepassados e melhorados com as vivências do dia a dia de cada benzedor e de cada benzedeira; b) a forte demonstração de fé nas suas crenças religiosas e símbolos; c) o vasto conhecimento sobre as plantas e suas propriedades, além da prática de produção de remédios a partir dessas plantas, bem como da sabedoria popular. DOSSIÊ AMAZÔNIA Segundo Ben (2015), normalmente os benzedores são pessoas já em idade avançada, experientes, alguns sem instrução formal, mas com um conhecimento amplo, prático e empírico espetacular, demonstrando enfim uma grande preocupação com a transmissão e DOSSIÊ AMAZÔNIA 151 continuidade de suas informações, uma vez que não possuem a quem ensinar seus conhecimentos. Com base nos relatos de experiência, diário de campo e registros foi possível produzir um fluxograma para cada benzedor e benzedeira desta pesquisa. O fluxograma é uma ferramenta da qualidade utilizada para representar a sequência e interação das atividades do processo por meio de símbolos gráficos. Os símbolos proporcionam uma melhor visualização do funcionamento do processo, ajudando no seu entendimento e tornando a descrição do processo mais visual e intuitiva. (PEINADO; GRAEML, 2007). O objetivo dos fluxogramas é demonstrar como o ato de benzer acontece e como são utilizados por cada benzedor. Figura 3 – Fluxograma de práticas de benzeção da Dona T. Fonte: elaborado pela Autora. Figura 3 – Fluxograma de práticas de benzeção da Dona T. Fonte: elaborado pela Autora. Figura 3 – Fluxograma de práticas de benzeção da Dona T. Figura 3 – Fluxograma de práticas de benzeção da Dona T. Fonte: elaborado pela Autora. Dona T. (Fig. 2) inicia o processo de benzimento a partir dos diagnósticos: Físico e Verbal. A partir deles, ela identifica se o paciente sofre de uma doença que pode ser facilmente identificada pelos médicos ou que seja somente identificada através de benzimentos. Dependendo da identificação afirmativa ou não, há um tipo de encaminhamento. Normalmente, para que Dona T consiga fazer esse diagnóstico, ela ouve o relato do paciente ou do responsável por ele (a mãe ou pai, quando se trata de crianças). Se a doença for de ordem médica, a conduta é indicação ao tratamento convencional. Se o diagnóstico for de doença onde o benzimento é necessário, dona T. realiza, conforme a necessidade, condutas distintas. Essas condutas são utilizadas conforme a especificidade da doença identificada, podendo ser orações/rezas, plantas/chá e objetos. Fonte: elaborado pela Autora. Fonte: elaborado pela Autora. Dona T. (Fig. 2) inicia o processo de benzimento a partir dos diagnósticos: Físico e Verbal. A partir deles, ela identifica se o paciente sofre de uma doença que pode ser facilmente identificada pelos médicos ou que seja somente identificada através de benzimentos. Dependendo da identificação afirmativa ou não, há um tipo de encaminhamento. Dona T. (Fig. DOSSIÊ AMAZÔNIA 2) inicia o processo de benzimento a partir dos diagnósticos: Físico e Verbal. A partir deles, ela identifica se o paciente sofre de uma doença que pode ser facilmente identificada pelos médicos ou que seja somente identificada através de benzimentos. Dependendo da identificação afirmativa ou não, há um tipo de encaminhamento. Normalmente, para que Dona T consiga fazer esse diagnóstico, ela ouve o relato do paciente ou do responsável por ele (a mãe ou pai, quando se trata de crianças). Se a doença for de ordem médica, a conduta é indicação ao tratamento convencional. Se o diagnóstico for de doença onde o benzimento é necessário, dona T. realiza, conforme a necessidade, condutas distintas. Essas condutas são utilizadas conforme a especificidade da doença identificada, podendo ser orações/rezas, plantas/chá e objetos. No caso de orações/rezas, são indicadas em caso de quebranto ou mau-olhado. As plantas/chá apresentam uma maior especificidade, pois cada parte da planta é utilizada para determinado caso, assim como seu modo de preparo e sua indicação terapêutica, seja para quebranto, inflamações e mau-olhado. Com o uso de objetos, dona T. os utiliza quando o paciente não pode chegar até ela. Nesse caso, ela utiliza objetos pessoais, roupas, terços etc. para realização do benzimento e para o fim específico que poderá ser quebranto, mau-olhado ou inflamações (femininas). Somente após o emprego das indicações terapêuticas, Dona T. finaliza o seu diagnóstico identificando o tipo de doença não natural. A benzedeira não cobra nem um tipo de valor ou gratificação ao paciente. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 152 Figura 4 – Fluxograma de práticas de benzeção do Sr. S. T. Fonte: elaborado pela Autora. Para o processo de benzimento, o Sr. S.T. (Fig. 3) é bem mais objetivo. Seu instrumento de benzimento rotineiramente inicia-se com uma planta (Pião-Roxo), que segundo ele, é bastante forte para o diagnóstico inicial, pois a planta murcha dependendo do problema apresentado. O diagnóstico inicial para ele consiste em sinais físicos que qualquer pessoa poderá ver no paciente. Posteriormente o Sr. S.T realiza o diagnóstico verbal que se dá a partir do relato do paciente sobre aquilo que somente ele sente e pode relatar. Após os diagnósticos, é sugerida a conduta que é utilizada conforme a especificidade do problema apresentado pelo paciente. Esse benzedor sempre realiza orações/rezas e utiliza Figura 4 – Fluxograma de práticas de benzeção do Sr. DOSSIÊ AMAZÔNIA S. T. Fonte: elaborado pela Autora. Para o processo de benzimento, o Sr. S.T. (Fig. 3) é bem mais objetivo. Seu instrumento de benzimento rotineiramente inicia-se com uma planta (Pião-Roxo), que segundo ele, é bastante forte para o diagnóstico inicial, pois a planta murcha dependendo do problema apresentado. O diagnóstico inicial para ele consiste em sinais físicos que qualquer pessoa poderá ver no paciente. Posteriormente o Sr. S.T realiza o diagnóstico verbal que se dá a partir do relato do paciente sobre aquilo que somente ele sente e pode relatar. p q q p Após os diagnósticos, é sugerida a conduta que é utilizada conforme a especificidade do problema apresentado pelo paciente. Esse benzedor sempre realiza orações/rezas e utiliza plantas/chá durante a benzeção. As orações/rezas são indicadas em caso de quebranto ou mau-olhado e as plantas/chá são utilizadas dependendo do caso, assim como seu modo de preparo e sua indicação terapêutica, para quebranto, inflamações e mau-olhado. Somente após o emprego das indicações terapêuticas, o processo de benzimento é finalizado com a identificação do tipo de doença de ordem não natural. O benzedor não faz qualquer menção a valores financeiros pelos benzimentos. 153 DOSSIÊ AMAZÔNIA Figura 5 – Fluxograma de práticas de benzeção do Sr. J.C. Fonte: elaborado pela Autora. Figura 5 – Fluxograma de práticas de benzeção do Sr. J.C. Fonte: elaborado pela Autora. Para explicação do fluxograma do Sr. J.C. (Fig. 4), há alguns detalhes mais específicos que precisam ser apresentados. Ele realiza benzimentos e trabalhos (espirituais) como tratamento para algumas doenças não naturais, mas somente após identificar a sua necessidade através de um diagnóstico inicial, seguido de diagnóstico físico e verbal. Em seguida, é apresentada a conduta, que consiste em orações/rezas, plantas/chás, defumações e objetos. As orações/rezas são indicadas por caso, mas em sua maioria são contra quebranto ou mau-olhado e em casos de cunhos espirituais (rituais, trabalhos etc.). Já as plantas/chá são utilizadas dependendo do caso, seu modo de preparo e sua indicação terapêutica, para quebranto, inflamações e mau-olhado. A defumação é utilizada principalmente em trabalhos espirituais, assim como os objetos. Após o emprego das indicações terapêuticas, o benzimento Figura 5 – Fluxograma de práticas de benzeção do Sr. J.C. Figura 5 – Fluxograma de práticas de benzeção do Sr. J.C. Fonte: elaborado pela Autora. Fonte: elaborado pela Autora. Fonte: elaborado pela Autora. Para explicação do fluxograma do Sr. J.C. (Fig. DOSSIÊ AMAZÔNIA 4), há alguns detalhes mais específicos que precisam ser apresentados. Ele realiza benzimentos e trabalhos (espirituais) como tratamento para algumas doenças não naturais, mas somente após identificar a sua necessidade através de um diagnóstico inicial, seguido de diagnóstico físico e verbal. Em seguida, é apresentada a conduta, que consiste em orações/rezas, plantas/chás, defumações e objetos. p q ç p ç j As orações/rezas são indicadas por caso, mas em sua maioria são contra quebranto ou mau-olhado e em casos de cunhos espirituais (rituais, trabalhos etc.). Já as plantas/chá são utilizadas dependendo do caso, seu modo de preparo e sua indicação terapêutica, para quebranto, inflamações e mau-olhado. A defumação é utilizada principalmente em trabalhos espirituais, assim como os objetos. Após o emprego das indicações terapêuticas, o benzimento ou o trabalho é finalizado com a identificação do tipo de doença de ordem não natural. Normalmente, como forma de garantia da profilaxia do benzimento ou do trabalho, o Sr. J.C pede que o paciente retorne após alguns dias para o acompanhamento. O Sr. J.C. é o único entre os benzedores que cobra pelo seu trabalho. Segundo ele, todas as profissões que requerem um tipo de saber cobram por tal conhecimento, sendo injusto ele não receber ou não poder cobrar por algo que ele pratica e possui que é o saber e a prática de cura através do ato de benzer. A partir do meu primeiro contato com o assunto, conforme observado na introdução, foram sugeridos um planejamento e a descrição da pesquisa, com a apresentação da proposta desta aos sujeitos em questão. A pesquisa bibliográfica e a etnografia sobre os benzedores serão os procedimentos metodológicos neste estudo. É importante discutirmos alguns traços que distinguem a experiência da observação participante e as dificuldades no estudo de algo tão subjetivo, sob as perspectivas do observador e dos observados. O tempo de ofício de cada participante variou. Dona T., por exemplo, já possui 61 anos como benzedeira, seu S.T. 50 anos, pois iniciou os benzimentos com 20 anos de idade e seu J.C. 52 anos como benzedor, visto que iniciou os benzimentos aos 7 anos após sempre renegá- los. Questionados com quem aprenderam a técnica, assim surgiram as respostas: O senhor S.T e J.C. afirmam que aprenderam com Deus, e Dona T., com membros da própria família. Para chegar aos benzedores de Tracuateua como pesquisadora, as conversas com minha família e com amigos foram fundamentais. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 154 benzia, só que “não era tão forte”, mas em Santa Maria tinha a Dona T., e ela era “forte”. Assim decidi que iria procurá-la em breve e convidá-la para participar da pesquisa. benzia, só que “não era tão forte”, mas em Santa Maria tinha a Dona T., e ela era “forte”. Assim decidi que iria procurá-la em breve e convidá-la para participar da pesquisa. A chegada ao Sr. S.T. também foi feita através de relatos e testemunhos de várias pessoas dentro da sede do município e na comunidade onde seu S.T. reside. Várias pessoas informaram que havia um benzedor “forte” na comunidade que cuidava muito bem de crianças. Passaram-se três dias para que eu finalmente encontrasse pessoalmente o benzedor. O terceiro benzedor pesquisado foi seu J.C. da comunidade quilombola do Jurussaca. Este já era por mim bastante conhecido devido aos relatos de meu pai que há muito tempo utilizava de seus benzimentos, em várias situações, mas os relatos das comunidades ao entorno sobre o benzedor me fizeram ir procurá-lo. Curiosamente, ao chegar à residência de seu J.C., ele já sabia da minha ida, mesmo sem qualquer aviso prévio. j q q p Ao chegar à residência de Dona T., era tardezinha, cheguei, bati palmas e pedi para conversar, ela logo pegou algumas cadeiras e começamos uma conversa informal. Ela começou relatando da facilidade e do prazer que sentia em ajudar ao próximo, mas que isso não acontecia dentro da família. Ela não conseguia realizar benzimentos com os familiares, apenas com pessoas desconhecidas. Nesse momento, chegou um casal em uma moto com uma criança, procuravam dona T. para benzer a filha. Ela pediu licença e se retirou. Não pude acompanhá- la durante este benzimento, pois ainda não havia apresentado meu objetivo ali. Aproximadamente uma hora após a chegada do casal, Dona T retornou a nossa conversa e começou a falar sobre sua trajetória como benzedeira, desde sua infância até a atualidade, contou sobre as sensações e situações que passava por possuir este dom, relatou também sobre o desaparecimento de um neto que dormia na rede enquanto ela fazia tarefas domésticas, e quando ela foi olhar a criança a mesma não estava mais na rede, quando sentiu sua falta, ela estava bem ao lado de um poço, dormindo. DOSSIÊ AMAZÔNIA Uma de minhas vizinhas relatou que ela também DOSSIÊ AMAZÔNIA 155 atrapalhar sua rotina, pois ele iria para sua roça, combinamos de acompanhá-lo outro dia, já com a permissão garantida. O encontro com seu J.C. na comunidade Quilombola de Jurussaca, no período da tarde, foi bem interessante. Ao chegar lá, seu J.C. estava sentado embaixo de uma grande mangueira, já esperando a visita. Me apresentei, embora ele já soubesse meu nome. Vale ressaltar que em 2004 trabalhei como professora na comunidade de seu J.C., no entanto, não possui contato com o mesmo. Porém, como ele e meu pai são amigos de longa data, imaginei que por isso ele soubesse meu nome e tivesse achado alguma semelhança entre meu pai e eu. Mas seu J.C. sabia que eu iria visitá-lo naquele dia e sabia meu nome, mesmo sem eu mencionar meu pai. que eu iria visitá-lo naquele dia e sabia meu nome, mesmo sem eu mencionar meu pai. Ele disse que sabia o porquê que eu estava lá e disse: “Tu pra mim é transparente. Eu te olho e te enxergo de um lado para o outro”, por isso eu estou aqui hoje e não sai. Eu sabia que tu vinha (sic) aqui hoje” (informação oral, 2018). Por esse motivo, a conversa com seu J.C foi bem mais direta, falei sobre meu interesse na pesquisa e logo ele se prontificou a contribuir. Foram feitas visitas às comunidades do município de Tracuateua que possuem benzedores. E na busca da análise epistemológica do ofício dos benzedores no município de Tracuateua – PA, este trabalho foi norteador para a abordagem a utilização da Etnografia. Para André (2007, p. 105), “o pesquisador que conhece um lugar, a partir do ponto de vista do grupo, dando volume às vozes locais, parte para uma pesquisa do tipo etnográfica”. Para que pudesse analisar o ofício dos benzedores no município de Tracuateua/PA, foi preciso estar lá, presenciar, acompanhar o cotidiano dos benzedores, as rezas, os doentes que chegam, e isso só pode ser descrito minuciosamente quando se está presente na realidade. Morar em uma das comunidades que residem os benzedores talvez seja uma das melhores ferramentas de pesquisa. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Segundo dona T., quem desejava levar a criança era o Curupira. Isso me fez recordar o quanto ouvi histórias desse tipo na infância. p q p Dona T. relatou que está disposta para o benzimento sempre que é procurada; ela costumava ir aos domingos à igreja, mas sempre que estava assistindo a missa ela era chamada para auxiliar alguém, e por esta razão ela deixou de ir à missa. Após os relatos de Dona T., comecei a falar sobre meu interesse pelo seu ofício, e o quanto ele era instigante e necessitava ser estudado, embora a complexidade do assunto seja tamanha. Perguntei sobre a possiblidade de acompanhá-la durante os benzimentos para entender melhor seu trabalho e ela concordou, sugeriu datas e locais para que então eu pudesse começar a pesquisa. A tarde findava e eu pedi licença para retornar no dia sugerido por ela e iniciar as pesquisas em campo. Em outra manhã de domingo, fui até a residência de seu S.T, cheguei lá por volta das 8h da manhã. Mas seu S.T. acorda muito cedo e já não estava mais em casa a essa hora. Retornei no dia seguinte. Ao chegar à comunidade da Tracuá, seu S.T. já estava de pé. Fui recebida com um abraço e em seguida ele me ofereceu um café. Sua esposa o acompanhava nesse dia. Comecei falando sobre o interesse em pesquisar os benzedores de Tracuateua e disse a ele o quanto havia ouvido seu nome entre as pessoas do município. Ele então relatou que outros pesquisadores já haviam procurado por ele. E que um deles havia lhe dado um diploma que respaldava seus conhecimentos e que era muito importante para ele. Fiquei curiosa em ver o documento, mas hesitei em pedir a seu S.T. Ele, porém, entrou em sua casa e em seguida surgiu com uma pasta munido de vários papéis. O documento apresentado por seu S.T. era uma declaração de determinado órgão que também realizava pesquisas sobre o trabalho de seu S.T, certificando-o quanto à prática de benzimentos. Seu S.T. também fez relatos das suas dificuldades, onde se encaixava a família e seus filhos, pois se tornaram fiéis de outras religiões e não gostavam que seu S.T praticasse benzimentos. Mesmo com o impasse, seu S.T concordou em participar da pesquisa, e para não DOSSIÊ AMAZÔNIA 3 O SEGREDO DO SAGRADO O ofício de benzeção e suas práticas ritualísticas, no conceito de Gomes e Pereira (2004, p. 19), “é uma linguagem oro-gestual com a qual algumas pessoas – detentoras de poder especial – controlam as forças que contrariam a vida harmoniosa do homem. Benzer é garantir o funcionamento da normalidade desejada e conter o mal”. O processo de benzeção pode apresentar vários sentidos, desde doar o ato de benzer (Eu te abençoo) até admirar-se, espantar-se, passar bons fluídos, produzir os benzimentos, dentre outras nomenclaturas (OLIVEIRA, 1985, p. 8-9). Neste momento; é importante ressaltar que a benzeção, os benzedores, as benzedeiras e seus trabalhos só podem ser compreendidos ao estudar aos fatos que as levaram a iniciar tal prática, isto é, o momento em que recebem o “dom”. Muitas vezes se transmite esse dom entre pessoas de uma mesma família, outras vezes, o indivíduo é identificado como tal por outra pessoa, por possuir uma característica de bondade ou de habilidade para ajudar as pessoas, ou se tem ainda todas essas situações combinadas de diversas maneiras entre si, o que leva a benzedeira a acreditar no seu potencial curativo (OLIVEIRA, 1985, p. 34). Sempre há uma espécie de sinal para a descoberta para a mulher ou o homem escolhido para o dom divino, normalmente relacionado a um episódio muito marcante na vida da pessoa. Segundo Dona T, seu dom foi recebido ainda criança: Aos 8 anos de idade, na volta para casa, um animal encontrava-se em agonia de parto, minha avó me pediu para pegar o azeite e começamos a benzer juntas, puxava a barriga do animal, até que consegui, e desde aí comecei a benzer (T., informação oral, 2018). Seu S. T. relatou que chorou no ventre de sua mãe, pois nasceu com o dom, e durante o crescimento sentia que era diferente: 156 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Existem doenças que não são para os médicos, têm que ser tratadas por nós, benzedores, estou cansado de ir a hospitais para benzer, eles vêm me buscar altas horas da noite para benzer as pessoas, e eu vou. É um dom que DEUS me deu, não posso negar ajuda a ninguém, e não cobro nenhum centavo (S.T., informação oral, 2018). 3 O SEGREDO DO SAGRADO O saber tradicional tende a se completar, em ambientes diferentes, mediante a relação daqueles que de algum modo carregam consigo este diário. Como exemplo, podemos citar os benzedeiros e benzedeiras, rezadores, erveiros, puxadores (que puxam barriga, costas, um tipo de massagem que alivia dores e outros espasmos musculares), e outros que vão muito além da interação com plantas medicinais. Em sua maioria, praticam as benzeções no dia a dia através de suas rezas, gestos e energização, mas há aqueles que também complementam seus atos de benzer agregados ao uso de ervas a partir do etnoconhecimento que é componente forte no saber tradicional, assim como a palavra dita aumenta a força do ato ao processo de tratamentos ao doente. É É nesse contexto que devemos entender a naturalidade do sobrenatural para o reestabelecimento da saúde física e mental, uma vez que a noção religiosa de corpo e alma, inseparáveis desde o instante do nascimento, comporta a existência de sintomas físicos oriundos de problemas espirituais, cuja cura exigia o conhecimento de produtos da fauna e da flora com orações e benzeduras (ANZAI, 1985, p. 129). É nesse contexto que devemos entender a naturalidade do sobrenatural para o reestabelecimento da saúde física e mental, uma vez que a noção religiosa de corpo e alma, inseparáveis desde o instante do nascimento, comporta a existência de sintomas físicos oriundos de problemas espirituais, cuja cura exigia o conhecimento de produtos da fauna e da flora com orações e benzeduras (ANZAI, 1985, p. 129). As benzeções começam a ser rezadas em voz baixa, em murmúrios e muitas às vezes, de forma quase que inaudível ou incompreensiva. O clima de segredo e mágico contribuiu para a preservação e para a variação regional nos textos sobre benzimento, assim como a inclusão de equivalências simbológicas e nativas distintas de região, muito embora venham a manter em sua estrutura o ritual e as rezas, e os elementos invariáveis e corriqueiros embora possuam ter significados distintos. g Referente às orações, Confortin (2005, p. 21) explica que “geralmente são expressas em termos simples, às vezes até mesmo um tanto obscuros e incompreensíveis, mesclam valores do cristianismo, do folclore e das religiões africanas e indígenas”. DOSSIÊ AMAZÔNIA DOSSIÊ AMAZÔNIA 157 Percebe-se que cada praticante de benzimentos possui seu próprio método de trabalho, sua técnica e seu meio de tratamento, trazendo consigo um marco próprio para suas práticas de cura. Os benzedores e benzedeiras sempre falarão das enfermidades como fruto advindos destes elementos, entendendo o adoecimento para além das explicações meramente convencionais, mas aliando elementos simbólicos e imaginários passíveis de cura pela reza, pelo mágico e por técnicas de tratamento empregadas por especialistas da medicina popular e da religião, muito embora não sendo reconhecidas pelos órgãos oficiais, mas sim por suas comunidades e pacientes que usufruem das benzeções (LOYOLA,1983). Prossegue a autora: E o que se pode perceber é que o benzimento, para essas pessoas detentoras de um conhecimento literalmente poderoso, é o ato de tornar benzido ou sagrado algo a ponto de curar. Em outras palavras, o que prevalece é sua função terapêutica aliada à sua função religiosa (LOYOLA,1983, p. 92). É no uso das palavras ditas pelos benzedores durante os benzimentos que percebemos a manifestação do “segredo”, é ali, junto a uma gama de técnicas ou sinais que vem até serem relatados, que eles acontecem. As palavras que somente eles detêm e somente eles ouvem, e que é característico somente daqueles que possuem o dom e o exerce como ofício para benzer e curar. É perceptível, ainda, que a reprodução de determinadas frases pode ser estimada como uma forma de manutenção da memória, característica de sociedades que se baseiam na oralidade. Os estudos realizados sobre o saber dos benzedores do município de Tracuateua vêm auxiliar sobre a importância de valorizar e contribuir com os estudos epistemológicos, expandindo e favorecendo a epistemologia do conhecimento e da natureza. Morin (2011) afirma que deve haver um diálogo entre a ciência e os saberes populares para que possa existir uma facilidade maior do indivíduo em se contextualizar, e que a construção do conhecimento deve considerar a interdependência de outros saberes, desconsiderando se é mais ou menos importante, mas que todo tipo de conhecimento deve estar presente dentro de um todo complexo. 3 O SEGREDO DO SAGRADO Por essa razão, muitas doenças tratadas pelas benzedeiras e benzedeiros quilombolas não constam com a mesma nomenclatura da lista de tratamentos da medicina institucionalizada, como mostra a Tabela 1: Tabela 1 – Doenças identificadas pelos Benzedores e Benzedeiras de Tracuateua Doenças Características/Sintomas Quebranto Olhos lacrimejantes; moleza por todo o corpo; tristeza; bocejar constante; espirros repetidos e inapetência. Mau-olhado Tristeza e moleza. Erisipela Febre alta e calafrios; dor de cabeça; náuseas e vômito; feridas vermelhas na pele; inflamadas e doloridas; sensação de queimação na região afetada; manchas vermelhas com bordas elevadas. Espinhela Caída Vômito; posição desconfortável; choros (em caso de bebês, pela sensação de dor); braço ou pernas mais longos do que o outro. Cobreiro Bolhas, formigamento ou dor na pele; vermelhidão e inchaço na pele; mal-estar. Fogo selvagem Bolhas e lesões na pele. Fonte: elaborada pela autora. Tabela 1 – Doenças identificadas pelos Benzedores e Benzedeiras de Tracuateua Doenças Características/Sintomas Quebranto Olhos lacrimejantes; moleza por todo o corpo; tristeza; bocejar constante; espirros repetidos e inapetência. Mau-olhado Tristeza e moleza. Erisipela Febre alta e calafrios; dor de cabeça; náuseas e vômito; feridas vermelhas na pele; inflamadas e doloridas; sensação de queimação na região afetada; manchas vermelhas com bordas elevadas. Espinhela Caída Vômito; posição desconfortável; choros (em caso de bebês, pela sensação de dor); braço ou pernas mais longos do que o outro. Cobreiro Bolhas, formigamento ou dor na pele; vermelhidão e inchaço na pele; mal-estar. Fogo selvagem Bolhas e lesões na pele. Fonte: elaborada pela autora. Tabela 1 – Doenças identificadas pelos Benzedores e Benzedeiras de Tracuateua 4 CONSIDERAÇÕES FINAIS Os estudos realizados entre as benzedeiras e os benzedores do município de Tracuateua são necessários para o conhecimento sobre o ofício dos benzedores e sobre a importância de valorizar e contribuir com os estudos e pesquisas na área da Antropologia da Saúde e em perspectivas interdisciplinares, expandindo e favorecendo a epistemologia do conhecimento e da natureza. Os registros, os relatos dos benzedores e dos pacientes que praticam e recebem o benzimento, demonstram experiências significativas sobre outra forma de conhecer e estar no mundo, como também as habilidades que vão experimentando ao longo da vida, que são fundamentais para os contextos sociais para que se possa conhecer e compreender como esses saberes estão relacionados à prática do benzimento. Espera-se que este estudo venha a viabilizar outras pesquisas, partindo de uma análise epistemológica dos benzedores, com registros e problematizações sobre o ofício de benzer e suas práticas de cura. Acredito que deva ocorrer um diálogo entre a ciência e essas outras formas de conhecimentos para que possa haver um entendimento maior para que o sujeito o entenda, e que a construção do conhecimento deve considerar a interdisciplinaridade e o diálogo entre outros saberes, não considerando se é mais ou menos relevantes e sim diferentes, e essa diversidade contribui para o avanço de um conhecimento compartilhado e não fragmentado. Entende-se que seja necessário haver uma conexão entre conhecimentos, onde o saber científico possa reconhecer que o benzimento não é uma “crendice”, mas sim um saber diferenciado, um conhecimento como outros que utilizam o sagrado, as orações e as plantas transmitindo transformações na vida daqueles que procuram alternativas ou elementos complementares na busca para a cura ou um infortúnio, contribuindo de forma acentuada para somar aprofundamento teórico sobre os elementos simbólicos e religiosos, mas também formas de habilidades que seguem resistindo, através de seus saberes e domínios diversificados de conhecimentos. É É importante reforçar a necessidade de diálogos entre os conhecimentos científicos e outras formas de saber, apresentando assim um desafio entre aqueles que têm uma percepção do ambiente em que todos os objetos, materiais e sujeitos, interagem entre si e produzem assim uma materialidade religiosa. Trata-se, por conseguinte de enraizar-se e corroborar o conhecimento dessas pessoas. Clarificar, não somente no meio acadêmico, mas na sociedade, a autoridade e precisão do ofício dos benzedores, no auxílio e nos processos de cura da população. DOSSIÊ AMAZÔNIA Nas palavras do autor: Complexus significa o que foi tecido junto; há complexidade quando elementos diferentes são inseparáveis constitutivos do todo (como o econômico, político, o sociológico, o psicológico, o afetivo, o mitológico), há um tecido interdependente, interativo e interretroativo entre o objeto de conhecimento e seu contexto, as partes e o todo, o todo e as partes, as partes entre si (MORIN, 2011, p.38). Conforme o autor entende-se que deva haver uma conexão entre conhecimentos, onde o saber científico possa reconhecer que o benzimento não é uma crendice, mas sim um saber único, um conhecimento como outros que utilizam o sagrado, as orações, as plantas, e energias, transmitindo a cura na vida daqueles que acreditam e dos que não acreditam no benzimento, contribuindo de forma acentuada para somar aprofundamento teórico sobre os elementos simbólicos e religiosos que seguem resistindo, através de seus saberes e domínios cheios de conhecimento popular. Sinaliza-se, assim, a necessidade de diálogos entre os conhecimentos científicos e populares, apresentando assim um desafio entre aqueles que acreditam que todos os objetos e sujeitos, interagem entre si. Trata-se, por conseguinte de enraizar-se e corroborar o conhecimento dessas pessoas. Clarificar, não somente no meio acadêmico, mas na sociedade, a autoridade e precisão do ofício dos benzedores, no auxílio e nos processos de cura da população. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 158 REFERÊNCIAS ANDRÉ, Marli Elisa Dalmazo A. de. Etnografia da Prática Escolar. 14. ed. Campinas: Papirus, 1997. ANZAI, Leni Caseli. Vida cotidiana na zona rural do município de Goiás 1888-1930. Goiânia, 1985. Dissertação – Faculdade de Ciências Humanas e Filosofia, Universidade Federal de Goiás. Goiânia, ago. 1985. CHARTIER, Roger. O mundo como representação. Estudos Avançados, São Paulo, v.5, n.11, p. 173-191, 1991. Trad. Andréa Daher e Zenir Campos Reis. Disponível em: https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-40141991000100010&ln. Acesso em: 06 ago. 2020. CONFORTIN, Helena. Benzeduras, superstições, simpatias... Mitos ou realidade. Erechim: EdiFAPES, 2005. 4 CONSIDERAÇÕES FINAIS Dialogar sobre os saberes de benzedores(as) e as consequências dessa apreensão de conhecimento ou reconhecimento e de suas afinidades com o conhecimento científico, terá reflexos diretos na implantação de políticas públicas que poderão contribuir significativamente para a valorização e interação entre conhecimentos. Deste modo, o conhecimento pode ser compartilhado e assim, não somente as pessoas que são tratadas pelos benzedores poderão vislumbrar de um saber relevante e que precisa ser estudado, enquanto muitos na sociedade e na academia ainda desconhecem ou sequer reconhecem o caráter epistemológico do ofício dos benzedores no tratamento de doenças. Estudos realizados sobre o conhecimento das benzedoras e dos benzedores do município de Tracuateua podem contribuir para a possibilidade de valorizar as práticas de benzeção favorecendo a epistemologia da natureza. Os registros, os relatos dos benzedores e dos pacientes que praticam e recebem o benzimento, demonstram experiências significativas sobre o saber chamado de “popular”, que são fundamentais para os contextos sociais para que se possa conhecer e compreender como esses saberes estão relacionados à prática do benzimento. DOSSIÊ AMAZÔNIA 159 Espera-se que este estudo venha viabilizar outras pesquisas, partindo de uma análise epistemológica dos benzedores, com registros e problematizações sobre o ofício de benzer e suas práticas de cura. MORIN, Edgar. Introdução ao pensamento complexo. 3. ed. Porto Alegre: Sulina, 2007 MOTTA-MAUÉS, Maria Angelica; VILLACORTA, Gisela Macambira. Matintapereras e pajés: gênero, corpo e cura na pajelança amazônica (Itapuá/PA). In: ENCONTRO ANUAL DA ANPOCS, n. XXIV, Belém, 2000. Anais [...]. GT11 - Pessoa, Corpo e Doença - Sessão 3, Doença, Gênero e Diferença Social. Belém, 2000. 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Conhecimento prudente para uma vida decente: um discurso sobre as ciências revisitado. São Paulo: Cortez, 2004. p. 777-821. SANTOS, Boaventura de Sousa. Para uma Sociologia das Ausências e uma Sociologia das Emergências. In: SANTOS, Boaventura de Sousa (Org.). Conhecimento prudente para uma vida decente: um discurso sobre as ciências revisitado. São Paulo: Cortez, 2004. p. 777-821. VALE, Giovanna Luiza Santos. A Epistemologia das garrafadas. In: ENCONTRO DE ANTROPOLOGIA VISUAL DA AMÉRICA AMAZÔNICA, n. III, Belém, 19 e 21 de SANTOS, Boaventura de Sousa. Para uma Sociologia das Ausências e uma Sociologia das Emergências. In: SANTOS, Boaventura de Sousa (Org.). Conhecimento prudente para uma vida decente: um discurso sobre as ciências revisitado. São Paulo: Cortez, 2004. p. 777-821. VALE, Giovanna Luiza Santos. A Epistemologia das garrafadas. In: ENCONTRO DE ANTROPOLOGIA VISUAL DA AMÉRICA AMAZÔNICA, n. III, Belém, 19 e 21 de setembro de 2018. Anais [...]. Belém: Universidade Federal do Pará, 2018. Disponível em: http:www.eavaam.com.br/anais/anais/2018/gt11/58.pdf. Acesso em: 07 ago. 2020. – VOLUME – – – SABERES NA AMAZÔNIA SABERES NA AMAZÔNIA SEÇÃO LIVRE SEÇÃO LIVRE 163 MARÇO 202 1 VOLUME 1 REVISTA GR ADUAÇÃO EM L INGU AGENS E 202 MARÇO NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 164 enquanto análise da Geografia Crítica, que é a ciência marcada por uma análise marxista baseada nas relações de trabalho, relações sociais, desprendendo-se apenas de visões descritivas da realidade. A Geografia, tal qual outras ciências de cunho social, tem uma relação de afinidade com outras ciências, como por exemplo a Economia e a Sociologia, o que faz dela uma ciência que busca explicar a realidade a partir de diferentes enfoques teórico- filosóficos. O objetivo deste artigo é discorrer sobre os espaços de representação em uma abordagem lefrebvriana, demonstrando sua importância na ciência geográfica. No que tange ao espaço enquanto categoria analítica, pode-se dizer que é um conceito complexo e de suma importância para a Geografia. Partindo dessa premissa, Carlos (2011, p.17) afirma que “o espaço, como categoria de pensamento e realidade prática, traz em si a ideia de referência para o ser humano, uma vez que é sua condição de existência, assim como as transformações da sociedade trazem como consequência modificações espaciais”. Diante disso, percebe que a compreensão de espaço perpassa a sua materialidade, atingindo um viés subjetivo que é o das relações sociais. A Geografia Crítica vem trazer, portanto, essa discussão tendo como base o materialismo histórico e dialético. O foco do debate é o entendimento dos espaços de representação, conceito desenvolvido por Lefebvre (1974) e que trouxe tanto para a Geografia como para a outras ciências uma forma de ver o espaço como conectado à sociedade na forma de representação do mundo, da vida e do cotidiano. Tal visão é importante pois não despreza a materialidade, mas entende que os símbolos e as formas mais subjetivas de construção social são componentes indivisíveis do espaço. O foco do debate é o entendimento dos espaços de representação, conceito desenvolvido por Lefebvre (1974) e que trouxe tanto para a Geografia como para a outras ciências uma forma de ver o espaço como conectado à sociedade na forma de representação do mundo, da vida e do cotidiano. Tal visão é importante pois não despreza a materialidade, mas entende que os símbolos e as formas mais subjetivas de construção social são componentes indivisíveis do espaço. A abertura política que se afirma no país, a descrença que vários geógrafos passaram a ter em relação à “nova geografia”, a própria falência do sistema de planejamento, a acentuação dos problemas nacionais que se tinha proposto solucionar, a volta de Milton Santos com sua riqueza e vigor intelectual, tudo isto iria contribuir para a emergência de uma “geografia nova” não comprometida com o aparelho ideológico do Estado, comprometida sim, com os interesses da maioria da população, com os trabalhadores rurais e urbanos, e com um projeto histórico que é o da transformação da sociedade. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Os autores utilizados como aporte teórico desse artigo foram escolhidos com base na corrente da Geografia crítica, optando por autores de referência nessa temática, diversificando entre estrangeiros e brasileiros e buscando embasar a discussão conceitual de espaço, tais como Lefebvre (1976), Harvey (2006), Santos (1996; 2008), Lacoste (1990), Moreira (1985), Carlos (2011), Gottdiener (2010), Brunet (1990) e Castells (2000). Na primeira seção, faz-se um breve histórico sobre a Geografia Crítica, trazendo sua base teórica e surgimento, assim como o conceito de espaço geográfico de modo diversificado, conforme a teoria de autores contemporâneos. Na segunda seção, apresenta-se diferentes concepções a respeito dos espaços de representação. SEÇÃO LIVRE SEÇÃO LIVRE 165 Essa nova Geografia vem responder a uma necessidade da sociedade em romper com a neutralidade e assumir um viés político, reproduzindo o ideal marxista da luta de classes. Uma característica notória dessa corrente é a criticidade com que analisa o espaço geográfico, saindo do âmbito tradicional com foco no empirismo e “pensando o espaço em sua complexidade” (LACOSTE, 1990, p.35). Esta ciência crítica surge em meio crise trazendo mudanças expressivas e respondendo a um descontentamento geral. Como afirma Moreira (1985, p.58), a Geografia é uma ciência de extrema relevância para a sociedade: A geografia é um saber vivido e aprendido pela própria vivência. Um saber que nos põe em contato direto com nosso mundo exterior, com o seu todo e com cada um de seus elementos, a um só tempo. Se nisto reside sua peculiaridade, da qual deriva sua natural popularidade, reside nisto igualmente seu amplo significado político. Ainda de acordo com Moreira, é importante ressaltar essa visão da Geografia enquanto ciência utilizada e vivida no cotidiano, muito mais que um conhecimento científico essa ciência é plural e intrínseca a vivência social, se materializando diariamente na ação de seus atores sociais. Segundo Lacoste (1997, p. 25): Na verdade, a função ideológica essencial do discurso da geografia escolar e universitária foi, sobretudo, a de mascarar por procedimentos que não são evidentes, a utilidade prática da análise do espaço, sobretudo para a condução da guerra, como ainda para a organização do Estado e prática do poder. É, sobretudo, quando ele aparece inútil que o discurso geográfico exerce a função mistificadora mais eficaz, pois a crítica de seus objetivos neutros e inocentes parece supérflua. Lacoste (1997), ainda afirma que durante muito tempo a Geografia foi utilizada para fazer a guerra, como estratégia militar e política de dominação, sendo utilizada para consolidar e estruturar o modo capitalista de produção e atender aos interesses da burguesia. A Guerra do Vietnã é um exemplo, uma vez que, em 1972, quando a partir de um estudo geográfico de análise espacial para verificar os melhores locais surgiu um plano de aniquilamento sistemático da rede de diques que protegem as planícies extremamente populosas do Vietnã do Norte (LACOSTE, 1997). Diferente da Geografia Tradicional, a Geografia Crítica traz a análise geográfica enquanto oportunidade de libertação do homem. 1 DA CRÍTICA DA GEOGRAFIA AOS ESPAÇOS DE REPRESENTAÇÃO A Geografia Crítica surgiu na França em meados da década de 1970, e em seguida espalhou-se por outros países como: Suíça, Itália, Alemanha, Brasil, México entre outros. Essa corrente da Geografia tem como base filosófica e teórica o materialismo histórico e dialético. Fundamentada em teorias marxistas, vai além do pensamento tradicional ligado fortemente ao positivismo lógico e ao empirismo, trazendo assim uma abordagem militante com foco em uma sociedade mais justa e igualitária. Partindo desse pressuposto de renovação e construção de uma nova geografia, Corrêa (1982, p.20) diz: Partindo desse pressuposto de renovação e construção de uma nova geografia, Corrêa (1982 20) di SEÇÃO LIVRE Ademais, a Geografia passa a ser práxis revolucionária com objetivo de transformar o espaço, estudando as práticas sociais e os sujeitos que compõem e constroem este espaço. A partir dessa corrente, o espaço passa a se tornar objeto principal de estudo da Geografia, pois é nele que se pode ver a materialização da sociedade em todas as suas nuances, uma vez que o espaço é verdadeiro e amplamente social, representando as dinâmicas sociais sem menosprezar as questões naturais e suas singularidades (SOUZA, 2013). O espaço geográfico é um dos conceitos mais importantes da Geografia, ele é analisado enquanto fenômeno social produzido na relação sociedade-natureza, é um conceito complexo exigindo um esforço de construção epistemológica devido a sua abrangência e os diversos debates teóricos acerca do tema. Segundo Santos (2012, p. 30), “o espaço não é nem uma coisa nem um sistema de coisas, senão uma realidade relacional: coisas e relações juntas”. O conceito de espaço perpassa o material e se dá principalmente nas relações de trabalho da sociedade, diante disso, não pode ser considerado estático, mas dinâmico e concreto. Segundo Carlos (2011, p.28): Segundo Carlos (2011, p.28): 166 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Todavia, o espaço guarda o sentido do dinamismo das necessidades e dos desejos que marcam a reprodução da sociedade em seu sentido mais amplo, a realização da vida para além da sobrevivência. Seguindo essa perspectiva, é visível o quanto o espaço se tornou dinâmico e local da produção e reprodução social desenvolvida cotidianamente pelos atores sociais em seus modos de apropriação do espaço, de forma tanto individual quanto coletiva envolvendo espaço e tempo delimitados (CARLOS, 2011). Santos (2008, p. 46), nos apresenta um conceito de espaço que bem caracteriza esse contexto da Geografia Crítica: [...] algo dinâmico e unitário, onde se reúnem materialidade e ação humana. O espaço seria o conjunto indissociável de sistemas de objetos, naturais ou fabricados, e de sistemas de ações, deliberadas ou não. A cada época, novos objetos e novas ações vêm juntar-se às outras, modificando o todo, tanto formal quanto substancialmente. Segundo este autor, o espaço é algo em movimento, vivo, sendo fruto da ação humana e uma soma do sistema de ações e objetos. Tais elementos são indissociáveis na formação do espaço. Do espaço não se pode dizer que seja um produto como qualquer outro, um objeto ou uma soma de objetos, uma coisa ou uma coleção de coisas, uma mercadoria ou SEÇÃO LIVRE Não se pode dizer que seja simplesmente um instrumento, o mais importante de todos os instrumentos, o pressuposto de toda produção e de todo intercâmbio. Estaria essencialmente vinculado com a reprodução das relações (sociais) de produção (LEFEBVRE, 1976, p. 34). um conjunto de mercadorias. Não se pode dizer que seja simplesmente um instrumento, o mais importante de todos os instrumentos, o pressuposto de toda produção e de todo intercâmbio. Estaria essencialmente vinculado com a reprodução das relações (sociais) de produção (LEFEBVRE, 1976, p. 34). Portanto, Lefebvre (1976) traz uma análise marxista do espaço, mostrando uma visão de espaço ligado à prática social, de modo que este tem um papel de estruturação de uma lógica capitalista. Partindo dessa premissa, pode-se perceber que na teoria do referido autor o espaço tem natureza multifacetada, ele é a parte material, como também o social e é lócus da produção e reprodução dos atores sociais, em todas as áreas do cotidiano humano. É essencialmente um produto social. p Deste modo, Lefebvre (1991) mostra uma visão de espaço ligado à prática social, uma visão dos sujeitos como construtores desse espaço que tem um papel de estruturação de um sistema, uma lógica capitalista. No que tange ao espaço de representação, será tomada a definição de Lefebvre (1991), que trata da tríade do espaço percebido (práticas espaciais), concebido (representações do espaço) e vivido (espaço representacional) e, diante disso, inspira a análise sobre os sujeitos em relação a produção do espaço. A tríade percebido-concebido-vivido (em termos espaciais: práticas espaciais, representações de espaço, espaço representacional) perde toda a sua força quando é tratada como um modelo “abstrato”. [...] ela (a tríade) tem sua fonte na história de um povo, assim como na história de cada indivíduo que pertence àquele povo. [...] Compreende os loci das paixões, das ações e das situações vividas e, portanto, implica em tempo. Consequentemente pode ser [...] direcional, situacional ou relacional, porque é essencialmente qualitativa, fluida e dinâmica. (LEFEBVRE,1991, p. 40-42). A análise tridimensional do espaço percebido, concebido e vivido constitui a autoprodução da sociedade. É perceptível que esses conceitos são compostos de subjetividade, individualidade e concomitantemente de uma conjuntura social, são processos ativos. Agora vamos conceituar esses espaços em suas particularidades. O espaço percebido é o que é relativo às práticas espaciais, pode ser apreendido por meio dos sentidos, além dos cinco sentidos estabelecidos pela ciência, ele está ligado aos elementos, aquilo que é material. SEÇÃO LIVRE Esse conceito vem se modificando com o passar do tempo, tem sofrido alterações que se desdobram como uma expressão da sociedade em seu processo histórico e são essenciais para responder a necessidade da época. No campo da sociologia, Castells (1984 apud GOOTDIENER, 2010 p.124) afir No campo da sociologia, Castells (1984 apud GOOTDIENER, 2010 p.124) afirma: Acredito que estamos à beira de uma catástrofe socioespacial gigantesca, mas não, como dizem os ecologistas, por causa do processo de metropolização e devastação dos recursos naturais. É porque os novos interesses dominantes e as novas revoltas sociais tendem a dissociar o espaço das organizações e o espaço da experiência, O referido autor traz uma abordagem marxista do espaço, conceituando-o como um produto material de uma determinada formação social. Segundo Gottdiener (2010), a teoria de Castells é formada por dois pilares: a aplicação da teoria de Althusser e a teoria da produção do ambiente construído além da utilização do conceito de urbano como uma unidade espacial dentro do sistema de ambiente construído. Ainda de acordo com Gottdiener (2015, p. 124): O aspecto mais importante da mudança de pensamento de Castells é que, deixando de aplicar Althusser a uma teoria do espaço, ele veio a desenvolver um aspecto específico dessa teoria, ou seja, a relação entre o Estado e o espaço de assentamento. A teoria de Castells traz os meios de produção e a força de trabalho como processos do espaço urbano, segundo o autor o urbano é uma unidade espacial da reprodução da força de trabalho. Castells acredita que o espaço é um elemento indispensável para a análise da sociedade atual, como afirma: “ao contrário da maioria das teorias clássicas, que supõem o domínio do espaço pelo tempo, proponho a hipótese de que o espaço organiza o tempo na sociedade em rede.” (CASTELLS 2012, p. 467). Nesse contexto, é notório que o espaço tem uma relação de interação com a sociedade, as tecnologias e principalmente pelo tempo enquanto fator organizado pelo espaço. Assim como Castells (2000), Lefebvre (1976) traz uma abordagem marxista do espaço, diretamente ligada as transformações da sociedade conforme citação abaixo: Do espaço não se pode dizer que seja um produto como qualquer outro, um objeto ou uma soma de objetos, uma coisa ou uma coleção de coisas, uma mercadoria ou SEÇÃO LIVRE SEÇÃO LIVRE 167 um conjunto de mercadorias. Assim como o espaço, o tempo e a matéria delineiam e abrangem as qualidades essenciais do mundo físico, a espacialidade, a temporalidade e o ser social podem ser vistos como as dimensões abstratas que, em conjunto, abarcam todas as facetas da existência humana. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 De acordo com Soja (1993), a existência humana se constrói por intermédio do imaginário, simbolismo, signos e todas as práticas temporais e espaciais presentes na sociedade. Aqui o importante é sinalizar que o pensamento crítico não se ocupa mais de representações apenas baseadas no aspecto empírico e na experimentação. Trata-se, para além disso, de expressar a vivência dos grupos sociais. Das práticas cotidianas, das iconografias e diferentes linguagens expressa no espaço. Revela, do mesmo modo, as necessidades (individuais e sociais), os desejos relacionados ao corpo e à memória (ou o inconsciente). Trata-se do espaço como forma de pensar, como expressão da consciência coletiva e, quando necessário, como transgressão às representações dominantes. É o lócus da ação e, de origem simbólico, tem na cotidianidade sua materialidade e no poder a sua maior expressão. Para Tuan (2013, p. 15): “[...] poucas obras tentam compreender o que as pessoas sentem sobre espaço e lugar, considerar as diferentes maneiras de experienciar (sensório-motora, tátil, visual, conceitual) e interpretar espaço e lugar como imagens de sentimentos complexos [...]”. Nesse sentido, entende-se que todo sujeito citadino tem uma ligação com a sua cidade, no que tange a memórias afetivas, experiências, signos, o que podemos chamar de relação de representação. De acordo com Lefebvre (1974), é na espacialidade que se percebe a total imbricação do percebido, concebido e vivido, além de transparecer que os fenômenos socioespaciais são tanto agentes quanto produtos da reprodução do espaço. Lefebvre (1974), deduz que o espaço traduz um conjunto de diferenças, ou seja, é o lócus de coexistência da pluralidade e das simultaneidades de padrões, de maneiras de viver a vida urbana. Contudo, não descarta a idéia de que o espaço também é o lugar dos conflitos, onde a exploração subordina não apenas a classe operária como outras classes sociais. Para tal, o espaço percebido refere-se as práticas espaciais, a materialidade dos objetos na relação com o sujeito, já o espaço concebido são as representações do espaço e seus signos concebidas pelos agentes. No que tange ao espaço vivido, trata-se do espaço representacional, as chamadas invenções mentais, o imaginário individual e coletivo. SEÇÃO LIVRE O espaço tem um aspecto perceptível que pode ser apreendido por meio dos sentidos. Em se tratando do espaço concebido, vê-se que ele está intrinsicamente ligado ao pensamento e não pode ser apreendido enquanto tal sem ter sido concebido previamente. Ele é a representação do espaço, ações ligadas ao conhecimento e ao pensamento. No que tange ao espaço vivido, por sua vez, pode-se dizer que ele é o espaço representacional. É a experiência vivida do espaço. Ele é o conceito que expressa a cotidianidade da vida humana, é a vivência que não se consegue definir apenas teoricamente (SCHMIDT, 2012). O espaço se tornou dinâmico e vivificado como local da produção e reprodução social desenvolvida cotidianamente pelos atores sociais em seus modos de apropriação do espaço de forma tanto individual quanto coletiva envolvendo espaço e tempo. Espaço e tempo são conceitos essenciais da vivência humana, que estão interligados no cotidiano social. Segundo Soja (1993, p.34): Assim como o espaço, o tempo e a matéria delineiam e abrangem as qualidades essenciais do mundo físico, a espacialidade, a temporalidade e o ser social podem ser vistos como as dimensões abstratas que, em conjunto, abarcam todas as facetas da existência humana. 168 2 ESPAÇO E REPRESENTAÇÃO: DIFERENTES CONTRIBUIÇÕES Uma contribuição significativa para a compreensão do espaço se dá a partir dos estudos de Santos (1998), através do estabelecimento do conceito de formação sócio-espacial e da concepção do espaço enquanto instância da sociedade: “o espaço organizado pelo homem é como as demais estruturas sociais, uma estrutura subordinada-subordinante. E como as outras instâncias, o espaço, embora, submetido a lei da totalidade, dispõe de uma certa autonomia” (SANTOS, 1988, p.145). É importante ressaltar que Santos (1996) apresenta o espaço enquanto fator social, organizado pelo homem e parte do processo de existência e reprodução social. O autor combate a ideia arcaica de espaço enquanto vazio, sem articulação, vácuo e traz uma análise critica da realidade baseado nas transformações da globalização que levaram a um estabelecimento de novas dinâmicas relativas ao espaço. Posteriormente, Santos (1996), em seu livro A Natureza do Espaço, traz uma nova conceituação para o espaço geográfico. Segundo o autor: “O espaço é formado por um conjunto indissociável, solidário e também contraditório, de sistemas de objetos e sistemas de ações, não considerados isoladamente, mas como o quadro único no qual a história se dá” (SANTOS, 2006, p.39). Para o autor, a natureza é que dá origem as coisas que posteriormente serão trabalhadas (transformadas) pelo homem através da técnica, por meio de uso de instrumentos e técnicas o homem constrói o espaço. E a partir da evolução das técnicas e relações sociais o espaço geográfico também sofre mudanças, o espaço seria então uma expressão da própria da sociedade. SEÇÃO LIVRE 169 Ainda sobre o conceito de espaço, o geográfo britânico David Harvey tece uma crítica sobre o que chama de falta de preocupação com a conceituação de espaço pelas ciências humanas, como pode ver a seguir: Marx, Marshall, Weber e Durkhein tinham isso em comum: davam prioridade ao tempo e à história e não ao espaço e à geografia, e, quando tratavam do espaço e da geografia, tendiam a considerá-los de modo não problemático, enquanto contexto ou sítio estável para a ação histórica. (HARVEY, 2006, p. 142). O autor enfatiza a necessidade e importância de se discutir o conceito de espaço enquanto teoria espacial de categoria de análise e investigação. Partindo do pressuposto de que para compreender a sociedade atual é necessário observar a relação entre tempo e espaço tomando cuidado para não priorizar um em detrimento ao outro. O espaço geográfico é formado pelo conjunto de populações, por suas obras, suas relações localizadas, pelo seu meio de vida [...]. Ele não pode ser confundido com os 2 ESPAÇO E REPRESENTAÇÃO: DIFERENTES CONTRIBUIÇÕES Além disso, Harvey (2006) faz uma abordagem tripartite sobre o espaço dividindo-o em espaço absoluto, relativo e relacional em uma teoria marxista que se soma a teoria lefebvriana da representação do espaço. Não existiria, por exemplo, nenhum valor no espaço-tempo relacional sem trabalhos concretos construídos em inumeráveis lugares no espaço e tempo absolutos. Nem o valor emergiria como um ‘poder imaterial mas objetivo’ sem os inumeráveis atos de troca, os processos de circulação contínuos que mantêm o conjunto do mercado mundial no espaço-tempo relativo. O valor é, portanto, uma relação social que internaliza toda a história e a geografia do trabalho concreto no mercado mundial; expressa as relações sociais (principalmente, mas não exclusivamente, de classe) construídas pelo capitalismo na escala mundial. (HARVEY, 2012, p. 26). De acordo com Harvey (2012), espaço absoluto é algo fixo onde acontecem planejamento de eventos ou registros, como por exemplo, cidades e condomínios fechados com espaços essencialmente cartesianos. Já o espaço relativo tem um caráter relacional de espaço-tempo inspirado nas contribuições de Albert Einstein. É o espaço do movimento, da circulação e dos fluxos. Por sua vez, o espaço relacional refere-se a relações internas, é o espaço dos sonhos, sendo inspirado em Leibniz. O autor também aborda questão da tensão dialética entre o material, concebido e vivido, articulando espaço e tempo e deixando claro o quanto esses espaços são interdependentes. Diante disso, é importante ressaltar a abordagem do espaço vivido enquanto local de produção de sensações e emoções, tendo como força analítica a tensão dialética e a interação. Espaço e tempo são conceitos essenciais da vivência humana, que estão interligados no cotidiano social. Segundo Soja (1993, p.34): Assim como o espaço, o tempo e a matéria delineiam e abrangem as qualidades essenciais do mundo físico. A espacialidade, a temporalidade e o ser social podem ser vistos como as dimensões abstratas que, em conjunto, abarcam todas as facetas da existência humana. De acordo com Soja (1993), a existência humana se constrói por intermédio do imaginário, simbolismo, signos e todas as práticas temporais e espaciais presentes na sociedade. O geógrafo francês Roger Brunet, por sua vez, traz contribuições sobre o conceito de que se assemelham a de Milton Santos no que tange a interação do homem com a natureza para criação e produção do espaço. De acordo com Brunet (2001, p. 2 ESPAÇO E REPRESENTAÇÃO: DIFERENTES CONTRIBUIÇÕES 15): O espaço geográfico é formado pelo conjunto de populações, por suas obras, suas relações localizadas, pelo seu meio de vida [...]. Ele não pode ser confundido com os 170 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 objetos que o povoam [...] Ele nasce com o trabalho das sociedades e só tem um fim com ele. O autor afirma que o espaço enquanto superfície é imprescindível para análise geográfica uma vez que, por intermédio dela pode-se constatar fatores como localização e distância. Brunet foi o criador da coremática, um estudo teórico-metodológico que objetiva a representação da análise do espaço geográfico utilizando modelos gráficos, uma forma de expressão dinâmica. Segundo Brunet (2001), os homens criam Coremas ao transformarem o espaço mediante as suas relações de trabalho. Tais estruturas se materializam por uma expressão cartográfica, que o autor as considera como abstrações ou um sistema de signos. Para entendimento dos espaços de representação será tomada a definição de Lefebvre (1991) que trata da tríade que envolve o espaço percebido (práticas espaciais), o concebido (representações do espaço) e o vivido (espaço representacional) e diante disso inspira a análise sobre os sujeitos em relação a produção do espaço, como pode-se ver abaixo: A tríade percebido-concebido-vivido (em termos espaciais: práticas espaciais, representações de espaço, espaço representacional) perde toda a sua força quando é tratada como um modelo “abstrato”. [...] ela (a tríade) tem sua fonte na história de um povo, assim como na história de cada indivíduo que pertence àquele povo. [...] Compreende os loci das paixões, das ações e das situações vividas e, portanto, implica em tempo. Consequentemente pode ser [...] direcional, situacional ou relacional, porque é essencialmente qualitativa, fluida e dinâmica (LEFEBVRE, 1991, p. 40;42). Para melhor exemplificação dessa relação, será utilizada a Figura 1: Para melhor exemplificação dessa relação, será utilizada a Figura 1: Para melhor exemplificação dessa relação, será utilizada a Figura 1: Para melhor exemplificação dessa relação, será utilizada a Figura 1: Figura 1 – Tripé sobre a produção do espaço Fonte: Cardoso e Cristiane, 2009 Conforme figura acima, a análise tridimensional do espaço percebido, concebido e vivido constitui a autoprodução da sociedade. SEÇÃO LIVRE SEÇÃO LIVRE 171 compostos de subjetividade, individualidade e, concomitantemente, de uma conjuntura social como processos ativos. Partindo desse pressuposto e consoante os postulados de Schmidt (2012), serão conceituados esses espaços em suas particularidades: o espaço percebido é relativo as práticas espaciais e pode ser visto por meio dos cinco sentidos estabelecidos pela ciência. Está ligado aos elementos, aquilo que é material, sendo que o espaço tem um aspecto perceptível que pode ser apreendido por meio dos sentidos. Ainda de acordo com Schmidt (2012), em se tratando do espaço concebido: vê-se que ele está intrinsicamente ligado ao pensamento. O espaço não pode ser percebido enquanto tal sem ter sido concebido previamente em pensamento, pois ele é a representação do espaço, de ações ligadas ao conhecimento. No que tange ao espaço vivido, pode-se dizer que ele é o espaço representacional, é a experiência vivida do espaço. Ele é o conceito que expressa a cotidianidade da vida humana, é a vivência que não se consegue definir apenas teoricamente. Concomitantemente a essa proposta, Lefebvre (1983) com sua tríade do espaço de representação, trata o aspecto da representação para além da dominação, como forma de quebrar certos paradigmas Aqui, o importante é sinalizar que o pensamento crítico não se ocupa mais de representações meio-mortas em estado de sobrevivência, entretanto que deveria surpreendê- las em sua vida, dentro da vivência e inclusive ao nascer, quando surge. De onde? Da prática. Através de que? Dos grupos sociais e da linguagem ou, mas bem das línguas, mas também das necessidades (individuais e sociais) dos desejos, do chamado ao corpo, e da memória: o “inconsciente” (LEFEBVRE, 1983). A representação é entendida, portanto, como forma de pensar, como expressão da consciência coletiva, como o lócus da ação de origem simbólico que tem na cotidianidade sua materialidade. Todo sujeito tem uma ligação como espaço em que vive, no que tange a memórias afetivas, experiências, signos, o que se pode chamar de relação de representação. Para tal, o espaço percebido refere-se as práticas espaciais, a materialidade dos objetos na relação com o sujeito, já o espaço concebido são as representações do espaço e seus signos concebidas pelos agentes conceituais, no que tange ao espaço vivido trata-se do espaço representacional, as chamadas invenções mentais, o imaginário individual e coletivo. Assim, é importante pensar sobre como esse espaço é produzido, de que forma se dão essas relações. 2 ESPAÇO E REPRESENTAÇÃO: DIFERENTES CONTRIBUIÇÕES É perceptível que esses conceitos são Figura 1 – Tripé sobre a produção do espaço Figura 1 – Tripé sobre a produção do espaço Fonte: Cardoso e Cristiane, 2009 Conforme figura acima, a análise tridimensional do espaço percebido, concebido e vivido constitui a autoprodução da sociedade. É perceptível que esses conceitos são Conforme figura acima, a análise tridimensional do espaço percebido, concebido e vivido constitui a autoprodução da sociedade. É perceptível que esses conceitos são NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Por fim, conclui-se que os espaços de representação são importantes para compreender o cotidiano humano, uma vez que envolvem as dimensões física, mental e social dos sujeitos. Partindo dessa premissa, é notório destacar a força e a importância dessa dimensão espacial para a Geografia diante do fenômeno da globalização, é necessário pensar em modelos para melhor entendimento do conceito, além de sobrepor limitações na sua interpretação. 3 CONSIDERAÇÕES FINAIS Abordar o espaço geográfico segundo a corrente da Geografia Crítica é um desafio devido as diversas abordagens epistemológicas realizadas sobre o conceito. Nesses termos, é fato que o espaço geográfico é um conceito de suma importância para a Geografia, isso explica as inúmeras teorias sobre essa categoria analítica. Partindo desse pressuposto, conclui- se ser de extrema importância entender as implicações do espaço na sociedade, uma vez que ele é produto das relações sociais. Ademais, reconhecer a espacialização na sociedade atual é entender todo um modo de vida e a ação dos atores sociais cotidianamente de forma muito diversificada, uma vez que o espaço se transforma conforme as mudanças na sociedade. Ele é produzido e reproduzido por ela em todas as suas nuances, tendo como foco as relações de trabalho. Esse estudo mostra o espaço em um contexto de uma sociedade de classes subordinada a um sistema capitalista e revela o quanto as ações dessa sociedade dinamizam o espaço de forma a modificá-lo conforme suas ações. Não se tratam, portanto, de ações naturais, mas daquelas que são fruto de conflitos. Conclui-se, portanto, que o espaço geográfico é um conceito complexo e dinâmico e que deve ser amplamente discutido objetivando o avanço da ciência geográfica frente as necessidades da sociedade. BRUNET, Roger. Le déchiffrement du monde: théorie et pratique de la géographie. Paris: Belin, 2001 [1990]. BRUNET, Roger. Le déchiffrement du monde: théorie et pratique de la géographie. Paris: Belin, 2001 [1990]. ARLOS, Ana Fani Alessandri. A condição espacial. São Paulo: Contexto, 2011. 157 p. CASTELLS, Manuel. A sociedade em rede. São Paulo: Editora Paz e Terra, 2000. CORRÊA, Roberto Lobato. Geografia brasileira: crise e renovação. In: MOREIRA,Ruy (org). Geografia: teoria e crítica. O saber posto em questão. Petrópolis: Vozes,1982. GOTTDIENER, Mark. A produção social do espaço urbano. 2.ed. São Paulo: Edusp, 2010. SEÇÃO LIVRE Segundo a teoria de Lefebvre (1984) acontece de três maneiras: prática espacial, representações do espaço e espaços de representação, diante disso, Lefebvre (1984) faz uma análise tridimensional do espaço em sua obra como podemos ver a seguir: A prática espacial, que engloba produção e reprodução, lugares especificados e conjuntos espaciais próprios de cada formação social, que assegura a continuidade numa relativa coesão. Essa coesão implica, no que concerne ao espaço social e a relação de cada membro de determinada sociedade ao seu espaço, ao mesmo tempo de uma competência certa e uma certa performance; as representações do espaço, ligadas às relações de produção, a “ordem” que elas impõem e, desse modo, ligadas ao conhecimentos, aos signos, aos códigos, às relações “frontais”; os espaços de representação, apresentam (com ou sem códigos) simbolismos complexos, ligados ao lado clandestino e subterrâneo da vida social, mas também a arte que eventualmente poder-se-ia definir não como código do espaço, mas como código de representação (LEFEBVRE, 1984,p.36) Os espaços de representação estão imbricados a vida cotidiana dos sujeitos, diante disso, para compreensão dessa questão é necessário compreender as relações de espaço e tempo que compõem essa produção social, e tal espaço social é a expressão das normas sociais, costumes, valores construídos em um determinado ambiente. Para Lefebvre (1984), o espaço é parte do processo e produto das relações sociais, fazendo parte dos meios e forças de produção. 172 TTDIENER, Mark. A produção social do espaço urbano. 2.ed. São Paulo: Edusp, 2010 HARVEY, David. O espaço como palavra chave. Revista GEOgraphia. V. 14, n. 28, 2012, p. 8 – 39, 2012. Original: HARVEY, D. Space as a keyword. In: CASTREE, N. e GREGORY, D. (Orgs.) David Harvey: a critical reader. Malden e Oxford: Blackwell, 2006. Tradução livre: Letícia Gianella. Revisão técnica: Rogério Haesbaert e Juliana Nunes. HARVEY, David. A produção capitalista do espaço. 2. ed. São Paulo: Annablume, 2006. ARVEY, David. A produção capitalista do espaço. 2. ed. São Paulo: Annablume, 200 LACOSTE, Yves. A Geografia isso serve, em primeiro lugar, para fazer a guerra. Trad. M. C. França. 4. ed. Campinas: Papirus, 1997. LACOSTE,Yves. Paysages politiques. Paris:Biblio Essais,1990. SEÇÃO LIVRE 173 LEFÉBVRE, Henri. A produção do espaço (1ª ed. 1974) Paris, Anthropos, 1984. LEFÉBVRE, Henri. Espaço y política. (1ª ed. 1973) Barcelona. Ediciones Peninsula, 1976 LEFEBVRE, Henri. La presencia y la ausência: contribucion a la teoria de las representaciones. México, DF: Fondo de Cultura Económica, 1983 LEFEBVRE, Henri. La production de l’espace. Paris: Ed anthropos, 1974. LEFEBVRE, Henri. O direito à cidade. 1° ed. São Paulo: Moraes, 1991. MOREIRA, Ruy. O que é geografia. 5. ed. São Paulo: Brasiliense, 1985. SANTOS, M. Metamorfoses do Espaço Habitado: fundamentos teóricos e metodológicos da geografia. 6ª ed. São Paulo: Edusp, 2012. SANTOS, Milton. Por Uma Geografia Nova. São Paulo: Hucitec, 1988. SANTOS, Milton. Espaço e método. (1ª ed. 1985) 5ª Ed. EDUSP, São Paulo, 2008. SANTOS, Milton. Técnica, Espaço, Tempo. (1ª ed. 1994) 5ª Ed. EDUSP, São Paulo, 2008. SANTOS, Milton. A Natureza do Espaço. Técnica e Tempo. Razão e Emoção. 2º Edição. São Paulo: Hucitec, l996. SCHMIDT, Christian. A teoria da produção do espaço de Henri Lefebvre: em direção a uma dialética tridimensional. Trad. Marta Inez Medeiros Marques; Marcelo Barreto. GEOUSP – espaço e tempo, São Paulo, n. 32, p. 89-109, 2012 SOJA, Edward Willian. Geografias pós-modernas: a reafirmação do espaço na teoria social crítica. Rio de Janeiro: Jorge Zahar 1993.323 p. SOJA, Edward Willian. Le développement des territoires: formes, lois, aménagement. La Tour D’Aigues, 2004. SOUZA, Marcelo Lopes de. Por uma Geografia nova. São Paulo: Hucitec-Edusp, 1978. NÚMERO 01 – – CAMPUS BRAGANÇA VOLUME VOLUME VOLUME REVISTA DO PROGRAMA DE PÓS GRADUAÇÃO EM LINGUAGENS E SABERES NA AMAZÔNIA OUZA, Marcelo Lopes de. Por uma Geografia nova. São Paulo: Hucitec-Edusp, 1978. SOUZA, Marcelo Lopes de. Os conceitos fundamentais da pesquisa sócio-espacial. Rio de Janeiro: Bertrand Brasil, 2013. TUAN,Yi-fu. Espaço e lugar: a perspectiva da experiência. Tradução: Lívia de Oliveira. Londrina: Eduel, 2013. SEÇÃO LIVRE SEÇÃO LIVRE 175 – NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 176 um ser biológico exclusivo com suas peculiaridades. Na nossa pesquisa, o objeto de estudo está relacionado à velhice dos homens (OLIVEIRA, et al, 2011). um ser biológico exclusivo com suas peculiaridades. Na nossa pesquisa, o objeto de estudo está relacionado à velhice dos homens (OLIVEIRA, et al, 2011). Numa análise sociológica, a velhice humana apresenta-se de forma ambígua, ou seja, é vista como sinônimo de sabedoria e prestígio, mas também como fase de decrepitude e uma fonte de sofrimentos, evidenciada como um momento de felicidade para alguns e de tristeza para outros. Esta dualidade do próprio conceito do que é a velhice na sociedade humana se dá de forma diferente em cada ser, logo, concluímos que o envelhecimento se reificará no indivíduo conforme os seus hábitos e costumes particulares no decorrer de sua existência e, dependendo principalmente do seu estado de saúde, do seu modo de viver a vida, poderá ser algo positivo ou negativo no seu existir, o que dependerá em parte do próprio indivíduo em suas escolhas, enquanto ser vivo, participante no e do mundo físico (CÍCERO, 1999; 2006). q p p Desta forma, o animal racional, em sua formação humana, tem um tempo de vida neste mundo, com início, meio e fim, o que podemos resumir em duas etapas: a primeira é o nascimento e a segunda a morte. A formação humana se dá nas quatro fases do desenvolvimento: na infância (do nascimento até os 11 anos de idade), em seguida, na adolescência (que vai dos 12 aos 20 anos de idade), posteriormente, na fase adulta (a partir dos 21 anos de idade) e, finalmente, na velhice, fase considerada, nos países em desenvolvimento, a partir dos 60 anos e nos desenvolvidos, iniciando aos 65 anos. Em outras palavras, vivemos uma vida biológica constituída de quatro fases distintas, com suas características físicas e/ou psicológicas diferentes, sendo elas: a infância, em seguida a adolescência, a idade adulta e a velhice. Como podemos perceber o homem, desde o seu nascimento até a sua morte, é um ser inserido em um processo de formação do caráter humano, de sua identidade cultural e social. Portanto, o envelhecimento humano é universal e inevitável, processo que é vivido e sentido de formas diferentes por cada ser vivo. A velhice é intrínseca à própria natureza humana, e todo e qualquer ser humano logo se deparará com esta fase da vida. Conseguir atravessar a infância, a juventude, a fase adulta e assim chegar à maturidade é muito relevante, entretanto, a grande maioria não se preocupa e nem se prepara para a chegada da velhice e, sobretudo, para viver um envelhecimento ativo e saudável. Nas civilizações antigas, havia uma preocupação com a velhice e com o rejuvenescimento. Essa apreensão persiste até a contemporaneidade, todavia, apesar do mundo ter evoluído cientificamente e tecnologicamente, ainda não se encontrou a “fórmula milagrosa”, ou a tão sonhada “fonte da juventude” que é vislumbrada em diversos filmes hollywoodianos. Portanto, não há como determos a velhice. Precisamos entender que é um processo natural e biológico da criação dos seres vivos, em especial, dos humanos, que devem aceitá-la e prepararem-se para um envelhecimento ativo e digno na sociedade, haja visto que este processo humano é inevitável. 2 O FILÓSOFO CONFÚCIO E A VALORIZAÇÃO DA VELHICE HUMANA NA CHINA. Na China, também, há uma filosofia com base nos ensinamentos do Mestre Confúcio que valoriza os velhos e ensina que todas as famílias deveriam obedecer aos idosos, manifestando respeito pelas gerações mais velhas. Confúcio (551 - 479 a.C.) era um filósofo da civilização oriental, considerado, por muitos, um homem sábio, com enorme conhecimento sobre a alma humana e que construiu conceitos filosóficos relacionados à moral, à ética e à sabedoria. O Confucionismo não pode ser confundido com religião, haja visto que se trata de uma organização nacionalista da sociedade, respaldada no princípio da simpatia universal, que deve ser atingida pela educação e prolongando-se do ser humano à família e da família ao Estado, sendo este último a maior família (CONFÚCIO, 1999). Nesta perspectiva, os ensinamentos de Confúcio, os Lun Yú, vulgarmente conhecidos por "Anacletos" foram compilados pelos seus discípulos após a sua morte. O objetivo essencial da vida humana é, segundo Confúcio, a perfeição, partindo do desenvolvimento da natureza individual e atingindo a criação de Estado bem ordenado e um mundo pacífico. Esta ideia é o "jen", a palavra que significa "benevolência", "bondade", "verdadeira humanidade". A humanidade é o pilar central do Confucionismo, no qual se acredita que todos os humanos são naturalmente bons, sendo a educação o fator principal que irá determinar a condição humana. Assim, o Confucionismo trabalha uma filosofia, enquanto doutrina que conciliará a natureza humana com teorias políticas e sociais, visando o bem viver na sociedade. Na estrutura da Filosofia de Confúcio, a família é a base e todos os membros do grupo familiar devem obediência ao ser humano masculino mais velho. Desta forma, a autoridade do patriarca mantém-se com o passar dos anos e até mesmo a mulher, que sempre foi subordinada, em sua velhice, ganha poderes maiores do que os jovens masculinos, exercendo grande influência na educação dos netos. Confúcio (1999) percebia os velhos como pessoas com grande autoridade e sabedoria e declarava que aos 60 (sessenta) anos o ser humano passa a compreender sem a necessidade de refletir tudo o que ouve e, a partir dos 70 (setenta) anos, o indivíduo segue os desejos de seu coração, sem violar regra alguma. SEÇÃO LIVRE SEÇÃO LIVRE 177 verdadeiro seria Li Er ou Lao Dan, mas ele se fez conhecido mundialmente através da sua obra intitulada “Da Razão Suprema e da Virtude, o Tao te King”, livro escrito entre 350 e 250 a. C. e que faz parte da literatura chinesa (TAO TE CHING, 2011; LAO TZU, 2014). Para Lao-Tsé, a velhice era entendida como uma vivência, um momento supremo de alcance espiritual máximo e que o ser humano, ao chegar à idade de 60 anos, estaria próximo do momento de liberta-se do seu corpo físico, para viver o êxtase da sua passagem à santidade (a dimensão espiritual). De acordo com o Filósofo Lao-Tsé, a velhice é reconhecida apenas no outro indivíduo humano e não em quem a vivencia, o detentor do corpo que envelhece, conectado na dimensão temporal da existência que se vislumbra a cada instante, de forma renovada, vive uma transformação, visando novos limites, no sentido de adentrar com a alma pura no mundo metafísico (da santidade), o que seria uma evolução transcendental nesta etapa da vida (TAO TE CHING, 2011; LAO TZU, 2014). 1 LAO-TSÉ (604 – 531 A. C.), O “VELHO MESTRE” E A VELHICE. Para aprofundarmos a temática do envelhecimento humano, verificamos, por meio de pesquisas, que na China, desde a antiguidade até a contemporaneidade, velhice é sinônimo de sabedoria e respeito à família, segurança dos anciãos, o que evidencia uma educação e cultura milenar (tradição) de cuidar bem de seus velhos que são considerados como pessoas de imenso valor na sociedade chinesa. Dessa forma, na antiga China, destacamos alguns filósofos que conceituaram a velhice, dentre os quais Lao-Tsé (604 - 531 a. C.) que foi o fundador do Taoísmo, doutrina mística e filosófica (formulada no séc. VI a.C.) que enfatiza a vida em harmonia com o Tao (termo chinês que significa "caminho", "via" ou "princípio"), o todo e único. Pouco se conhece sobre a história da vida do Filósofo chinês Lao-Tsé ou Lao-Tzy, nome que, em sua tradução mais adequada, significa “ancião”, “velho sábio”, “grande senhor” ou “velho mestre”, porém seu nome 3 A VELHICE NA SOCIEDADE JAPONESA No Japão, da antiguidade até a modernidade, a velhice sempre foi e continua sendo vista, assim como na China, sinônimo de sabedoria e respeito aos velhos. Estes são vistos, de fato, como pessoas de grande autoridade na sociedade japonesa. Dessa forma, a velhice e o envelhecimento humano, para os japoneses, são fenômenos naturais e inerentes à nossa espécie, que devem ser avaliados, cuidados e estudados com muita atenção pelos governantes, ressaltando que as pessoas idosas devem ser tratadas com carinho, amor, respeito e dignidade, devido à vasta experiência acumulada em suas existências. A família é considerada como referência fundamental no cuidado dos velhos, haja vista ser o porto seguro do idoso, sendo os familiares: os filhos, os netos e os mais jovens, aqueles que irão ser educados para cuidar bem de seus pais e avós, o que significa, ainda, tratar deles próprios, já que mais tarde serão velhos também. Os jovens japoneses são inseridos pela família em um processo de ensino-aprendizagem construído nos lares que os educa para viverem e demonstrarem sempre alegria, fraternidade, festa e plenitude pela presença dos velhos em seu meio familiar e social (CONFÚCIO, 1999). A Sociedade Japonesa, particularmente, apresenta uma educação de dignidade e respeito, uma cultura milenar, passada de geração a geração, no sentido de cuidar bem, glorificar e cultuar seus velhos, o que sem dúvida, é algo muito valioso e de prazer para as pessoas da chamada terceira idade. Destaca-se que os japoneses antes de tomarem qualquer decisão acerca de determinadas situações, aconselham-se com os anciãos, por considerarem seus conselhos sábios e experientes e estes, também, têm grande influência e atuação nas decisões importantes nos grupos sociais em que fazem parte, inclusive, alterando as agendas e o destino da política de seu país (PESSANHA, 2006). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 178 em virtude da morte e posterior a ela, reverenciando-os, como culto aos antepassados, o que seria a única ligação dessa doutrina com o mundo espiritual. em virtude da morte e posterior a ela, reverenciando-os, como culto aos antepassados, o que seria a única ligação dessa doutrina com o mundo espiritual. Nesta perspectiva, o amor dos filhos aos pais envelhecidos é algo divino, especial, que compreende maior proteção e segurança na última fase da vida humana, o que se considera um dos mais admiráveis atos da raça humana consigo mesma e para com seus semelhantes, em destaque, com a sua geração e para com as futuras gerações (SANTOS, 2001). Para Confúcio (1999), o homem velho é um ser imerso no conhecimento e com grandes experiências vividas a serem repassadas aos mais jovens que lhes devem respeito. Assim, a velhice pressupõe autoridade que se justifica pela sabedoria presente nos velhos que têm muito a ensinar aos novos. 2 O FILÓSOFO CONFÚCIO E A VALORIZAÇÃO DA VELHICE HUMANA NA CHINA. Confúcio orientava a sociedade a prestigiar os velhos e a velhice e afirmava que o processo de envelhecimento do homem deveria ser com paz e felicidade, exigindo dos mais jovens, o amor e o respeito para com os anciãos, os antigos, os seus pais e avós no seio familiar e na sociedade. Confúcio (1999) dizia que no mundo não há nada maior do que a criação humana e, no ser humano, nada é maior que a piedade filial. A filosofia Confucionista afirma que os filhos têm deveres e obrigações perante seus pais, o que pressupõe: amá-los, cuidá-los com carinho e atenção durante a vida inteira, visando torná-los pessoas felizes, de diversas formas e em todos os momentos em suas existências no bojo familiar e, ainda, demonstrarem saudade e dó deles, SEÇÃO LIVRE 179 SEÇÃO LIVRE pelo poeta e filósofo Ptah-Hotep que evidenciou o envelhecimento como um estado de dependência dos idosos em relação aos jovens, e afirmou: Como é penoso o fim de um velho! Ele se enfraquece a cada dia; sua vista cansa, seus ouvidos tornam-se surdos; sua força declina; seu coração não tem mais repouso; sua boca torna-se silenciosa e não fala mais. Suas faculdades intelectuais diminuem, e lhe é impossível lembrar-se hoje do que aconteceu ontem. Todos os seus ossos doem. As ocupações que até recentemente causavam prazer só se realizam com dificuldade, e o sentido do paladar desaparece. A velhice é o pior dos infortúnios que pode afligir um homem. O nariz entope, e não se pode mais sentir nenhum odor (BEAUVOIR, 2018, p. 97). Verificamos que Ptah-Hotep sinaliza e considera que a velhice é uma fase cruel da vida do ser humano, a qual o indivíduo enfrentará, quando chegar nela, haja vista que passará por momentos de dependência, dificuldades físicas, biológicas e mentais, terá diversas enfermidades e não terá boas condições e saúde. A visão deste filósofo não é muito agradável aos nossos olhos e pode até ser vista de forma preconceituosa, já que, em sua época, se privilegiava a beleza, o vigor físico e a juventude. Porém, não podemos negar em nossa contemporaneidade a velhice, fenômeno do envelhecimento que é uma realidade concreta em todos os continentes e em evolução constante no mundo. Desta forma, em todas as sociedades, o número de pessoas velhas com sessenta ou mais anos de idade cresce rapidamente, ano após ano, conforme estatísticas. Percebe-se que há um grande contingente de velhos na modernidade que vivem em condições semelhantes às vislumbradas no texto do poeta e que a velhice organicamente representa declínio, mas que ocorre de formas diferentes nos indivíduos e de acordo com a condição social e econômica nas quais se encontram, sendo a pobreza extrema (vivenciada por grande parte dos velhos) um dos fatores determinantes para um processo de envelhecimento penoso, não saudável, não ativo e temido pela humanidade. Na Antiga Grécia, dos anos de 4.000 a 1.000 a.C., o país de relevo montanhoso e com planícies entre as montanhas, com diversas ilhas e de clima árido, as cidades do mundo grego se desenvolviam, entretanto, duas cidades-estados das mais importantes brigaram pela supremacia dessa civilização e destacaram-se neste período: Atenas e Esparta. 4 A VELHICE E O ENVELHECIMENTO NAS SOCIEDADES OCIDENTAIS A palavra Ocidente vem do latim occidens, que significa “pôr do sol”, oeste, que difere do oriente. O chamado “Mundo Ocidental”, “Civilização Ocidental” ou “Ocidente”, são nomenclaturas que abrangem um conceito geopolítico e cultural, associadas primeiramente à Europa Ocidental e que, posteriormente, se estendeu a outras regiões do mundo com o processo de colonização, onde essas áreas têm uma população representativa de ascendência europeia, de forma que suas culturas e sociedades são fortemente influenciadas pela Europa. Nesta perspectiva, na contemporaneidade, o mundo ocidental inclui a maior parte da Europa, além de muitos países de origem colonial europeia nas Américas e na Oceania, como Estados Unidos, Canadá, México, Argentina, Brasil, Chile, Austrália, Nova Zelândia, entre outros. Beauvoir (2018), em seu livro “A Velhice”, destaca que, na civilização ocidental, por volta do ano 2.500 a. C., a beleza e o vigor físico eram atributos humanos poetizados e aclamados, mas foi nesse período que surgiu o primeiro texto sobre a velhice, escrito no Egito, NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 180 saiu vitoriosa, o que coincidiu com o declínio de Atenas. Todavia, Atenas ofertou ao mundo a Filosofia, a Democracia e outros conhecimentos, deixando-nos um grande legado. Atenas foi considerada o centro cultural e intelectual do Mundo Ocidental e, ainda, nos presenteou com os grandes filósofos, dentre eles: Sócrates, Platão e Aristóteles (PLATÃO, 1985). saiu vitoriosa, o que coincidiu com o declínio de Atenas. Todavia, Atenas ofertou ao mundo a Filosofia, a Democracia e outros conhecimentos, deixando-nos um grande legado. Atenas foi considerada o centro cultural e intelectual do Mundo Ocidental e, ainda, nos presenteou com os grandes filósofos, dentre eles: Sócrates, Platão e Aristóteles (PLATÃO, 1985). A história do mundo grego evidencia a diversidade cultural entre as cidades-estados da Grécia Antiga, bem como, a política, a filosofia e a educação no bojo das cidades, a luta pela conquista de territórios, poder e direitos, em uma civilização que se tornou modelo para outras sociedades na modernidade. Entretanto, naquela época, a sociedade vivia com seus preconceitos em relação à velhice e cultuava a juventude, o corpo e a beleza. A senescência, ou seja, o processo do envelhecimento era algo detestável, deplorável, não desejável e pouco abordado na sociedade grega, o que não é tão diferente da nossa contemporaneidade, apesar da evolução da Ciência, da Medicina e da Tecnologia, em especial, da Gerontologia e da Geriatria. A Gerontologia é a área da saúde multidisciplinar, onde diferentes profissionais da saúde desenvolvem estudos sobre o envelhecimento humano, levando em consideração as consequências físicas, emocionais, econômicas e sociais intrínsecas ao desenvolvimento da velhice no decorrer da vida e, deste modo, contribuindo para a prevenção e intervenção na saúde do idoso, com o objetivo de garantir uma melhor qualidade de vida para os velhos. A Geriatria é uma especialidade médica que estuda e trata das doenças ligadas ao envelhecimento. O médico geriatra é o profissional responsável pela promoção da saúde, prevenção e tratamento de enfermidades de pessoas que estão na chamada terceira idade. p Na Jônia voluptuosa e hedonista, Minermo, sacerdote em Cólofon, exprime, por volta de 630 a.C., os sentimentos de seus concidadãos; ele canta os prazeres, a juventude, o amor; e detesta a velhice: “Que vida, que prazeres, sem Afrodite de ouro?” Ele lamenta Títono: “Infeliz dele! Foi com um mal imortal que os deuses o atingiram!” Repete sem cessar que preferiria morrer a envelhecer: “Como as folhas que a estação Segundo Homero, entretanto, a velhice está associada à sabedoria, e é encarnada em Nestor, o conselheiro supremo; o tempo lhe conferiu a experiência, a arte da palavra, a autoridade. Entretanto, ele aparece como fisicamente enfraquecido. E não é ele quem assegura aos gregos à vitória. Só um homem na força da idade seria capaz de inventar um artificio mais eficaz do que todas as táticas tradicionais. [...]. Por outro lado, Homero zomba dos demogerontes de Troia. Ele evoca o “limiar maldito da velhice”. Num hino que lhe é atribuído, Afrodite diz: “Os deuses também odeiam a velhice.” (BEAUVOIR, 2018: p. 104). SEÇÃO LIVRE Estas tinham hábitos e costumes próprios, economias independentes e diferentes culturas, que geraram uma rivalidade entre essas cidades-estados, porém, formavam um povo que falava a mesma língua e que cultuava os mesmos deuses. Nesta perspectiva, a principal diferença entre Atenas e Esparta está no Modelo de Governo da cidade-estado, o que representa a diversidade cultural grega: Atenas consolidava a Democracia, enquanto Esparta efetivava uma Monarquia (Aristocracia). Em relação à educação dos jovens, tanto os atenienses como os espartanos possuíam a mesma preocupação, mas realizavam-na de forma diferente. Atenas priorizava na educação o estudo da filosofia, da matemática e outras áreas do conhecimento. O saber era vital para toda e qualquer ação. A educação ateniense refletia os anseios e valores daquela sociedade, que tinha como objetivo principal a formação de indivíduos completos, ou seja, com bom preparo físico, psicológico e cultural. Por volta dos sete anos de idade, o menino ateniense era orientado por um pedagogo. De outro modo, Esparta efetivava uma educação rudimentar nos seus colégios militares, onde se aprendia a ler e escrever o básico e o necessário para a vida cotidiana, haja vista que se priorizava o corpo, visando transformar os jovens em guerreiros para as batalhas. q p p j g p Atenas e Esparta travaram, entre os anos de 431 a 404 a.C., a Guerra do Peloponeso (conhecida como Guerra Mundial da Antiga Grécia) que foi detalhada em relatos de dois historiadores da Grécia Antiga: Xenofonte e Tucídides. Os espartanos deram suporte a um golpe oligárquico em Atenas, derrubando o Sistema Democrático e implantando um Sistema de Governo autoritário, conhecido como Tirania dos Trinta. Após muitos anos de batalhas, Esparta SEÇÃO LIVRE Já vimos: à atitude dos poetas diante dessa aventura individual que é, para eles, a velhice, opõem-se as ideologias que consideram esta última uma categoria social. É assim Sólon rejeita a ideia melancólica que Minermo faz da idade avançada. Ele lhe responde que é desejável viver até 80 anos: “Eu não cesso de aprender, enquanto avanço na minha velhice.” É que seu sistema de valores era bem diferente. A volúpia dos prazeres pouco contavam, para ele. Seu problema era político. Pretendia arbitrar entre os eupátridas e os tetas: na verdade, ele favoreceu a aristocracia. Como todos os conservadores, desejava apoiar-se nos anciãos e reservar a estes um grande espaço na construção da Pólis. (BEAUVOIR, 2018, p.106). Não há como negar que a velhice, tanto no passado, como no presente, em sua concreticidade, representa, para muitos, uma fase terrível da vida humana, que inclusive é negada e invisibilizada pela maioria da população e por grande parte dos governos conservadores. Geralmente, a velhice é vista nos outros, jamais em nós mesmos. Entretanto, outros conceitos e de forma positiva foram sendo criados, no decorrer do tempo e conforme o avanço da humanidade, por aqueles que enxergam a velhice e o envelhecimento como etapas da vida humana absolutamente normal, como processos aos quais temos que aceitá-los. Desse modo, adaptarmo-nos a esta nova fase do existir humano requer cuidados e mudanças de hábitos, em especial, no tratamento da nossa saúde e, além disso, em outras áreas: sociais, esportivas e educativas, nas quais a participação e inserção dos velhos são essenciais para o bem-estar social e para a melhoria da qualidade de vida deles. Atualmente, chamam-nos a atenção algumas áreas específicas da Medicina e da Educação, cujo foco está relacionado à preparação das gerações para a velhice, para a construção de um envelhecimento digno, participativo e ativo na sociedade: a Gerontologia, a Geriatria, as Universidades da Terceira Idade - UNATIs e a Universidade da Maturidade - UMA. O filósofo Platão (427 a.C. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Dessa forma, mesmo com todos esses avanços no que tange aos cuidados com os velhos, desde a Grécia Antiga até a modernidade, a questão social do envelhecimento ainda é pouco trabalhada, estudada e pesquisada nas sociedades capitalistas, haja vista que estas também cultuam a juventude, a beleza, o corpo escultural, o novo, em detrimento ao velho, e por considerarem os velhos como peças descartáveis no processo da produção e reprodução do capital, como se esses fossem engrenagens que não servem mais para a acumulação de riquezas, por não serem mais belos e por não produzirem, conforme as exigências e os desejos das grandes corporações ou empresas, ou seja, por serem vistos não mais produtivos para a enorme máquina do Capital globalizado. Assim, a velhice e o envelhecimento, tanto nas cidades da antiga Grécia, como nas cidades do atual mundo moderno colonizado e capitalizado, são vistos de forma idêntica: algo negativo, uma coisa ruim e que para muitos expressam terror, como se a velhice não fosse atingir a todos. Por outro lado, existem outras visões de caráter positivo, exemplificados a seguir: A filósofa, intelectual, ativista e professora Simone Beauvoir (1908 – 1986) em sua obra “A velhice”, descreve o fenômeno social do envelhecimento de forma profunda. A escritora avalia e sinaliza diversos conceitos de importantes pensadores e poetas daquele período na Antiga Grécia, onde a velhice era vista dialeticamente de forma ambivalente, isto é, tanto positiva como negativa: SEÇÃO LIVRE SEÇÃO LIVRE 181 das flores faz brotar sob os raios do sol, durante um fugidio instante gozamos a flor de nossa juventude, e logo as negras Parcas nos cercam, uma trazendo a dolorosa velhice e a outra a morte. O fruto da juventude não tardou a apodrecer: mal dura o tempo da luz do dia. (BEAUVOIR, 2018, p. 105). Como Minermo, Anacreonte, também ele originário da Jônia, cantou, no Século VI, o amor, o prazer, o vinho, as mulheres; envelhecer é perder tudo o que fazia a doçura de viver; ele descreve com dor o reflexo que seu espelho lhe devolve: cabelos fanados, têmporas grisalhas, dentes estragados, e se lamenta da morte próxima. [...]. Já vimos: à atitude dos poetas diante dessa aventura individual que é, para eles, a velhice, opõem-se as ideologias que consideram esta última uma categoria social. É assim Sólon rejeita a ideia melancólica que Minermo faz da idade avançada. Ele lhe responde que é desejável viver até 80 anos: “Eu não cesso de aprender, enquanto avanço na minha velhice.” É que seu sistema de valores era bem diferente. A volúpia dos prazeres pouco contavam, para ele. Seu problema era político. Pretendia arbitrar entre os eupátridas e os tetas: na verdade, ele favoreceu a aristocracia. Como todos os conservadores, desejava apoiar-se nos anciãos e reservar a estes um grande espaço na construção da Pólis. (BEAUVOIR, 2018, p.106). das flores faz brotar sob os raios do sol, durante um fugidio instante gozamos a flor de nossa juventude, e logo as negras Parcas nos cercam, uma trazendo a dolorosa velhice e a outra a morte. O fruto da juventude não tardou a apodrecer: mal dura o tempo da luz do dia. (BEAUVOIR, 2018, p. 105). das flores faz brotar sob os raios do sol, durante um fugidio instante gozamos a flor de nossa juventude, e logo as negras Parcas nos cercam, uma trazendo a dolorosa velhice e a outra a morte. O fruto da juventude não tardou a apodrecer: mal dura o tempo da luz do dia. (BEAUVOIR, 2018, p. 105). Como Minermo, Anacreonte, também ele originário da Jônia, cantou, no Século VI, o amor, o prazer, o vinho, as mulheres; envelhecer é perder tudo o que fazia a doçura de viver; ele descreve com dor o reflexo que seu espelho lhe devolve: cabelos fanados, têmporas grisalhas, dentes estragados, e se lamenta da morte próxima. [...]. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 182 de forma feliz e harmoniosa na família e diante da sociedade, o que significa, parafraseando o filósofo Platão, viver na paz e na tranquilidade, longe das coisas ruins do mundo. de forma feliz e harmoniosa na família e diante da sociedade, o que significa, parafraseando o filósofo Platão, viver na paz e na tranquilidade, longe das coisas ruins do mundo. Sócrates (469 a.C. – Atenas, 399 a.C.) ensina-nos com a sua filosofia educativa, sendo homens ou mulheres, que devemos vivenciar uma conduta virtuosa e honesta na sociedade, buscando sempre o Conhecimento, a Cultura e a Educação, para atuarmos no cotidiano problemático dos espaços terrestres, nas relações sociais de poder e de conflitos, agindo com sabedoria e prudência e estando-nos bem preparados para as situações adversas do existir humano. Desse modo, para essas pessoas que se encontram num nível elevado de consciência espiritual, política e educacional, o envelhecimento não seria problema algum, apenas um estágio da vida no qual temos que, desde a juventude, prepararmo-nos para vivermos no futuro, uma velhice de forma feliz e sadia, em um processo de envelhecimento ativo e participativo na sociedade (PLATÃO, 1985; BEAUVOIR, 1990). Em “A República”, destacamos que Platão faz referência a um diálogo de Sócrates com Céfalo, tendo como tema a velhice: na ocasião Sócrates é acompanhado por diversos jovens que o admiravam e um deles, Polemarco, convida-o para ir à casa de seu pai Céfalo. Ao chegarem à casa, Céfalo fica surpreso com a presença de Sócrates e pede desculpas por não o visitar, devido a sua velhice e por quase não sair de sua casa. Céfalo convida Sócrates para visitá-lo e afirma a seu amigo que “quanto mais os prazeres do corpo emurchecem, tanto mais crescem o desejo e o prazer da conversação”. Sócrates aceitou o convite e respondeu que gostava de conversar com os velhos, pois acreditava que com os antigos estavam a resposta, ou as respostas, para a compreensão do envelhecimento, já que estes vivenciam um caminho no qual teremos que caminhar provavelmente no futuro. Sócrates questionava “o que é este caminho: será áspero e difícil, ou cômodo e fácil.” Sócrates questionou ao seu amigo Céfalo como estando velho, sente-se ao alcançar esta etapa da vida que os poetas denominam de “limiar da velhice”? SEÇÃO LIVRE – 347 a.C), que foi discípulo de Sócrates, a quem considerava o mais sábio e o mais justo dos homens, contempla a velhice em seu pensamento filosófico afirmando que o processo de envelhecimento é uma etapa da vida repleta de paz e tranquilidade, e em sua obra “A República”, Platão revela passagens nas quais o seu mestre Sócrates faz referências ao envelhecimento, citando “que para os seres humanos prudentes e bem preparados, a velhice não constitui peso algum.” Na modernidade do mundo capitalizado, percebemos que a velhice, fenômeno mundial do envelhecimento, está em evolução e tem sido cada vez mais abordado pelas mídias, em manchetes de jornais, programas televisivos e na Internet. A velhice também é um tema evidenciado na área educacional, em especial, nas Universidades, no ensino superior, onde diversos especialistas e estudiosos têm chamado a atenção, reivindicando das autoridades governamentais planejamento e efetivação de políticas públicas direcionadas à população idosa, o que tem contribuído significativamente para a melhoria da qualidade de vida dos velhos e para a realização de debates no âmbito da comunidade acadêmica e na sociedade, em destaque, a brasileira. Desta forma, entendemos que é fundamental e benéfico tanto para os governos, como principalmente, para os velhos e a sociedade, o desenvolvimento de projetos educacionais tendo como objeto de estudo o envelhecimento, no sentido de preparar as gerações do presente e as do futuro para um envelhecer ativo, saudável e com dignidade no bojo das cidades, para ainda aceitar a velhice como parte de um processo natural da vida, que deve ser vivido com sabedoria, SEÇÃO LIVRE SEÇÃO LIVRE 183 compreender o organismo humano, incluindo a personalidade na teoria dos quatro humores corporais (sangue, fleuma ou pituíta, bílis amarela e bílis negra) que, consoante às quantidades relativas presentes no corpo, levariam a estados de equilíbrio (eucrasia) ou de doença e dor (discrasia). Com Hipócrates, foram elaborados os primeiros procedimentos médicos direcionados para os velhos e cuidados com a saúde na velhice. Ele determinou normas assistenciais, no que tange à higiene corporal; recomendou atividade física e mental e alimentação adequada, bem como moderação nas ações do cotidiano, do existir humano, o que entendemos ser o nascimento da abordagem geriátrica na Medicina e da Gerontologia. Os gregos ofertaram ao mundo grandes conhecimentos e conceituaram o processo do envelhecimento a partir de diversas concepções. O pensamento aristotélico contribuiu para os estudos e avanços da Medicina em relação à velhice no período de transição da Antiguidade para a Idade Média (séculos II a V d. C.). Os ensinamentos medicinais de Hipócrates sobre o envelhecimento influenciaram o filósofo e médico romano Claudio Galeno, que expandiu a teoria dos humores, ligando-a a personalidade. Para ele, havia uma relação direta entre os níveis de humores no corpo e as inclinações emocionais e comportamentais, que chamou de “temperamentos”, sendo estes formados por quatro elementos: fleumático/terra (fria e seca), sanguíneo/ar (quente e úmido), colérico/ fogo (quente e seco) e melancólico/água (fria e úmida). O equilíbrio dos humores determina o tipo de personalidade da pessoa, além de indicar a tendência a sofrer certas doenças. Se algum dos humores se desenvolver mais do que outros, passará a dominar a personalidade da pessoa, o que certamente alteraria o comportamento dos indivíduos, sejam novos ou velhos e, desse modo, a velhice ocorreria de forma determinada pelos temperamentos humanos. p p Nessa perspectiva, sabemos que os procedimentos de ensino-aprendizagem da medicina de Hipócrates foram fundamentais para a compreensão da velhice, do envelhecimento humano e para o cuidado com a saúde dos indivíduos nesta fase da vida. Estes procedimentos proporcionaram à humanidade a estruturação da Geriatria e da Gerontologia na modernidade e, a partir desses conhecimentos, Vieira (2011) aborda sobre temas e problemas para pensar o envelhecimento: Até recentemente, acreditava-se que grande número dos problemas encontrados no idoso doente eram consequências normais do envelhecimento. Somente há poucos anos a abordagem geriátrica ampla e a medicina geriátrica começaram a ser estruturadas. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Céfalo respondeu que se sentia ótimo, pois o triste poema ou canção, retratado por muitos, penalizando a velhice por todos os males, para ele era proveniente do próprio existir e não da idade avançada e que a velhice significava muita paz e liberdade. Disse-lhe, ainda, que se os homens tiverem bom caráter e alma equilibrada, a velhice não será problema algum (PLATÃO, 1985; SANTOS, 2001). O filósofo Aristóteles (384 – 322 a. C.), nascido em Estagira, Macedônia, vai aos 18 anos para Atenas e ingressa na Academia de Platão, onde se torna discípulo deste, tendo sido considerado um dos principais filósofos da época e com grande influência na organização do saber grego que colaborou e impactou a história do pensamento ocidental. Aristóteles, em relação às concepções acerca do envelhecimento, é bem diferente de seu mestre Platão, haja vista que entendia que uma boa velhice seria aquela sem enfermidades e que ser velho é extremamente deplorável, o que confirmou em sua obra “Ética a Nicômaco”, onde afirma que os velhos são pessoas reticentes, hesitantes, lentas, desconfiadas, não virtuosas, que só imaginam o mal e carentes de generosidade, vivendo das lembranças do seu passado e não considerando a opinião dos outros. Aristóteles considerava, ainda, que o ser humano evoluía até os 50 anos. Desta forma, segundo ele, tais características colocavam os velhos como não capacitados, tanto para o poder na pólis (cidade-estado), como para cargos de relevância política (PLATÃO, 1985; SANTOS, 2001). Não podemos deixar de falar do médico Hipócrates (460 – 370 a. C.), celebridade da Antiga Grécia, que é considerado “Pai da Medicina”, e por trabalhar a saúde a partir de uma visão racional e científica, não considerando as superstições, crendices populares e práticas mágicas da “saúde” primitiva. Para Hipócrates, a velhice inicia-se a partir dos 50 anos e associada ao desequilíbrio dos humores. Ele estabelece diferentes características nos estados clínicos entre idosos e jovens, apontando que nos velhos, a temperatura não tende a ser alta, estes têm doenças crônicas, os hábitos intestinais transformam-se, bem como, o diagnóstico das enfermidades é diferente e difícil. Hipócrates fundamentou a sua prática e a sua forma de NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 184 No Século I, antes da Era Cristã, destaca-se a figura do emblemático romano Marco Túlio Cícero (106 – 33 a. C.) que foi advogado, político, escritor, orador e filósofo da gens Túlia (família da Roma) da República Romana, que se elege cônsul em 63 a. C. A sua família lhe proporcionou uma educação de qualidade. Marco Túlio estudou na escola pública e ao completar a maioridade, passou a vestir a toga virilis (traje masculino). O seu pai Cícero (o velho) lhe entrega aos cuidados do célebre Senador e jurista romano Quinto Múcio Cévola, que o pôs a par das Leis e das Instituições Políticas de Roma. Cícero é educado com base nos ensinamentos dos antigos filósofos, poetas e historiadores gregos, considerado o primeiro romano que chegou aos principais postos do Governo, com base na sua eloquência e ao mérito com que exerceu as suas funções de magistrado civil (PLATÃO, 1985, SANTOS, 2001). Cícero, na antiguidade (44 a. C.), em suas reflexões sobre o envelhecimento, acaba escrevendo o livro “De Senectute ou Catão, o velho”, que é apresentado em forma de dissertação e dedicado ao seu amigo Tito Pompônio Ático. A obra descreve um diálogo entre o idoso Marco Catão, considerado modelo máximo da cultura romana, e os jovens, homens públicos: Lélio e Cipião, em que a velhice é o tema abordado. Ressalta-se que Cícero e Tito estavam próximos de vivenciar a velhice, que é tratada no texto. Neste, Cícero defende e conceitua a velhice como algo bom ou ruim e isto, dependendo do agir do indivíduo na Natureza. Demonstra, também, a dualidade existente no processo do envelhecimento humano, a oposição dialética entre velhice e juventude que poderá ocorrer no ser de forma positiva ou negativa (PLATÃO, 1985, SANTOS, 2001). g ( ) Em “De Senectude”, os jovens Cipião e Lélio interrogam Marco Pórcio Catão (234 – 149 a.C.) sobre o envelhecimento, com relação às consequências e vantagens do envelhecer. O Senador Catão, já com 80 anos, reponde, derruba os tabus e preconceitos contra a velhice e coloca os velhos em posição de destaque, como grandes responsáveis pela restauração dos Estados quando arruinados pelos jovens (PLATÃO, 1985; SANTOS, 2001). SEÇÃO LIVRE Hoje se sabe que a tendência a rotular sintomas como “problemas da idade” é um dos principais obstáculos ao correto diagnóstico e tratamento das doenças comuns na velhice. A doença é aceita pelo paciente, pela família e muita das vezes até pelo médico como manifestação normal da idade. Os sintomas são considerados intratáveis ou não merecedores de atenção médica. Isto não é verdade. A medicina geriátrica é uma especialidade que envolve a prevenção ou a reabilitação destes problemas comuns no envelhecimento, indo além do diagnóstico e do tratamento isolados da doença. Nesta abordagem ampla, a medicina geriátrica enfatiza a qualidade de vida e a independência funcional do paciente idoso, trabalhando em conjunto com uma equipe multidisciplinar de profissionais, a equipe de gerontologia.” (VIEIRA, 2011, p.7). Vieira (2011) confirma, na modernidade, a relevância da Geriatria e da Gerontologia para a interpretação e conceituação da velhice e do processo de envelhecimento humano na sociedade, em especial, para o cuidado dos velhos e tratamento de suas doenças de forma preventiva e curativa, algo ainda muito recente na Medicina, mas, com o avanço da ciência e da tecnologia, o fenômeno do envelhecimento tem ganhado maior ênfase e sido foco de diversos estudos científicos, em diversas áreas do conhecimento da ciência que agora se expressa na Gerontologia. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Conforme Catão, a velhice é acusada, por muitos, de ser uma fase da vida deplorável pelos seguintes motivos: a) afasta o homem dos negócios; b) torna o corpo mais sujeito a doenças; c) priva o homem de quase todos os prazeres e d) não está muito distante da morte. Catão elaborou, então, a defesa da velhice e, pela lógica de sua argumentação, afirma que o envelhecimento poderá ocorrer de forma feliz, que dependerá exclusivamente do seu modo de vida e de viver na sociedade. Considerando, também, que a velhice digna é uma edificação árdua que exige bastante vigor e uma prática dialética virtuosa entre jovens, adultos e velhos e que todos da sociedade devem respeitar e aceitar as diretrizes da natureza para viverem a excelência de suas idades e com felicidade. A obra de Cícero (44 a. C) é um instrumento, um manual de orientação para jovens e velhos que objetivam uma existência feliz e harmoniosa com a natureza, livres do embate de gerações. Cícero considera o envelhecimento um processo fisiológico, uma fase natural da vida, na qual os indivíduos estão sujeitos à redução da memória e da capacidade funcional, como alteração nos órgãos dos sentidos e diminuição da capacidade laboral. Destacou que quando se chega à velhice, os prazeres corporais paulatinamente cedem lugar aos projetos intelectuais e enfatizou a necessidade de prestigiarmos os velhos e de prepará-los psicologicamente para o fim da vida. Entretanto, Cícero defendia a tese de que, para se viver uma velhice saudável e prazerosa, seria fundamental o cuidado corporal, nutricional, mental, a inserção em projetos educacionais e outras atividades que gerassem benefícios individuais e coletivos, todavia que fosse de acordo com a condição física de cada ser. Dessa forma, ele dizia que a arte de envelhecer está em descobrirmos a alegria que todas as fases da existência humana nos proporcionam, seja enquanto criança, jovem, adulto ou velho, haja vista que todas são cheias de virtudes. O processo de ensino-aprendizagem do envelhecer saudável, ativo e feliz se dá no convívio social, na coletividade e na participação com o outro, com seu irmão velho na comunidade, no trabalho, nas escolas e igrejas, conforme as suas SEÇÃO LIVRE 185 escolhas no meio natural, no espaço onde vive (urbano ou rural), e respeitando a Mãe Natureza (PLATÃO, 1985, SANTOS, 2001). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Cícero (1999; 2006), ainda em sua obra, afirma que não podemos atribuir à velhice todos os males da vida e que quem vive se lamentando, certamente fez o mesmo nas outras idades. Neste aspecto, ele refere-se que os humanos inteligentes devem sempre se distanciar da tristeza e do mau humor, desses temperamentos que são prejudiciais em qualquer fase da vida. Na concepção estóica de Cícero, a velhice deve ser aceita por todos, pois é uma determinação da natureza, processo fisiológico que abraçará a todos. Assim, estudar e pesquisar o fenômeno, a questão social e educacional do envelhecimento é fundamental para se pensar as condições da velhice na contemporaneidade, em especial, no Brasil, no Estado do Tocantins e na cidade de Araguaína. O romano Lúcio Aneu Séneca ou Sêneca (20 a. C – 65 d.C.) foi um dos mais relevantes advogados, escritores e intelectuais do Império Romano. Ele refletiu sobre o envelhecimento em seus escritos e destacou várias cartas destinadas a seu amigo Lucílio (no período entre 63 e 65 d. C.). Segundo Sêneca, a velhice não seria algo ruim, mas uma coisa boa, já que faz parte do processo da natureza humana que deve, com tranquilidade, ser aceita e ocorrer vivendo-a de forma feliz, com sabedoria, prudência e no culto às virtudes para o bem viver. Dessa forma, Sêneca respaldou o pensamento de Cícero em relação ao envelhecimento humano (PLATÃO, 1985). 5 CONSIDERAÇÕES FINAIS É de suma importância conhecer a visão dos antepassados por meio dos filósofos. A história oportuniza-nos enxergar o outro e a nós mesmos. Nesta perspectiva, o amor dos filhos aos pais envelhecidos é algo divino, especial, que compreende maior proteção e segurança na última fase da vida humana, o que se considera um dos mais admiráveis atos da raça humana consigo mesma e para com seus semelhantes, em destaque com a sua geração e para com as futuras gerações. Assim, a velhice e o envelhecimento, tanto nas cidades da antiguidade, como nas cidades do atual mundo moderno colonizado e capitalizado, são vistos de forma idêntica: algo negativo, uma coisa ruim que, para muitos, expressam terror e como se a velhice não fosse atingir a todos. Por outro lado, existem outras visões de caráter positivo e que devem ser consideradas. Desse modo, adaptarmo-nos a esta nova fase do existir humano, a velhice, é algo que requer cuidados e mudanças de hábitos, em especial, no tratamento da nossa saúde. Além da saúde, é preciso priorizar outras áreas: sociais, esportivas e educativas, nas quais a participação e inserção dos velhos são essenciais para o bem-estar social e a melhoria da qualidade de vida deles. É preciso, também, que as autoridades governamentais e o Estado brasileiro se responsabilizem pelas políticas públicas voltadas à população idosa. Atualmente, chamam-nos a atenção algumas áreas, específicas, da Medicina e da Educação, cujo foco é a preparação das gerações para a velhice, para a construção de um envelhecimento digno, participativo e ativo na sociedade: a Gerontologia, a Geriatria, as Universidades da Terceira Idade (UNATIs) e a Universidade da Maturidade (UMA). Portanto, a questão do envelhecimento humano e da velhice é temática viva e presente no século XXI e que precisa ser pesquisada e estudada profundamente em nossa Amazônia brasileira, em toda a região norte, em especial no Estado do Tocantins. REFERÊNCIAS NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 186 ALMEIDA, V. L. V. Imagens da velhice: o olhar antropológico. A terceira idade, São Paulo: v. 10, n. 15, p 35-40, dez. 1998. AMARO, Sarita. Serviço Social na Educação: bases para o trabalho profissional. 1ª reimpressão – Florianópolis: Ed. Da UFSC, 2012. ARROYO, Miguel G. Outros Sujeitos, Outras Pedagogias. Petrópolis, RJ: Vozes, 2012. ARROYO, Miguel G. Outros Sujeitos, Outras Pedagogias. Petrópolis, RJ: Vozes, 2012. ASLAN, Ana, 1898. Vencendo a velhice. Tradução José Augusto Carvalho. 5ª ed. Rio de Janeiro: Record, 1994. 127p. Tradução de: Contre la vieillesse. BEAUVOIR, S. de. A velhice: realidade incômoda. 2ª ed. DIFEL, São Paulo: 1976. BEAUVOIR, Simone de (1908 – 1986). A velhice. Tradução de Maria Helena Franco Martins. 2ª ed. – Rio de Janeiro: Nova Fronteira, 2018. BRANDÃO, Carlos Rodrigues. O que é educação. 33 ed. – São Paulo: Brasiliense, 1995. – (Coleção primeiros passos; 20). BRASIL. Constituição da República Federativa do Brasil: promulgada em 05 de outubro de 1988. São Paulo: Saraiva, 1988. BRASIL. Estatuto da Pessoa Idosa. – Lei 10.741 de 1/10/2003 – Dispõe sobre o Estatuto do Idoso e dá outras providências. Autor da lei: Senador Paulo Paim PT/RS – Brasília, 2003. CÍCERO, M. T. Saber envelhecer. Tradução. Paulo Neves. Porto Alegre: L & PM, 1999. CÍCERO, M. T. A Velhice Saudável: O Sonho de Cipião. Tradução Luiz Feracine. São Paulo: Escala, 2006. 112 p. Tradução de: De senectude. CONFÚCIO. Vida e doutrina, Os Analectos. Tradução de Múcio Porphyrio Ferreira. São Paulo: Pensamento, 1999. FALEIROS, Vicente de Paula. Cidadania e direitos da pessoa idosa. Ser Social. In: Revista do Programa de Pós Graduação em Política Social. Brasília, UNB, n. 20, jan./jun. 2007. 35-61 p. LAO TZU. Tao te Ching. (Tradução e comentários de Laércio B. Fonseca. (2.ed) Editora: Lenon Tree, 2014 / Limeira –SP. OLIVEIRA, Rita de Cássia da Silva; SCORTEGAGNA, Paola Andressa; OLIVEIRA, Flávia da Silva. O envelhecimento e a velhice: teorias, demografia e política. 1ª ed. Curitiba, PR: CRV, 2011. 100p. PESSANHA, Clarice C. Franco. Breves considerações sobre a evolução histórica da imagem do Idoso na Civilização Oriental. Revista de Trabalhos Acadêmicos - Universo Campos dos Goytacazes, n. 6, Vol. 2, 2016. Disponível em: http://www.revista.universo.edu.br/index.php?journal=1CAMPOSDOSGOYTACAZES2&pa ge=issue&op=view&path%5B%5D=176&path%5B%5D=showToc. Acesso em: 14 de jun. 2019. SEÇÃO LIVRE 187 PLATÃO (427 – 347 a. C.). A República. Platão; tradução de Carlos Alberto Nunes. – 3. Ed. – Belém: EDUFPA, 2000. PLATÃO. A República. Tradução de Elza Moreira Marcelina. Brasília: UnB, 1985. Livro 7. SANTOS, S. S. C. Envelhecimento: visão de filósofos da antigüidade oriental e ocidental. Rev. Rene., Fortaleza, v.2, n.1, p. 88-94, jul./dez. 2001. 1Graduação em História pela Universidade Federal do Pará UFPA (1999). Mestrado em Antropologia pela Universidade Federal do Pará UFPA (2002). Doutorado em Ciências Sociais na especialidade Antropologia Cultural e Social pelo Instituto de Ciências Sociais da Universidade de Lisboa (ICS) Professor no Campus De Bragança da UFPA, Ligado a Faculdade de Educação e Professor da Pós-Graduação em Linguagens E Saberes Na Amazônia. E-mail: LUISJSARAIVA@GMAIL.COM TAO TE CHING. Tradução de Wu Jyh Cherng. Rio de Janeiro: Mauad, 2011. TAO TE CHING. Tradução de Wu Jyh Cherng. Rio de Janeiro: Mauad, 2011. VIEIRA, Cláudia Caciquinho. IDOSO E AMEDICINA: diálogo com a abordagem geriátrica ampla. In: MOREIRA, Jacqueline de Oliveira (Org.). Gerontologia e cuidado: temas e problemas para pensar o envelhecimento. Gerontologia e cuidado: temas e problemas para pensar o envelhecimento. Curitiba, PR: CRV, 2011. SEÇÃO LIVRE SEÇÃO LIVRE 189 ALIMENTAÇÃO E RELIGIOSIDADES NO ROMANCE MENINA QUE VEM DE ITAIARA DE LINDANOR CELINA Luis Junior Costa Saraiva1 Paulo Sergio Oeiras da Silva2 Jéssica do Socorro Leite Corrêa3 RESUMO O trabalho traz reflexões sobre a relação entre alimentação e saúde no romance Menina que vem de Itaiara de Lindanor Celina. A personagem principal, Irene, descreve parte da sua infância e início de sua adolescência, com um cotidiano bem típico amazônico, entre os saberes culturais presente em toda obra, tendo como cenário a cidade de Itaiara, nome fictício da cidade de Bragança. Destacaremos alguns segmentos da narração que apresentam elementos importantes sobre a relação entre alimentação e religiosidades. A narrativa da obra é em primeira pessoa e a personagem principal descreve momentos de sua memória, momentos em que retrata a cultura patriarcal da época, apresentando valores e costumes religiosos locais. Como resultado da análise, percebe-se que a autora, deixa informações importantes já esquecidas, uma memória literária rica em conhecimento sobre alimentação e religiosidades e os diversos processos de cura. Palavras-Chave: Lindanor Celina. Alimentação. Saúde. Religiosidades. 2 Graduado em Letras - Língua Portuguesa pela Faculdade de Letras do Campus Universitário de Bragança da Universidade Federal do Pará (2009-2013). Mestre em Linguagens e Saberes na Amazônia- UFPA. E-mail: oeirasufpa@gmail.com p g 3 Graduada em Pedagogia pela Universidade Federal do Pará (2015). Mestra em Linguagens e Saberes na Amazônia- UFPA. E-mail: etieljessica@gmail.com 1Graduação em História pela Universidade Federal do Pará UFPA (1999). Mestrado em Antropologia pela Universidade Federal do Pará UFPA (2002). Doutorado em Ciências Sociais na especialidade Antropologia Cultural e Social pelo Instituto de Ciências Sociais da Universidade de Lisboa (ICS) Professor no Campus De Bragança da UFPA, Ligado a Faculdade de Educação e Professor da Pós-Graduação em Linguagens E Saberes Na Amazônia. E-mail: LUISJSARAIVA@GMAIL.COM 2 Graduado em Letras - Língua Portuguesa pela Faculdade de Letras do Campus Universitário de Bragança da Universidade Federal do Pará (2009-2013). Mestre em Linguagens e Saberes na Amazônia- UFPA. E-mail: oeirasufpa@gmail.com 3 Graduada em Pedagogia pela Universidade Federal do Pará (2015). Mestra em Linguagens e Saberes na Amazônia- UFPA. E-mail: etieljessica@gmail.com INTRODUÇÃO A presente análise foi desenvolvida durante a produção acadêmica para a construção da dissertação de mestrado do Programa de Pós-Graduação em Linguagens e Saberes na Amazônia da Universidade Federal do Pará, sob o tema “Reflexões sobre alimentação e saúde no romance Menina que vem de Itaiara de Lindanor Celina”. A narrativa da obra é em primeira pessoa e a personagem principal, Irene, descreve momentos de sua memória, na qual retrata a cultura patriarcal da época. Nesta obra, Irene descreve parte da sua infância e início de sua adolescência, cotidiano, inquietações e traquinagens, que tem como pano de fundo a cidade de Itaiara, nome fictício da cidade de Bragança, local no qual é ambientado o romance. A cidade de Bragança-PA, fica localizada a aproximadamente 210 km da capital do estado, muito conhecida pela produção de farinha d’água, além de destaque na atividade pesqueira e nas manifestações culturais. Muitas características da cidade são apresentadas no romance, as manifestações culturais, características sociais e estruturais da cidade, e muitos elementos de caráter alimentar. No presente artigo, destacaremos algumas narrativas que apresentam as variadas propriedades da cultura alimentar, saúde e religiosidade, e suas diferentes significações. g ç A cozinha sempre se configurou como um amplo espaço de análise sociocultural, um conjunto de elementos que perpassam pelas significações dos produtos utilizados, seu modo de preparado e o ritual de produção e consumo. Na obra Menina que vem de Itaiara, Irene narra diversas situações vivenciadas por ela e nos apresenta alguns desses elementos característicos do contexto amazônico, na qual percebemos bem forte a presença da tradição alimentar, principalmente por conta da época em que o romance foi escrito, período de pouca tecnologia, baixa influência da mídia televisiva com suas propagandas alimentares bem atrativas, seduzindo para as refeições rápidas, porém de baixo poder nutricional e alimentar, e consequentes prejuízos à saúde e ao bem estar. Assim, os hábitos alimentares tradicionais do romance não possuíam a concorrência que facilmente visualizamos nos dias atuais, proporcionando uma alimentação bem mais saudável, os alimentos usados, em sua maioria, eram oriundos de recursos naturais, por vezes, colhidos no pomar do quintal da casa de Irene. Os recursos naturais não apresentavam substâncias que hoje causam mal à saúde, como agrotóxicos, produtos utilizados na agricultura para controlar insetos, doenças, ou plantas daninhas que causam danos às plantações. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 190 ABSTRACT The work brings reflections on the relationship between food and health in the romace Menina que vem de Itaiara by Lindanor Celina. The main character, Irene, describes part of her childhood and early adolescence, with a very typical Amazonian life, among the cultural knowledge present in every work, has the city of Itaiara as a scenario, fictitious name of the city of Bragança. We will highlight some segments of the narration that present important elements about the relationship between food and religiosities. The narrative of the work is in first person and the main character describes moments in his memory, moments in which he portrays the patriarchal culture of the time, presenting local religious values and customs. As a result of the analysis, it is clear that the author leaves important information already forgotten, a literary memory rich in knowledge about food and religiosities and the various healing processes. Keywords: Lindanor Celina. Food. Cheers. Religiosities. Keywords: Lindanor Celina. Food. Cheers. Religiosities. Data da submissão 18.11.2020 Data da aprovação 07.12.2020 Data da submissão 18.11.2020 Data da aprovação 07.12.2020 2 Graduado em Letras - Língua Portuguesa pela Faculdade de Letras do Campus Universitário de Bragança da Universidade Federal do Pará (2009-2013). Mestre em Linguagens e Saberes na Amazônia- UFPA. E-mail: oeirasufpa@gmail.com INTRODUÇÃO Era bem mais fácil encontrar casas com suas próprias criações, tais como criações de galinhas, de patos, de perus, de porcos etc. Em todo romance, isso é algo bem presente, principalmente quando se reuniam para certas ocasiões em que eram servidos pratos típicos da Amazônia. Esta é a diferença atual para o que era frequente durante a construção do romance, os elementos que foram inseridos, algo daquele momento vivido, os tipos de alimentos, as configurações das casas, com espaços maiores em que se podia plantar, o espaço envolvido, pois de lá saía o leite, a carne, os grãos, as frutas e as verduras e, ocasionalmente, funcionavam as trocas. Se alguém matava um porco, era repartido com os outros vizinhos. Trata-se de um costume que muitos conheceram em alguns interiores e, ainda, em ocasiões a prática continua em datas específicas. Os elementos culturais na culinária herdada dos nossos antepassados estão presentes também nas manifestações religiosas. Esta situação, de alguma forma, propicia uma sintonia com o ser divino e deixa claro que a religião é um dos sustentáculos de uma cultura. As manifestações religiosas demonstram a necessidade do homem para se comunicar com o sobrenatural, para auxiliá-los em suas necessidades. Atualmente, algumas características permanecem. Portanto, fatores religiosos influenciaram profundamente em certos hábitos alimentares. Trocas sobre experiências alimentares ocorrem quando diversas culturas se encontram no mesmo espaço, quando relacionadas a diversidade entre natureza e sociedade, resultando SEÇÃO LIVRE 191 em combinações que reproduzem emoções nas degustações, nas diferentes formas de práticas alimentares. Alimentos que possuem, para determinado grupo social, propriedades curativas e míticas. em combinações que reproduzem emoções nas degustações, nas diferentes formas de práticas alimentares. Alimentos que possuem, para determinado grupo social, propriedades curativas e míticas. A alimentação é um tema presente em livros religiosos sagrados, como na Bíblia, os quais destacam os elementos alimentares ou ainda as proibições alimentares, como no Livro de Gênesis. E disse Deus: Produza a terra erva verde, erva que dê semente, árvore frutífera que dê fruto segundo a sua espécie, cuja semente esteja nela sobre a terra. E assim foi. E a terra produziu erva, erva dando semente, conforme a sua espécie, árvore frutífera, cuja semente está nela conforme a sua espécie. E viu Deus que era bom. (GÊNESIS, 1,11-12). INTRODUÇÃO Ainda em um outro livro sagrado da Bíblia, temos a seguinte passagem, “E seu fruto servirá de alimento e a sua folha de remédio” (EZEQUIEL, 47, 12). Segundo Balbach (1968), essa ciência terapêutica vem desde a Antiguidade. Claro que no início não existiam tantas enfermidades, mas, com o decorrer do tempo, a força física do homem foi diminuindo, em virtude das consequências das leis da natureza que foram sofrendo mudanças causadas pelas mãos do homem. Assim, os homens foram tratando das enfermidades como melhor podiam, e nada melhor para isso do que recorrer à natureza, que foi o primeiro médico e remédio da humanidade. Em Menina que vem de Itaiara, Lindanor Celina retrata a infância da personagem numa cidade interiorana, que ainda tem nos saberes tradicionais a solução para diversas situações relacionadas à saúde, típicas de comunidades tradicionais, que buscam em ervas e chás a cura para diversos males, dos mais leves aos mais complexos. Esse conjunto de saberes está muito presente na experiência dos personagens criados por Lindanor ao longo da obra, como, por exemplo, no Marreca, rezador de garganta, ou na Vijoca, experiente em remédios naturais, e outros que serão tratados ao longo do presente trabalho. Sobre o saber tradicional de comunidades amazônicas Lima (2009) diz que são características de componentes de populações tradicionais, a percepção dos sons da mata, sejam diurnos, noturnos, rastros, odores, conhecimento das espécies da fauna e flora e o uso adequado que se faz dessas espécies” e ainda “muitas habilidades presentes nas populações tradicionais, como o conhecimento das funcionalidades de ervas e sua manipulação para confecção de ungüentos, xaropes” (LIMA, p. 4). Analisando a relação entre alimentação e saúde na obra, percebemos que Lindanor Celina concilia essa variedade de alimentos regionais com hábitos, costumes e culturas, pois está interiorizada em seu trabalho. Hábitos adquiridos por crenças, valores e agentes sociais, que praticam uma vivência de tradições, uma ciência tradicional que está inserida nas personagens, transmitindo a importância dos saberes históricos culturais. Apresenta também soluções por meio de elementos religiosos acompanhados de alimentos, ervas e outras práticas. Assim, os hábitos e práticas alimentares produzidos historicamente se transformam em hábitos culturais que integram o modo de viver deste grupo social ou povo. Considera-se também que numa sociedade capitalista não existem hábitos e práticas alimentares homogêneas, pois existem hábitos que, mesmo desejados por todos, não podem ser transformados em práticas por grande parte da população. 4MANDINGA - termo de origem africana para feitiço. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 192 O conceito de cultura apresenta, de alguma forma, um padrão que deve ser respeitado, pois está representando significados que dominam certos grupos de pessoas, símbolos que historicamente se perpetuaram, mas que nem sempre são bem vistos por todas as pessoas. E essa cultura vem junto com a religiosidade de cada um. Existe uma religião dominante, o Cristianismo, que, talvez por isso, nem sempre são aceitos outros tipos de crenças, ou seja, mais difíceis de segui-las, porém, há um sincretismo de costumes, que vêm sendo repassados em cada cultura. Na crença e na prática religiosa, o ethos de um grupo torna-se intelectualmente razoável porque demonstra representar um tipo de vida idealmente adaptado ao estado de coisas atual que a visão de mundo descreve, enquanto essa visão de mundo torna-se emocionalmente convincente por ser apresentada como uma imagem de um estado de coisas verdadeiro, especialmente bem arrumado para acomodar tal tipo de vida. (GEERTZ, 2012, p. 67). O que seria essa visão de mundo para este autor? Como são vistas as práticas religiosas nas culturas? Veremos, a seguir, um pouco desta prática religiosa relacionada a elementos alimentares tratados pela autora no romance, que destaca informações importantes da visão de mundo do povo amazônico, e como cada atitude, cada alimento, se torna tão aceitável pelas pessoas. Se percebe como a religião toma forma nas ações humanas, permitindo mudanças em determinados padrões de vida. O sincretismo religioso afro-brasileiro como aspecto cultural foi - e continua a ser - uma estratégia de sobrevivência transnacional, pois teve sua origem exatamente no desarraigamento de milhões de africanos pela via da chamada passagem do meio. Também o entendemos como transnacional, porque sua complexidade temática se estendia - e ainda se estende - às mais diversas áreas da convivência e das vivências humanas: rituais religiosos, conteúdos históricos, práticas sociais, lendas e mitos como substratos culturais, e toda uma gama de outros fatores (ROMÃO, 2018, p. 368). INTRODUÇÃO (PACHECO, 2008, p. 222). 5Tribunal de Santo Ofício, foi um movimento da Igreja Católica Romana, criado para combater a heresia, em que os supostos hereges eram julgados, torturados e até a morte na fogueira. SEÇÃO LIVRE SEÇÃO LIVRE 193 Esses grupos de pessoas não tinham a liberdade para cultuarem suas divindades e, para enganar seus senhores, criaram as próprias estratégias para persuadi-los. Começaram, então, a usar as imagens de santos católicos, como táticas de sobrevivência, para encobrir seus orixás, caso não procedessem assim seriam pegos e advertidos, até por causa de elementos como a Inquisição5 que no Brasil teve início no período colonial, pois a Igreja jamais aceitaria e nunca aceitou tal ato. Então, surgiram santos que transitaram, e até hoje transitam, pelas religiões africanas e católica, o São Sebastião, São Benedito, São Cosme e Damião, santos de origem católicas, mas que também são cultuados nas religiões de origem africana. Com certa facilidade encontramos pessoas em eventos católicos, por exemplo, na festividade de São Benedito em Bragança, promesseiros, marujos, que vêm pagar suas promessas no período da festa, mas que raramente puseram seus pés em outros momentos na igreja católica, exceto neste período. Justamente porque tais promessas foram feitas em terreiros, durante rituais de candomblé ou de umbanda etc. Pois tais imagens de santos estão presentes nas outras religiões. p g Há um filme nacional chamado O Santo Milagroso, de 1967. É um filme de humor, no qual dois personagens retratam a fé em diferentes situações, um padre e um pastor que disputam seus fiéis, e um coroinha que se apaixona pela irmã do pastor. No decorrer do filme o coroinha é proibido de se encontrar com a mocinha da história, mas acaba fazendo uma promessa em um terreiro de umbanda, no intuito de conseguir a jovem, porém o padre e o pastor se envolvem num conflito que finda fazendo uma imagem do Santo Antônio “falar”. No final da trama, os jovens ficaram juntos e o milagre foi atribuído ao santo. O filme retrata essa realidade de mistura, esse sincretismo entre religiões, adquirindo costumes, mas que não fazem parte de certas doutrinas religiosas, são aspectos adquiridos pelo próprio indivíduo. É importante lembrar que no Brasil raramente se encontrará um panteão de origem africana totalmente baseado apenas em uma etnia. Costuma-se encontrar em um mesmo candomblé uma divindade de origem ioruba, p. ex. Oiá, também conhecida como Iansã, ao lado de outras divindades assimiladas, à guisa de exemplo, das práticas religiosas da omeanas, como Oxumarê, Omulu e Nanã Buruku. 1 PRÁTICAS ALIMENTARES E CURATIVAS NO HIBRIDISMO RELIGIOSO A narrativa na obra Menina que vem de Itaiara organiza um conhecimento social daquele determinado momento, que explica um pensamento comum do período. No romance, encontramos características marcantes da religião africana, que poderia ser candomblé ou umbanda. É o caso da palavra mandinga4, que é uma forma de exercer uma medicina mágica e simpatias, crenças herdadas de homens escravizados, oriundos da África. Encontramos também elementos da religião católica em sincronismo com outras religiões, ou ainda da forte presença do espiritismo. A obra termina por trazer ao longo das páginas a presença do sincretismo religioso tão presente em nosso país. Com a presença dos africanos no Brasil Colônia, as diferentes religiões e/ou os cultos tradicionais dos diversos grupos étnicos foram-se assimilando, para, num primeiro passo, paulatinamente constituírem o que se costuma chamar de candomblé e, num segundo instante, estabelecerem um diálogo também com a religião católica e os cultos indígenas, adotando elementos destes. (ROMÃO, 2018, p. 362). Como a Religião Católica era a dominante, imposta durante a “catequização” feita pelos primeiros colonizadores, após o comércio de pessoas escravizadas trazidas para o Brasil do continente africano, estes criaram estratégias para a sobrevivência de suas religiões maternas. O sincretismo religioso afro-brasileiro como aspecto cultural foi - e continua a ser - uma estratégia de sobrevivência transnacional, pois teve sua origem exatamente no desarraigamento de milhões de africanos pela via da chamada passagem do meio. Também o entendemos como transnacional, porque sua complexidade temática se estendia - e ainda se estende - às mais diversas áreas da convivência e das vivências humanas: rituais religiosos, conteúdos históricos, práticas sociais, lendas e mitos como substratos culturais, e toda uma gama de outros fatores (ROMÃO, 2018, p. 368). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 194 rústica em medicina mágica, medicina religiosa e a medicina empírica, pois para cada tipo de doença há um determinado remédio, enfim, a causa do mal está sempre no sobrenatural, daí haver uma certa unidade entre essa classificação. rústica em medicina mágica, medicina religiosa e a medicina empírica, pois para cada tipo de doença há um determinado remédio, enfim, a causa do mal está sempre no sobrenatural, daí haver uma certa unidade entre essa classificação. Em Menina que vem de Itaiara, temos, então, o personagem Marreca, exímio rezador de garganta e alguém que representa bem essa junção entre religiosidade, medicina e poderes de cura no sobrenatural. Abaixo temos a descrição do trabalho do benzedor, o qual percebemos que nesse tipo de ofício não tem hora para ser chamado, todo momento torna-se oportuno, precisou logo era chamado, à semelhança dos profissionais da saúde pública, a necessidade de socorrer fazia parte dos padrões de cultura, e a fé depositada no benzedor ia além de um simples conhecimento, a crença regia toda a comunidade. Tudo estava voltado para o sobrenatural. A mistura das religiões era bastante comum. Não se percebia as diferenças, preconceitos ou algo similar, e sim algo corriqueiro, faziam parte das narrativas populares. Era comum esse tipo de prática. As doutrinas religiosas não eram de conhecimento do povo. Tudo era mais simples, e se algo era proibido, por exemplo, pela Igreja Católica, certos rituais, não eram de conhecimento ou não se aplicava tanta importância. Fazia parte dos costumes do povo. Em toda região sempre havia mais de um benzedor, alguém que portava tais poderes. Era muito importante a presença de uma pessoa que pudesse aliviar o sofrimento alheio. Sempre era bem-vindo um benzedor. Ele gozava de um respeito, assim como também podia ser temido, uma vez que algumas pessoas confundiam suas práticas como o mundo obscuro do sobrenatural. Uma vez, no jantar, engoli uma espinha de peixe. De tainha. Era de noite, grande foi a agonia. Debalde me fizeram cuspir no prato que rodaram três vezes, à toa ingeri punhados e punhados de farinha enxuta, da bem brolô, pirão, banana. Em vão mamãe bradou: “São Brás, valei-me” não sei quantas vezes. São Brás, nem, nem. Certamente operava gargantas noutro canto do mundo, distante demais da rua do Capim. Pois dois dias passei naquela situação. SEÇÃO LIVRE Com o tempo, o sincretismo originou uma religião tipicamente brasileira, mesclando diversos elementos de outras religiões e crenças, mas com base primordialmente nos cultos africanos, nos rituais indígenas e no catolicismo: a umbanda. (ROMÃO, 2018, p. 368). Em se tratando de religiosidade, não há prática totalmente pura, mas um hibridismo religioso, que é muito presente no contexto brasileiro de um modo geral e algo muito presente na obra de Lindanor. Vamos explorar um pouco mais a medicina rústica (práticas médicas populares), como resultado de uma série de aculturações da medicina popular da cultura portuguesa, indígena e africana. Para tanto, é necessário o conhecimento das influências que ela sofreu dos antecedentes pré-ibéricos, lusos, ameríndios e africanos. Não devemos olvidar os antecedentes que a medicina popular negra recebeu, quando estiveram em contato com a África branca – os mouros. Todavia, o novo ambiente e novos contatos proporcionaram, para ambas as partes, novas experiências como os elementos que o ameríndio, e o novo habitat, lhes ofereceram. (ARAÚJO, 1959, p. 43). Araújo diz que “o pajé ameríndio, o feiticeiro negro, o bruxo europeu, certamente tiveram suas técnicas de lidar com o sobrenatural, num dado momento em contato” (1959, p. 44). Ainda, segundo o mesmo autor, ele faz uma classificação para o estudo da medicina NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Resolveu, então, tornar-se um eremita e ficar em constante oração. Assim, viveu numa gruta por muitos anos. São Brás nasceu na cidade de Sebaste, Armênia perto do ano 300. Num certo tempo, começou a questionar sobre sua profissão de médico, pois queria servir a Deus, mas não sabia como. Resolveu, então, tornar-se um eremita e ficar em constante oração. Assim, viveu numa gruta por muitos anos. Um dia, uma mãe desesperada o procurou porque seu filho estava quase morrendo com um espinho encravado na garganta. São Brás olhou para o céu, rezou e, em seguida, fez o sinal da cruz na garganta do menino. No mesmo instante, ele ficou milagrosamente curado. Por esse milagre, até os dias de hoje São Brás é invocado para curar os males da garganta. Em todos os lugares do mundo, quando uma criança ou qualquer pessoa se engasga, a invocação direta ao Santo logo é rezada: "São Brás te proteja." Ou simplesmente: "São Brás."6 Um dia, uma mãe desesperada o procurou porque seu filho estava quase morrendo com um espinho encravado na garganta. São Brás olhou para o céu, rezou e, em seguida, fez o sinal da cruz na garganta do menino. No mesmo instante, ele ficou milagrosamente curado. Por esse milagre, até os dias de hoje São Brás é invocado para curar os males da garganta. p g g Em todos os lugares do mundo, quando uma criança ou qualquer pessoa se engasga, a invocação direta ao Santo logo é rezada: "São Brás te proteja." Ou simplesmente: "São Brás."6 São Braz era um santo conhecido por curar gargantas, conforme comprova o relato acima citado. A mãe de Irene ao vê-la em sofrimento com a espinha na garganta, clama ao Santo (São Braz), na busca de um repentino milagre, porém o socorro por parte do santo não vem, embora a mãe clame inúmeras vezes. Só que logo que chegou o Marreca, o responsável de fazer a espinha sumir da garganta, utilizando uma faca e uma reza, que o texto não deixa claro se essa reza foi a São Braz ou direcionada a outro santo, mas a cura da garganta é atribuída ao benzedor. A espinha sumiu durante a reza que Marreco fez no momento em que “passava a faca” na garganta de Irene e balbuciava orações, que a personagem não conseguia compreender o que ele pronunciava, embora se esforçasse para isso. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Dormia uns pedaços, quando o sono podia mais que o incômodo, a dor, isso mesmo de boca aberta sobre um travesseiro, babando sem destino. Os vizinhos acudiram, era mais quem ensinava mezinhas, simpatias. Mas a espinha saiu quando bem quis, aliás, quando o Marreca chegou ...” (CELINA, 1997, p. 115). Fui-me deixando operar. Ergui o queixo, conseguinte ele mandou, e Marreca começou a encostar a lâmina em minha garganta, calcando-a de leve, não pelo fio, mas pelo lado, enquanto pronunciava sua reza, baixinho, aos cochichos. Debalde busquei entender palavra que fosse da benzedura, ele falava mais para dentro, muito balbuciado. Só conto de certo isto: no medo, depois atenção em que fiquei, esqueci a espinha, o mal-estar, a dor na garganta. Quando dei por mim, cadê espinha? Fora-se, descera evidentemente, eu nem sentira. Eu que há quarenta e oito horas só falava por mímica e boca não tinha senão para babar. Engoli em seco repetidas vezes, experimentando, e falei alto: “Mamãe, a espinha passou”. “Desceu mesmo, minha filha, verdade? Deus seja louvado!” (Idem, p. 117). Nos trechos acima, alguns elementos merecem análise. Um deles, refere-se ao consumo alimentar de peixes, um dos peixes que ainda hoje é bastante consumido em Bragança é a tainha, e é exatamente uma espinha desse peixe que engata na garganta de Irene. Toda alimentação deve ser bem mastigada, para não correr o risco de se ingerir uma espinha inteira. O peixe é um dos alimentos que requer um cuidado maior ao ingerir. Qualquer descuido pode resultar no perigo da espinha ingerida ficar presa na garganta, causando grande desconforto e consequente dor naquela região. A espinha pode ficar presa na garganta por dias. Daí a importância ou necessidade de alguém com habilidades para retirá-la. O peixe é um dos alimentos preferidos das comunidades da região bragantina, por ser esta uma região costeira, tão próxima da costa oceânica. Cabe notar que a personagem não é de imediato levada ao hospital, para que seja submetida a algum tratamento médico, e sim submetida aos 195 SEÇÃO LIVRE cuidados do Marreca, um benzedor conhecido pelos personagens do romance por suas habilidades de benzimento, reza e cura; Um pouco antes, a mãe de Irene, clama por São Braz, São Brás nasceu na cidade de Sebaste, Armênia perto do ano 300. Num certo tempo, começou a questionar sobre sua profissão de médico, pois queria servir a Deus, mas não sabia como. 6Informações disponíveis no site: https://cruzterrasanta.com.br/historia-de-sao-bras/132/102/. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 196 rezas e palavras com as quais as pessoas procuram alcançar a cura, proteção da saúde ou prevenção dos males. rezas e palavras com as quais as pessoas procuram alcançar a cura, proteção da saúde ou prevenção dos males. Fica entendido que a benzedura só é feita por oficiais especializados. Desta forma, teremos o curador de cobras (especialista), o curador e o benzedor (clínica geral). Já a simpatia é uma forma de secularização da benzedura. A sua execução não depende de um oficial especializado, qualquer pessoa que queira se disponibilizar poderá executá-la. Por isso, a simpatia passa a sensação de cura, proteção e prevenção. A simpatia não precisa ser executada como a benzedura, qualquer pessoa leiga pode fazê-la. Já a benzedura não pode ser feita por qualquer pessoa. O benzedor não ensina sua prática, a não ser em dias especiais, para alguém que esteja iniciando, se o benzedor pressentir sua morte na sexta-feira Santa, dia 25 de março, dia dos mortos ou dia de Natal. A importância desses dias é que, fora dessas datas, perderá a força para realizá-las, ficará ‘quebrado’. Ao passo que as simpatias podem ser ensinadas a qualquer momento, e não implicam a perda de forças. Por isso, a benzedura e as simpatias são empregadas por meio das rezas e as características dessas ações são os gestos, as palavras, que fazem desaparecer o motivo ou a própria doença. “O curador, rezador, ou “benzinheira” tem o poder de fazer passar de um ser humano para animal ou daquele para uma cousa, é a transferência, técnica muito usada” (ARAÚJO, 1959, p. 49). ( p ) Na região bragantina os mais velhos costumavam chamar para essas pessoas de benzedeiras, muitas já falecidas, geralmente usavam para as suas rezas um tipo de mato chamado “vassourinha”, outras vezes pião roxo e, na ausência dessas plantas, usavam o alho. Os gestos eram em cruzes ou o sinal da cruz (símbolo do cristianismo). Geralmente durante essas benzeções tais plantas murchavam, era o sinal que o quebranto saiu e aquelas folhas teriam absorvido todo o mal do corpo. Pois, como bem afirma Araújo sobre a magia a sua razão de ser repousada na lei da magia da transferência. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Cada pessoa tem dentro de si um conceito de religião e crendices. Por isso, alguns são capazes de conhecer e colocar em prática maneiras particulares de resolverem problemas de imediato. Percebemos que no romance é muito forte essa presença mítica. A fé depositada em certas simpatias, alimentos, frutas e ervas, se fazem muito presentes no momento tratado na obra. Sempre era mais comum primeiro recorrer a pessoas como o Marreca, com costumes e culturas herdadas pelas raízes étnicas, do que recorrer a um médico, visto que, nesse momento, era mais difícil o acesso à medicina canônica. Os meios naturais eram os mais acessíveis e utilizados. As doenças e suas causas nos induzem a saber melhor qual tipo de medicina onde as classificamos. Assim a cura de uma doença provocada pela quebra de um tabu (medicina religiosa) terá que se processar através de um ritual. Não importa que entre nesse ritual, por exemplo, o uso de um vomitório ou chazinho (Medicina empírica). (ARAÚJO, 1959, p. 45). O personagem Marreca era um “rezador contra espinhas de peixe, maus-olhados, quebrantos, esipras” (CELINA, 1997, p. 116). Mas, conforme está no romance, não é um simples rezador, devido as suas atividades, mas alguém respeitado por seus poderes de cura. Antigamente, encontravam-se muitas pessoas com essa denominação. Eram apenas rezadores ou benzedores. Hoje, a maioria são idosos, muitos já morreram, alguns chegaram até passar esses ensinamentos e práticas para os seus filhos ou netos, mas estes, na maioria das vezes, rejeitaram dar continuidade. “No candomblé a medicina é mística, é sacerdotal, sua terapêutica não é mágica e sim religiosa” (ARAÚJO, 1959, p. 46). Vamos entender um pouco mais sobre essa medicina mágica, uma vez que, segundo Araújo (1959), há uma diferença entre benzedura e simpatia, embora ambas sejam do ritual protetivo, ambas também apresentam um conjunto de gestos, SEÇÃO LIVRE 197 SEÇÃO LIVRE qualquer doença, diarreia ou vômito em uma criança jamais vai se dizer que foi algo provocado por certos comportamentos humanos, o que se vai dizer é que pode ter adquirido alguma bactéria, e a criança é levada imediatamente para o hospital, jamais vai se dizer que uma dor de cabeça foi causada pelo sol, a não ser uma insolação que é visível. A questão aqui de todo esse conhecimento e sabedoria, os mais antigos dirão que foi por “mau olhado”, ventre caído ou outras doenças que somente uma benzedeira pode tirar, caso contrário, vão levar para um médico, não vai ficar curada, pois não é doença causada por bactérias, e não acreditavam em superstições. Na citação a seguir, temos no exemplo da curandeira, mais uma personagem do romance que utiliza seus saberes sobre plantas e alimentos que curam. Pelo décimo segundo dia da doença, apareceu em casa a Vijoca. Vijoca, tempos em tempos vinha à cidade, ali se demorava mais ou menos um mês, receitando, pondo emplastros, benzendo e fomentando, dando passes, com suas artes de curar. Era aqui e acolá, dum lado e doutro, Itaiara, Bragança, Carutapera, Viseu, não parava, sempre chamada para ver doente. Não chegava para as encomendas. Mamãe, que padecia de umas dores de madre, há dias mandara chama-la. Procurada como era, a Vijoca demorou-se. Quando veio para ver mamãe, achou foi o povo todo fazendo quarto à minha irmã desenganada. g - Ai, dona Vijoca, lhe chamei para mim, mas quem está mal é minha filha! B l f l il Vij lh l i - Ai, dona Vijoca, lhe chamei para mim, mas quem está mal é minha filha! Bem calma, a fala tranquila, a Vijoca olhou longo e atentamente a menina, e pediu um dente de alho. Com o dente de alho benzeu Stela, acabou, parou, assim uns instantes, como quem escuta. Falou, sempre o mesmo descanso na voz: “Eles estão dizendo que foi vento na nuca que ela pegou”. A Vijoca, sem dizer palavra, atenta, como se escutasse ainda as suas vozes: “Eles estão dizendo que se fizerem o remédio direito, como eu vou ensinar, ela fica boa, e fica perfeita”. Houve um silêncio. E pela primeira vez mamãe sorriu. A curandeira pediu papel e lápis. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Outras vezes é a ‘benzinheira’ a pessoa que tem a força para mandar o quebranto para as areias gordas do mar sagrado. (ARAÚJO, 1959, p. 49). As personagens presentes na obra ainda hoje se configuram no cotidiano de cidades como Bragança. Em Aráujo, a oração se escutava um pouco o que rezava, mas as da região bragantina, não se escutava nada, apenas usavam gestos com as mãos fazendo cruzes na testa, na costa, nas mãos, e elas bocejavam bastantes dizendo que a criança estava com muito mal olhado, lacrimejavam bastante. Quando isso acontecia, geralmente, as pessoas iam em busca das benzedeiras, que passavam mais credibilidade durante as sessões de benzimento. Sobre a cabeça da criança com um raminho verde (o que vimos casualmente era jumeira, poderia ser de arruda, guiné, etc.), enquanto fazia cruzes, rezava. O benzimento para surtir efeito deve ser executado durante três dias seguidos de manhã com a criança ainda em jejum. No primeiro dia, com uma folha, no segundo com duas, no terceiro com três. Quando é arruda ou guiné pó benzimento é feito com um pequeno galho. Deve-se ter cuidado de lança-lo na água corrente, atirando com as costas voltadas para o rio, sem olhar para traz, mandando que em nome de Deus, dos santos e da Virgem Maria que o mal siga para as águas do mar sagrado, deixando a vitima da moléstia sã e sossegada. (ARAÚJO, 1959, p. 52). Naquela época se a criança estivesse com a cabeça quente, o benzedor tirava o sol da cabeça, com uma fralda enxuta, ou guardanapo, na cabeça e um copo cheio de água. Colocava os dois juntos na cabeça. Enquanto não parasse de borbulhar, ela rezava. Segundo Araújo (1959, p. 50) “coloca na cabeça do doente um pano branco e uma garrafa branca cheia de água até o gargalo; vai rezando; benzendo, quando a doença começa a sair a água ferve”. Também tirava o ventre caído, quando as crianças apresentavam diarreia e febre repentina, após alguém ter levantado a criança acima da cabeça. Para o que conhecemos atualmente, 7HORTELÃ – Mentha piperita – Família: Labiadas. Outros nomes: Hortelã- pimenta, menta. Descrição: Planta de 30 a 60 cm, ligeiramente aveludada. USO MEDICINAL: Na hortelã estão reunidas, em elevado grau, as propriedades antiespasmódicas, carminativas, estomáquicas, estimulantes, tônicas, etc. Prescreve-se a hortelã como remédio na atonia das vias digestivas, flatulências, timpanite (especialmente a de causa nervosa), cáuculos biliares, icterícia, palpitações, tremedeiras, vômitos (por nervosidade), cólicas uterinas, dismenorréia. É um medicamento eficaz contra os catarros das mucosas, já porque favorece a expectoração, já porque combate a formação de novas matérias a expulsar. Aplica-se o sumo embebido em algodão para acalmar as dores de dente. (BALBACH, 1968, p. 252). SEÇÃO LIVRE Sentou-se, muito lenta, junto à mesa da sala de jantar, apanhou a xícara de café quentinho que a Rita lhe trouxera, pediu a papai que escrevesse (era analfabeta) e foi ditando: “Urina de três meninos “Urina de três meninos hortelã de três qualidades: de panela, hortelã-pimenta7 e verga-mota (erva morta) tabaco torrado sal na mãozinha...” sal na mãozinha...” - Sal na mãozinha? – interrompeu mamãe. “Sim. Senhora pega um pouco de sal que dê para encher a cova da mão da criança, sal esse que se torra e se mistura à hortelã para ser socado”. Prosseguia: “Álcool canforado gergelim mamona azeite doce óleo de amêndoas também doces. óleo de amêndoas também doces. Soque tudo, ponha numa cuia de coco, junte a urina dos três meninos, e leve ao fogo para esquentar. Com a mistura, bem aquecida, faça três fricções, uma por noite, três noites seguidas. Depois nove pílulas contra estupor, uma banda por noite, são dezoito noites. Dê as pílulas desmanchadas no leite de mulher que estiver criando um menino. Ao fim dos dezoito dias, fazer, três dias seguidos, três chás de palha de 7HORTELÃ – Mentha piperita – Família: Labiadas. Outros nomes: Hortelã- pimenta, menta. Descrição: Planta de 30 a 60 cm, ligeiramente aveludada. USO MEDICINAL: Na hortelã estão reunidas, em elevado grau, as propriedades antiespasmódicas, carminativas, estomáquicas, estimulantes, tônicas, etc. Prescreve-se a hortelã como remédio na atonia das vias digestivas, flatulências, timpanite (especialmente a de causa nervosa), cáuculos biliares, icterícia, palpitações, tremedeiras, vômitos (por nervosidade), cólicas uterinas, dismenorréia. É um medicamento eficaz contra os catarros das mucosas, já porque favorece a expectoração, já porque combate a formação de novas matérias a expulsar. Aplica-se o sumo embebido em algodão para acalmar as dores de dente. (BALBACH, 1968, p. 252). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 198 alho, bem fortes. Que é para cortar o mal pela raiz, eles estão dizendo (CELINA, 1997, p. 104-105). No romance Vijoca representa uma figura popular. As pessoas depositavam uma confiança em função dos favores e conhecimentos curativos, suas ações de curandeira, esse mistério envolvendo o espiritual e as crendices mais conhecidas. Pois as pessoas presentes na história, no caso a família que aceita essa ajuda, essa oração, ou remédios, como emplastos, banhos receitados por curandeiros, representam pessoas reais que, pela religiosidade, são mais fáceis de aceitar ou de serem manipuladas. SEÇÃO LIVRE Aqui, no entanto, não se trata de manipulação, mas de fé. Algumas pessoas que se dedicam a esses trabalhos não cobram qualquer valor em dinheiro, mas podem receber ajudas ou troca de favores, até porque diziam que era um dom recebido de Deus. Atualmente, os curandeiros são conhecidos por praticarem medicina tradicional. Segundo Araújo (1959), o curandeiro não ensina suas rezas, geralmente seguem o calendário religioso. Em determinadas épocas recorrem à religião para adquirir mais forças pela confissão. Jajaba, pai-de-terreiro do candomblé, no dia 15 de agosto confessou. “Pernambucano, assim era chamado Artur Francisco da Cruz, o presidente do tore que funciona à Rua Socorro, também confessou nesse dia. Entrevistados, ambos disseram o que resumimos: quando a gente tem um trabalho grande e precisa de mais forças é bom confessar e comungar”. Santos e divindades seriam então os oficiais da medicina mágica. E muitos padres, sem o saber, se tornam os ministradores das “forças” e “virtudes” a muitos dirigentes de tore, candomblé ou curadores que na igreja aparecem em determinados dias com fito exclusivos de recebê-las (ARAÚJO, 1959, p. 74). O benzedor ou curandeiro tem seus segredos e seguem rituais religiosos próprios, e buscam reforços no Catolicismo no intuito de intervenções nas coisas sagradas, e de certa forma proibidas pela Igreja, quando usadas indevidamente. Dentro dessa perspectiva, os trabalhos, as benzeções têm resultados imediatos pelo sobrenatural com auxílio de elementos da natureza, como ervas e outros tipos de alimentos, pois junto com tais elementos, recebem em oração como um encantamento, como uma fórmula mágica, uma espécie de energia benéfica no que será ingerido ou utilizado para expulsão da doença ou outros males no corpo. - Mamãe, que dores são estas, tão fortes, da Rita? É Dr. Oscar foi das personagens dessa época uma que se plantou nas minhas lembranças para nunca mais. Não posso sentir o cheiro de chá de erva-cidreira que não o veja diante de mim, ele e seus purgantes pra vermes. Cada mês, aquele dia de - É dor de madre, dor nos intelitos (mamãe assim denominava tudo quanto ficasse dentro da barriga, estômago etc.), moça que não se cuida, em certos dias, que anda na terra quente, come tamarindo, fruta ácida, carrega peso, toma banho de igarapé gelado sem poder, dá nisso. Vai botando tuas barbas de molho, estás vendo!” (CELINA, 1997, p. 196). SEÇÃO LIVRE SEÇÃO LIVRE SEÇÃO LIVRE 199 A coitada engolia o chá pelando, mais as descomposturas, calada, fungando o choro, bufando de dor.” (CELINA, 1997, p. 195). A coitada engolia o chá pelando, mais as descomposturas, calada, fungando o choro, bufando de dor.” (CELINA, 1997, p. 195). Neste trecho, enfatizamos a natureza feminina, contada de uma forma mais popular e a solução caseira para o desconforto causado neste período eram os chás, que segundo a narrativa, eram tomados sem muitas regalias. A forma de tratamento apresentada no discurso retrata algo típico das mulheres interioranas, alguns cuidados nesta fase eram recomendados, e deveriam ser cumpridos rigorosamente. Os tabus estão presentes nas mais diferentes fases da vida e são significativos para o bem-estar do corpo e a preservação da saúde. Por exemplo: mulheres durante a menstruação devem evitar o consumo de abacaxi, melancia e jaca dura para não inflamar o útero; outra regra geral é a proibição de comer com “cisma” (aborrecido), pois pode causar amolecimento do corpo e febre (SANTOS, 2008, p. 33). Ainda hoje a mulher durante a menstruação segue regras que foram passadas de geração a geração. Há diversas crenças sobre esse delicado período feminino, nas quais não se sabe quando nem onde começaram. Vejamos algumas relacionadas à alimentação: a) Não se deve comer ovo, que causa mau cheiro; b) Evitar comer abacate, que é gorduroso e pode dar pus; c) Evitar tomar suco de cupuaçu, pode matar, aumentar a cólica ou causar hemorragia; d) Evitar qualquer fruta cítrica, aumenta o fluxo de sangue e pode causar hemorragia; e) Evitar tomar açaí, faz mal! Vai mudar a cor do sangue e causar inflamações. Estes são alguns exemplos de saberes populares entre as mulheres, mesmo as mulheres que tenham alcançado um grau de estudo formal mais avançado, muitas delas continuam a “obedecer” essas regras. g ç g Ainda no caso das mulheres na fase do ciclo menstrual, no romance Menina que vem de Itaiara (CELINA, 1997) podemos verificar a dificuldade que certas mães tinham, na época, de falar claramente sobre esse assunto com as suas filhas, a citação a seguir denota essa realidade. - Mamãe, que dores são estas, tão fortes, da Rita? 2 ALIMENTOS QUE CURAM As ervas medicinais presentes na obra em questão são bem frequentes e corriqueiras na vida dos bragantinos. Na época retratada pelo romance, nas zonas rurais era muito comum as casas terem em seus quintais ou terrenos plantações ou hortas medicinais, com um variado tipo de ervas de poder curativo, que serviam para diversos tipos de doenças, tais como: gripes, resfriados, dor de cabeça, cólicas etc., conforme afirma Balbach “Assim, as plantas têm esta tríplice tarefa: Alimentam e curam o homem e purificam o ar” (BALBACH, 1968, p. 52). Vejamos o exemplo da personagem Rita que, com os efeitos do período menstrual, recorria-se ao uso de chá de ervas para amenizar o incômodo destes dias. A Rita todo santo mês, ô martírio! Três dias malacafenta, o primeiro então, certo passá-lo inteirinho deitada, gemendo, na rede, no quartinho aos fundos do alpendre, uma garrafa de água quente na barriga. E suava, ensopava camisões, se torcia de dores. Mamãe fazia chá de sene e lhe levava, quente, ralhando: - “Tá pensando que é canja, dona moça? Vá, vá andar na terra escaldando, vá dançar no morro até essas tantas, vá! Pensa que é brincadeira de menina fêmea? Mulher é bicho fino, qualquer coisinha se arrebenta. SEÇÃO LIVRE - É dor de madre, dor nos intelitos (mamãe assim denominava tudo quanto ficasse dentro da barriga, estômago etc.), moça que não se cuida, em certos dias, que anda na terra quente, come tamarindo, fruta ácida, carrega peso, toma banho de igarapé gelado sem poder, dá nisso. Vai botando tuas barbas de molho, estás vendo!” (CELINA, 1997, p. 196). - É dor de madre, dor nos intelitos (mamãe assim denominava tudo quanto ficasse dentro da barriga, estômago etc.), moça que não se cuida, em certos dias, que anda na terra quente, come tamarindo, fruta ácida, carrega peso, toma banho de igarapé gelado sem poder, dá nisso. Vai botando tuas barbas de molho, estás vendo!” (CELINA, 1997, p. 196). A citação acima mostra como a mãe de Rita usa algumas figuras de linguagem para ensinar a filha sobre o ciclo menstrual e suas consequências, expressando a dificuldade que as mães tinham de tratar claramente de assuntos dessa natureza com a própria filha. A citação acima mostra como a mãe de Rita usa algumas figuras de linguagem para ensinar a filha sobre o ciclo menstrual e suas consequências, expressando a dificuldade que as mães tinham de tratar claramente de assuntos dessa natureza com a própria filha. Outro exemplo são os purgantes, eles nos fazem lembrar o terror da infância de nossos pais, como o caso da famosa “mamona”, bastante presente em Menina que vem de Itaiara. Veremos alguns casos: “Sei de um pé de cajarana no quintal, um dia levei umas lambadas porque, às escondidas, me fartei de cajaranas, depois de um bruto purgante de mamona” (CELINA, 1997, p. 11). p Ainda em Menina que vem de Itaiara (CELINA, 1997) temos o purgante sendo receitado por um médico, como na transcrição a seguir. 200 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 juízo, mamãe me agarrando à força, o copo de mamona8 dissolvida em chá de cidreira na mão: “Engula, engula, senão apanha!”. Tive pena dele, abri a boca, ele virou a mamona com quenopódio, dissolvida no chá de cidreira. (CELINA, 1997, p. 15). 8MAMONA- Ricinus communis – família: Euforbiáceas. Outros nomes: Ricino, carrapateiro, palma Cristo. Descrição: A mamona, quando nova, é uma planta herbácea que se lignifica com a idade, assumindo o aspecto de uma árvore pequena e muito esgalhada. USO MEDICINAL: das sementes, limpas das cascas, se obtém um óleo de efeito purgativo. O mesmo azeite também se presta para combater os vermes intestinais. As sementes são Tóxicas. “vinte delas, ingeridas por um adulto, geralmente lhe conduzem a morte. Seis a sete já liquidam uma criança. (BALBACH, 1968, p. 280). SEÇÃO LIVRE Provavelmente um dos primeiros métodos de cura que veio das plantas, o animal, instintivamente, quando adoece recorre às ervas curativas, o homem notou este gesto instintivo dos animais e, orientado por observações próprias, verificou-se que nas ervas há poder curativo. Desta forma, iniciaram-se as pesquisas em torno da natureza, especificamente com as ervas. “Tão perfeita é a natureza que provê tudo que necessitamos, pelas plantas todas doenças são curáveis, porém muitas curas ainda não foram descobertas”. (BALBACH, 1968, p. 45). p Para Balbach (1968), o mal muitas vezes se manifesta aos mais vulneráveis, por isso ficam propícios às enfermidades. Antes que uma doença possa ser classificada como diabetes, tuberculose, etc., por exemplo, há processos anormais que entorpecem o metabolismo nutritivo, mesmo outras enfermidades não podem surgir sem encontrarem um caminho preparado, se a pessoa estiver com suas funções orgânicas normais, primeiro a pessoa se predispõe para o mal, em seguida surgem, então, as desordens em todo mecanismo funcional, acumulam-se no organismo humores mórbidos, repletos de toxinas. Os micróbios encontram então um terreno propício para a sua atuação, se alastram, apesar da resistência que encontram da parte das defesas naturais do organismo enfraquecido. Há que se falar também nas ervas com poder de danificar os órgãos, algumas com poder de até mesmo de levar a óbito, ervas com alto poder degenerativo. Ervas venenosas. No romance, a protagonista Irene, diante do sofrimento da prima Diquinha, pensa que se conhecesse alguma erva, poria fim ao sofrimento da prima. Porém o sentido de acabar o sofrimento seria através da morte, não pela cura, mas o que importa entender no presente contexto, é a importância que as ervas têm no cotidiano das pessoas. Conforme retrata o texto a seguir. Falei que tia Helga tinha uma filha aleijada. Não saía da cama e comia num prato de madeira ... vivia num quarto à parte, escondida. Não falava, não andava, tudo era pela mão dos outros ... Foi crescendo, foram percebendo que ficara inutilizada. Quando queria comer, chamar alguém, batia os pés ... Quando tive a certeza de que prima Diquinha jamais ficaria boa, nunca iria tomar um banho no rio, brincar, falar, correr como a gente ... Um dia, tive um pensamento horrível: soubesse eu de um mato, umas ervas, um chá, umas gotas ou pílulas, lhe faria beber. Falei que tia Helga tinha uma filha aleijada. Não saía da cama e comia num prato de madeira ... vivia num quarto à parte, escondida. Não falava, não andava, tudo era pela mão dos outros ... Foi crescendo, foram percebendo que ficara inutilizada. Quando queria comer, chamar alguém, batia os pés ... Quando tive a certeza de que prima Diquinha jamais ficaria boa, nunca iria tomar um banho no rio, brincar, falar, correr como a gente ... Um dia, tive um pensamento horrível: soubesse eu de um mato, umas ervas, um chá, umas gotas ou pílulas, lhe faria beber. Soubesse dar injeção, possuísse uma dessas ampolas de matar, aplicaria, às escondidas, na minha pobre prima, assim que ela dormisse. Para que nunca mais acordasse (CELINA, 1997, p. 94). SEÇÃO LIVRE 201 “Astésia, esta criança tem febre”. Astésia palpou-a: “Tem não, madrinha, é que estava dormindo”. “Sim, mas não no abafado, com tudo aberto...” Conversa continuou, a Rita daí a pouco trouxe café com macaxeira, merendamos. Logo Deusarina, ama de Stela, apareceu com o prato com banana e aveia. Porém, ela recusou-se a comer. Mamãe tornou a palpá-la: “Astésia, Stela está doente, olha só a quentura, aumentou”. Trataram de vesti-la, agasalha-la, fazer-lhe um chá de sabugueiro que teve que engolir bem quente ... Quando papai entrou, pelas dez horas, Stela tinha tombado num agitado sono, cheio de tremores, entrecortado de breves, inarticulados gritos. Mamãe com dificuldade deu-lhe água açucarada, e ela pegou noutro soninho, ligeiramente mais calmo. (CELINA, 1997, p. 101). Em algumas ocasiões, as ervas deixam de trazer o êxito esperado no tratamento. Por vezes o paciente pode se sentir melhor e acabar abandonando o tratamento antes de alcançar uma cura total, pois o segredo da cura está no uso ordeiro e perseverante das plantas medicinais até que todo o mal seja extinto. Outras situações, é quando acontece um efeito contrário do que o paciente espera. Nesse caso, sobrevém uma aparente piora, e o mal parece agravar-se. Muitos, quando isso ocorre, ficam desesperados e abandonam o tratamento. Mas é justamente esta crise um sinal de que o organismo começou a reagir, expulsando as substâncias morbosas. O remédio está fazendo efeito. (BALBACH, 1968, p. 55). p O sabugueiro9 (Sambucus nigra) é uma das plantas medicinais mais utilizadas no mundo. É bastante utilizado para tratar os sintomas da gripe e do resfriado. Na narrativa, aconteceu justamente esta reação com o chá, como se tivesse agravado a febre com o sabugueiro. Essa reação é natural quando o organismo começa a reagir. O simples ato de dar água açucarada é como se fosse a solução, de cortar efeito ou superstição da mãe em depositar sua fé. No romance ainda temos a receita de Vijoca “não tardou a aparecer quem pedisse a fórmula da garrafada. Mamãe, prestativa, fez ela própria uma porção, que pela vizinhança distribuiu” (CELINA, 1997, p. 106). SEÇÃO LIVRE Soubesse dar injeção, possuísse uma dessas ampolas de matar, aplicaria, às escondidas, na minha pobre prima, assim que ela dormisse. Para que nunca mais acordasse (CELINA, 1997, p. 94). Por isso, é um perigo certas porções de ervas ou acessos a ervas desconhecidas, ou até mesmo ingeri-las acidentalmente, como o uso prolongado e persistente. Por isso, é um perigo certas porções de ervas ou acessos a ervas desconhecidas, ou até mesmo ingeri-las acidentalmente, como o uso prolongado e persistente. Certa vez, a mãe de Irene percebeu a alteração da pressão de uma criança, Stela, sua afilhada, que estava em visita à sua casa, e novamente recorreu aos conhecimentos tradicionais na busca pela recuperação da saúde da criança, conforme relato a seguir: NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 “Também a Vijoca me curou de uma tosse dos diabos. Nem precisou vir ter conosco. Concentrou-se mesmo onde estava dando consultas, na Boca-do-Caminho, e receitou: - Apeí, fedegoso, mussambê, flor de mamão macho, raiz e fruta de urucu, cebolinha, gengibre, mandacaru (ela chamava jaramacaru), jambu, laranja-da-terra, raiz de manjerioba (também chamada de pamarioba), açúcar branco, mel de abelha. Tudo cozido em lambedor. - Apeí, fedegoso, mussambê, flor de mamão macho, raiz e fruta de urucu, cebolinha, gengibre, mandacaru (ela chamava jaramacaru), jambu, laranja-da-terra, raiz de manjerioba (também chamada de pamarioba), açúcar branco, mel de abelha. Tudo cozido em lambedor. Receitas de Vijoca era, pá-casca. Diz mamãe que foi eu tomar a primeira colherada, e se notou melhora. Com tão pronto efeito, não tardou a aparecer quem pedisse a fórmula da garrafada. Mamãe, prestativa, fez ela própria uma porção, que pela vizinhança distribuiu. A asma de Tereza, filha de seu Guimarães, uma asma velha, de dez anos, curou-se. A tosse de cachorro de João Mandu, idem. João Mandu habitava bem um quarteirão abaixo de nossa casa, na rua do Capim. Pois sua tosse muitas noites impedia o sono de meus pais, tão longe e tão fundo ecoava. E não é que o homem ficou bonzinho dessa bronquite de anos, apenas com uma garrafada? Só para mamãe, lambedor de Vijoca nunca fez efeito. Podia-o tomar às carradas, bem nenhum lhe advinha para uma rouquidão, um catarro, uma piema, antes piorava. Mas mamãe sempre afirmou que seu organismo era diferente do de todo mundo. Magnésia que para qualquer cristão é laxante, para ela, o mesmo que água, vidros inteiros que ingerisse. Produzia até efeito contrário...” (CELINA, 1997, p. 106) (Grifo nosso). No fragmento especificado, encontramos a citação de um composto de ervas, de plantas medicinais, de cascas e de outros ingredientes. Todos próprios da região bragantina. Quando em quantidade certa, um preparo especial, com minuciosa sequência e ações coordenadas, se transforma na chamada garrafada, que era muito usada naquela época e até hoje ainda o é, porém em menor frequência. Era usada para as mais variadas doenças e males, como no próprio texto cita alguns exemplos: asma, tosse e bronquite. Ainda hoje encontramos pessoas que utilizam este composto. Como exemplo, temos as profissionais do sexo que, na maioria das vezes, exercem a prostituição como meio de sustento familiar. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Após uma relação sexual com as pessoas que atendem profissionalmente, para que não adquiram doenças dos seus “clientes”, ingerem doses da garrafada para a limpeza e proteção do corpo. Algumas profissionais do sexo acreditam que a garrafada serve, inclusive, como material anticonceptivo, para evitar uma gravidez indesejada. Também é fácil encontrarmos esses compostos nas farmácias de manipulação, locais que funcionam como laboratórios de produção dessas combinações de ervas e plantas medicinais e as comercializam em garrafas de um litro, meio litro, ou frascos com doses pequenas. Alguns desses locais ainda oferecem o benzimento e rezas, porém com o nome de consulta ou exame. SEÇÃO LIVRE Esse relato refere-se à garrafada, termo usado para uma combinação de ingredientes, dos mais diversos possíveis, tais como: ervas, frutas, cascas, alguns chegam a conter até pedaços de animais etc., produzidos com algumas exigências de tempo, de preparo, de sequência etc., após a preparação o composto é guardado em uma garrafa (daí o nome garrafada), geralmente de vidro e, normalmente, combate para uma grande quantidade de doenças. Há diversos tipos de garrafadas, com os mais variados objetivos. A seguir, segue o relato de Irene sobre a garrafada: 9SABUGUEIRO – Sambucus nigra – Família: Caprifoliáceas. Outros nomes: Sabugueiro da Europa. Descrição: Pequena árvore, de 3 a 4 metros de elevação. Tronco de casca pardacento-acinzentada, verrugosa, folhas opostas, compostas imparipenadas, de 5 a 7 folíolos curtamente peciolados, oval-lanceolados, acuminados, serreados. Este é o sabugueiro da europa, o sabugueiro muito conhecido do Brasil é o Sambucus australis. O primeiro produz frutos comestíveis ao passo que este último nunca os produz. Na medicina caseira ambos têm a mesma aplicação. USO MEDICINAL: As flores são eméticas, catárticas. Porém, quando secas, perdem suas propriedades laxativas, secas empregam-se, em infusão, contra os resfriados, as anginas, as gripes, etc. A casca, a raiz e as folhas são indicadas na retenção da urina, na hidropisia, no reumatismo. Os reumáticos devem igualmente tomar banho com o cozimento das folhas. A infusão das folhas e cascas, em fomentações, é prescrita contra as inflamações superficiais da pele, furúnculos, erisipela, queimaduras etc. Neste último caso, também se aplicam diretamente as folhas machucadas. Tiram a dor em pouco tempo. Em banhos as folhas são boas para hemorroidas. Nas enfermidades eruptivas, como no sarampo, rubéola, escarlatina, varíola, etc. O chá das flores é muito indicado, porque provoca rapidamente a transpiração. A frutinha purifica o sangue e limpa os rins. Seca, tostada, moída e preparada como café, é boa para cortar a diarreia. Dose: Uso interno – flores, 8 gramas para 1 litro de água; folhas, cascas e raízes, 10 a 15 gramas para 1 litro de água; 4 a 5 xícaras por dia. Uso externo – flores, 30 gramas para 1 litro de água; folhas, cascas e raízes, 50 gramas para 1 litro de água. (BALBACH, 1968, p. 315). 202 SEÇÃO LIVRE SEÇÃO LIVRE 203 Consideramos que a presente análise foi importante para alcançarmos um maior conhecimento sobre certos alimentos nutritivos para a saúde do corpo, assim como plantas medicinais. Esperamos que esta pesquisa contribua para futuros estudos, por meio desta obra que nos possibilitará entender um pouco mais da literatura paraense pela composição de Lindanor Celina. O trabalho pode trazer mais discussões sobre elementos não explorados do romance, assim como poderá servir como inspiração para futuras pesquisas, quiçá seja de grande importância para a comunidade acadêmica amazônica. REFERÊNCIAS ARAÚJO, Alceu Maynard. Medicina Rústica. Brasiliana. Vol. 300 - 3 ed. São Paulo: Nacional, 1959. CELINA, Lindanor. Menina Que Vem de Itaiara. Rio de Janeiro: Conquista, 1997. CELINA, Lindanor. Menina Que Vem de Itaiara. Rio de Janeiro: Conquista, 1997. BALBACH, A. As Plantas Curam. 24ª ed. Editora: M.V.P. São Paulo: 1968. BALBACH, A. As Plantas Curam. 24ª ed. Editora: M.V.P. São Paulo: 1968. BALBACH, A. As Plantas Curam. 24ª ed. Editora: M.V.P. São Paulo: 1968. Bíblia Sagrada. Ezequiel 47,12. A fonte maravilhosa que jorra do templo. Tradução Monges Beneditinos de Maredsous (Bélgica). ed:195ª São Paulo: Editora Ave-Maria, 2011. Bíblia Sagrada. Ezequiel 47,12. A fonte maravilhosa que jorra do templo. Tradução Monges Beneditinos de Maredsous (Bélgica) ed:195ª São Paulo: Editora Ave Maria 201 Bíblia Sagrada. Ezequiel 47,12. A fonte maravilhosa que jorra do templo. Tradução Monges Beneditinos de Maredsous (Bélgica). ed:195ª São Paulo: Editora Ave-Maria, 2011. Monges Beneditinos de Maredsous (Bélgica). ed:195ª São Paulo: Editora Ave-Maria, 201 Bíblia Sagrada. Genêsis, 1,11-12. A criação. Tradução Monges Beneditinos de Maredsous (Bélgica).195ª ed. São Paulo: Editora Ave-Maria 2011. 3 CONSIDERAÇÕES FINAIS A pesquisa empreendida no romance Menina que vem de Itaiara (CELINA, 1997), trouxe-nos um olhar mais cuidadoso com a alimentação, com a saúde, e a relação entre elas, como a primeira influencia a segunda numa relação complexa de troca, parceria ou adversidade. Trouxe também a presença de certos remédios caseiros, como purgantes, chás e a garrafadas, estas usadas quando as mulheres estão com problemas de útero, também há outros tipos de garrafada muito usada para curar problemas nos pulmões, gripes, tosses etc. Explora também o lado feminino, quando as mulheres no período menstrual não podem comer certos alimentos. Também tem grande destaque os diversos tipos de ervas, que são benéficas para muitos problemas de saúde, sem deixar de mencionar os curandeiros e benzedeiros presentes no romance, e que ganha grande destaque em Bragança, a qual ainda tem benzedeiras com tais habilidades para curar, de maneira mais prática certas doenças. ARAÚJO, Alceu Maynard. Medicina Rústica. Brasiliana. Vol. 300 - 3 ed. São Paulo: Nacional, 1959. ARAÚJO, Alceu Maynard. Medicina Rústica. Brasiliana. Vol. 300 - 3 ed. São Paulo: Nacional, 1959. GEERTZ, Clifford. A interpretação das culturas. Rio de Janeiro: LTC, 2012. GEERTZ, Clifford. A interpretação das culturas. Rio de Janeiro: LTC, 2012. LIMA, Walter Chile Rodrigues. Saber Tradicional: Suporte para o exercício da Territorialidade de uma Comunidade no Estuário Amazônico. In: Revista Ensaio Geral. Belém, v.l, n. l, Jan-jun. 2009. PACHECO, Sandra Simone Moraes. O hábito alimentar enquanto comportamento cultural produzido. In: FREITAS, Maria do C. Soares de; FONTES, Gardênia A. V.; OLIVEIRA, Nilce. Escritas e narrativas sobre alimentação e cultura. Salvador: EDUFBA, 2008. 422 p. ROMÃO, Tito Lívio Cruz. Sincretismo Religioso como Estratégia de Sobrevivência Transnacional e Translacional: divindades africanas e santos católicos em tradução. In: Trabalhos em Linguística Aplicada. On-line version ISSN 2175-764X. Trab. linguist. apl. vol. 57, n.1. Campinas: Jan./Abr. 2018, disponível: https://doi.org/10.1590/010318138651758358681. Universidade Federal do Ceará, Fortaleza (CE), Brasil. cruzromao@terra.com.br. Acesso em 13/04/2020. vol. 57, n.1. Campinas: Jan./Abr. 2018, disponível: https://doi.org/10.1590/010318138651758358681. Universidade Federal do Ceará, Fortaleza (CE), Brasil. cruzromao@terra.com.br. Acesso em 13/04/2020. SANTOS, Fábio Rodrigo dos. Práticas alimentares em Ilha de Maré, Salvador, Bahia. In: FREITAS, Maria do C. Soares de; FONTES, Gardênia A. V.; OLIVEIRA, Nilce. Escritas e narrativas sobre alimentação e cultura. Salvador: EDUFBA, 2008. 422 p. , g , , FREITAS, Maria do C. Soares de; FONTES, Gardênia A. V.; OLIVEIRA, Nilce. Escritas e narrativas sobre alimentação e cultura. Salvador: EDUFBA, 2008. 422 p. SEÇÃO LIVRE 205 SEÇÃO LIVRE – – INTRODUÇÃO O educador tem um papel de suma importância ao instigar, aguçar e estimular o processo de ensino-aprendizagem, isto é, ele é essencial na criação de mecanismos metodológicos diversos que possam abordar a construção da identidade individual e coletiva. Uma dessas maneiras é o trabalho com a realidade da cidade onde se localiza a escola, englobando questões culturais, sociais, econômicas, históricas e ambientais. Para tanto, a compreensão do papel do professor está na sua relação direta com a forma como desenvolve a mediação entre o estudante e o conhecimento, possibilitando o pensamento e a reflexão sobre aquilo que deseja e precisa ensinar para que o estudante, efetivamente, possa aprender. Dessa forma, o professor de Geografia precisa dialogar com a análise da leitura espacial do mundo, algo que não é tão simples assim, pois requer a construção de maneiras de fazer os estudantes aguçarem suas capacidades críticas. Então, para que isso seja concretizado, a proposta curricular da disciplina de Geografia precisa estar voltada para conteúdos e conceitos que estimulem o desenvolvimento do raciocínio e da espacialidade e que valorizem os alunos como principais sujeitos do processo de ensino-aprendizagem. A construção crítica do raciocínio geográfico possibilita ao educando o desenvolvimento de um pensamento mais complexo que irá reelaborar e dinamizar as suas relações espaciais. Tal complexidade intelectual permite que o educando compreenda diversas questões como locais-globais relativas à organização de novas estratégias para pensar e analisar os conceitos científicos. Aprender significa oferecer ao sujeito um tipo de conhecimento relevante já existente na estrutura cognitiva de quem aprende. Isto é, para que um novo tipo de conhecimento atinja o sujeito de forma positiva, é necessário que esse conhecimento tenha significado lógico, o qual pode ser apresentado ou descoberto pelo próprio sujeito. Porém, esse conhecimento deve contribuir para a criação de novos significados e corroborar com os já existentes. A vida cotidiana de muitos desses estudantes está baseada nas suas práticas socioespaciais, nos seus deslocamentos diários adquiridos ao longo do tempo. Os estudantes da Educação de Jovens e Adultos (EJA), em sua maioria, são trabalhadores que trazem consigo uma bagagem de ricas informações sobre as relações de trabalho e as cadeias produtivas e exploratórias. CAMPUS BRAGANÇA IX OVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 206 Data de submissão: 21.09.2020 Data de aprovação: 15.12.2020 INTRODUÇÃO É relevante compreender as práticas socioespaciais da EJA para refletir sobre a aprendizagem significativa com intuito de possibilitar que os alunos discutam a rotina, o cotidiano, a realidade a partir das suas espacialidades, o que pode ser um estímulo importante para o processo de construção dos saberes. Este artigo apresenta a importância da aprendizagem significativa e como a prática docente influencia diretamente no processo de ensino-aprendizagem, e, por fim, discute a Geografia apresentada no Documento Curricular da Secretaria Municipal de Educação de Duque de Caxias (2012) das etapas IV e V da EJA, utilizada até os dias atuais. Essa discussão possibilita compreender a maneira como o ensino da Geografia pode contribuir para pensar as práticas socioespaciais dos estudantes, de modo que os conteúdos trabalhados pelos docentes possam ser mais significativos. A escolha do município de Duque de Caxias para a realização dessa pesquisa se deu porque o referido município apresenta uma riqueza econômica significativa, porém outros aspectos como saúde, segurança e educação ainda necessitam de muitos investimentos, pois deixam a desejar. 207 SEÇÃO LIVRE Figura 1 - Localização do município de Duque de Caxias, RJ Fonte: Organizado por Seabra (2016, apud SOUZA, 2017, p. 46). Figura 1 - Localização do município de Duque de Caxias, RJ Fonte: Organizado por Seabra (2016, apud SOUZA, 2017, p. 46). Duque de Caxias está localizada na Região Metropolitana do Rio de Janeiro, na Baixada Fluminense, ocupando uma área de 442 km². O território está dividido em quatro distritos: 1º Distrito – Duque de Caxias (41 km²); 2º Distrito – Campos Elísios (989 km²); 3º Distrito – Imbariê (64 km²) e o 4º Distrito – Xerém (239 km²). A população do município de acordo com o IBGE (2010) era de 855.048 habitantes, com a estimativa de 924.624 (IBGE, 2020). No território caxiense, são notadas desigualdades sociais bem aparentes, o que deixa patentes os contrastes e as injustiças demográficos. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 208 aprendizagem significativa é uma teoria que tem como critério abordar o conhecimento atrelado àquilo que já é conhecido pelo sujeito, valorizando suas experiências e mostrando significados no que vai ser aprendido posteriormente. É importante ressaltar que o significativo não está necessariamente relacionado a um novo conhecimento, e sim ao novo sentido que o indivíduo dará ao adquirir outras informações do conhecimento que já tinha, ou seja, fazendo uma interlocução entre a estrutura de conhecimento fomentada pelos conceitos e a cognição. aprendizagem significativa é uma teoria que tem como critério abordar o conhecimento atrelado àquilo que já é conhecido pelo sujeito, valorizando suas experiências e mostrando significados no que vai ser aprendido posteriormente. É importante ressaltar que o significativo não está necessariamente relacionado a um novo conhecimento, e sim ao novo sentido que o indivíduo dará ao adquirir outras informações do conhecimento que já tinha, ou seja, fazendo uma interlocução entre a estrutura de conhecimento fomentada pelos conceitos e a cognição. q ç q j , j , terlocução entre a estrutura de conhecimento fomentada pelos conceitos e a cognição. O professor tem um papel essencial na construção de novas aprendizagens, pois mediante os conteúdos a serem desenvolvidos, é possível fazer as análises da realidade dos estudantes motivando-os a criar possibilidades, argumentações, comparações, de modo que os mesmos consigam trocar experiências, ouvir outros pontos de vista, agregar novas formas de conhecimento, facilitando, assim, a aprendizagem significativa. O conhecimento pode ser mediado por ambos, tanto o professor quanto os estudantes têm a possibilidade de aprender e construir juntos, afinal todos os indivíduos têm suas próprias histórias. Moraes (2004, p. 60) afirma que: “Nessa perspectiva, a função do educador é gerar atividades por meio de procedimentos concretos que facilitam a motivação para o aprendizado e a de ser um processo de construção do conhecimento”. A aprendizagem não pode ser vista como um processo mecânico, por isso o professor, com suas técnicas e metodologias, possibilita ao aluno perceber que os conceitos científicos podem ser incorporados ao seu cotidiano, aplicando nas concepções variadas situações. 1 A APRENDIZAGEM SIGNIFICATIVA COMO PROCESSO DE CONSTRUÇÃO DO CONHECIMENTO GEOGRÁFICO A pesquisa em questão almejou trabalhar de forma que o processo de ensino- aprendizagem tivesse relação com a prática social do estudante, objetivando mediar os conhecimentos e conceitos apreendidos ao longo da vida para uma abordagem que buscasse entender conteúdos significativos para os estudantes. Trabalhar a prática social do estudante significa compreender a importância de utilizar o conhecimento já trazido por ele para pensar as diferentes abordagens dos conteúdos escolares, promovendo os conceitos espontâneos e científicos nos quais o estudante aprende a desenvolver suas habilidades intelectuais para a construção de uma aprendizagem dos conteúdos geográficos. É É nesse sentido que o presente estudo pode contribuir para a teoria da aprendizagem significativa, discutidos nos textos de Moraes (2004), Alegro (2008), Tomita (2009), Morais e Cavalcanti (2011). Assim, objetiva-se trazer para o cotidiano um trabalho pedagógico e didático que valoriza os conhecimentos prévios dos discentes como troca de saberes e diálogos, pois a 2 O CURRÍCULO E A PRÁTICA DOCENTE EM GEOGRAFIA NA EDUCAÇÃO DE JOVENS E ADULTOS O currículo é um documento importante para o educador, pois contém os conteúdos que serão supostamente trabalhados ao longo do ano letivo, tendo o professor a possibilidade de selecioná-los. É importante ressaltar que o currículo não é só conteúdo, mas relação de poder, cultura e conhecimento, já que está relacionado à forma como a educação se consolida dentro e fora da sala de aula; às relações entre professor e educando, professor e escola, professor e comunidade; aos conceitos e conteúdos propostos; à forma ideológica que este documento é construído, dentre tantas outras situações às quais o documento se associa. Diante disso, o professor tem um papel importante, pois se faz necessário entender as contradições cotidianas, ter consciência política do seu papel enquanto educador, debater, buscar e romper com as desigualdades e a exclusão social. É importante ressaltar que as experiências cotidianas dos estudantes não estão necessariamente organizadas no currículo formal a ser seguido, e, por isso, cabe ao docente fazer as articulações dos conteúdos e dos conceitos científicos. Compreender a função social da disciplina de Geografia permite que o conhecimento construído entre professor e aluno em sala de aula seja um instrumento essencial para o processo de aprendizagem, uma vez que, para Kaercher (2003), a Geografia é feita no nosso dia a dia e deve partir do cotidiano do educando, cuja realidade torna-se objeto de reflexão e, assim, deve estar presente na espacialidade costumeira de cada aluno em sua plenitude e complexidade. Assim, o professor, ao organizar seu material para uma determinada aula, tem a possibilidade de criar a disciplina de inúmeras formas, pois, segundo Castrogiovanni (2003), o ensino da Geografia deve priorizar a análise do espaço vivido e oportunizar a compreensão da riqueza espaço-temporal e da própria existência humana. Portanto, neste estudo, incentiva-se o uso das práticas socioespaciais como uma abordagem. Santos (2008, p. 146) afirma: “[...] as práticas sociais, portanto, necessitam de um conhecimento da espacialidade das coisas, um conhecimento geográfico, ainda que não sistematizado”. A prática docente deve ser construída também através da realidade escolar, articulando teoria e prática. Logo, o professor pode aprofundar discussões, trocar experiências, instigar os conhecimentos adquiridos no trabalho, na vida, na escola com o intuito de debater, analisar, questionar, dentro do seu campo disciplinar. Por conseguinte, o educando pode fazer associações com o vivido, fomentando as múltiplas influências sociais presentes nas relações do aluno com a escola e com o mundo. SEÇÃO LIVRE SEÇÃO LIVRE 209 c) O indivíduo precisa se permitir a aprender novos conhecimentos e, com o auxílio de materiais e métodos, possibilitar ainda mais a apreensão do que já sabe para atrelar ao novo conhecimento; c) O indivíduo precisa se permitir a aprender novos conhecimentos e, com o auxílio de materiais e métodos, possibilitar ainda mais a apreensão do que já sabe para atrelar ao novo conhecimento; c) O indivíduo precisa se permitir a aprender novos conhecimentos e, com o auxílio de materiais e métodos, possibilitar ainda mais a apreensão do que já sabe para atrelar ao novo conhecimento; d) A organização do professor é fundamental, uma vez que ele apresenta oportunidades e várias alternativas para que os alunos possam refletir a partir dos conhecimentos prévios e do novo conhecimento. d) A organização do professor é fundamental, uma vez que ele apresenta oportunidades e várias alternativas para que os alunos possam refletir a partir dos conhecimentos prévios e do novo conhecimento. Tomita (2009, p. 63) afirma ainda que a “aprendizagem significativa pode ser entendida como uma possibilidade de aprender por caminhos múltiplos, porque envolve a razão pessoal de forma individualizada”. A organização dos procedimentos deve estar pautada em objetivos e metodologias bem coerentes aos conceitos, pois assim os estudantes compreendem a função das atividades e são estimulados a refletir. Em vista disso, o professor obterá as respostas expressivas no processo de ensino-aprendizagem. O obstáculo que enxergamos para a construção do conhecimento está situado na maneira como aprendemos a pensar sobre o mundo, isto é, como concebemos a NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 ç No entanto, quando há a ausência de uma formação continuada ou até mesmo exista uma formação inicial precária, o professor apresentará dificuldades para trabalhar determinados conteúdos e conceitos, fazendo com que não haja uma reflexão crítica daquilo que está sendo ensinado; e os discentes passam, então, a ser prejudicados à medida que não são estimulados a refletir. Tomita (2009) afirma que a aprendizagem deve ser significativa, mas, para isso, as experiências e vivências anteriores precisam ser consideradas como fatores relevantes para a construção desse processo. Daí é possível que os alunos consigam compreender melhor e solucionar problemas do seu cotidiano, o que pode ocasionar, ainda de acordo com a autora, novas ações, atitudes e comportamentos e, consequentemente, novas aprendizagens. Nessa expectativa e com base em Ausubel (1980), é recomendável incorporar na prática de ensino, a ideia de que o fator mais importante e que influencia na aprendizagem é aquilo que o aluno já sabe, isto é, a aprendizagem ocorre a partir do que o aluno já conhece e do que já se aprendeu. Assim, no processo de ensino- aprendizagem é importante levar em conta a soma acumulada da educação formal, sistematicamente ocorrida, e também da não formal ocorrida assistematicamente no convívio familiar e social. (TOMITA, 2009, p. 61). O conhecimento científico é um instrumento de “poder” daquele que o detém. De acordo com Moraes (2004), é importante que o indivíduo consiga perceber que a aprendizagem tem um significado e, para que tenha relevância, são necessários quatro fatores: a) O material disponível aos indivíduos deve ser significativo, de modo que haja relevância em aprender determinado conteúdo, não apenas a memorização de estados e capitais no ensino de Geografia, caso contrário, não haverá uma aprendizagem significativa; a) O material disponível aos indivíduos deve ser significativo, de modo que haja relevância em aprender determinado conteúdo, não apenas a memorização de estados e capitais no ensino de Geografia, caso contrário, não haverá uma aprendizagem significativa; b) O indivíduo necessita ter vontade própria de aprender e também utilizar materiais que podem ser um estímulo maior no processo de aprendizagem. Para ilustrar essa afirmação, citam-se dois exemplos de uma mesma aula de Geografia com a temática de problemas ambientais da cidade: uma em que o professor só ilustra a aula com a sua própria fala, e outra em que há a exposição oral, porém, com o auxílio de vídeo, imagens e comparações de linguagem; 2 O CURRÍCULO E A PRÁTICA DOCENTE EM GEOGRAFIA NA EDUCAÇÃO DE JOVENS E ADULTOS Silva aponta que: 210 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 articulação das disciplinas na escola. Isso não significa desconhecer as decisões políticas que vão desde como o corpo docente é formado, assim como se dá a escolha dos objetivos pedagógicos, quais linguagens serão desenvolvidas assim como os conteúdos (tanto aqueles que os alunos portam como aqueles que são consagrados nas disciplinas). (SILVA, 2012, p. 111). As atividades didático-pedagógicas produzidas por meio de uma leitura de mundo mais crítica facilitam e contribuem bastante para a construção de conhecimentos, conteúdos e conceitos científicos, pois é o momento em que o educador direciona e possibilita o amadurecimento do raciocínio, o pensamento reflexivo e as manifestações empíricas através do que foi trabalhado teoricamente. Para Cavalcanti, [...] professores abertos e sensíveis ao diálogo com seus alunos buscam contribuir para o processo de atribuição de significados aos conteúdos trabalhados, baseados em cada contexto específico, de acordo com as representações dos alunos, considerando suas capacidades individuais e de grupo, mas dirigindo o processo a fim de promover a aprendizagem-formação de conceitos – ‘adiantando’ seu desenvolvimento buscando ‘quebrar’ barreiras entre conhecimentos científico, escolar e cotidiano. (CAVALCANTI, 2012, p. 173). Nesse sentido, é necessário ter um conhecimento mínimo da comunidade escolar de forma a reconhecer a espacialidade na qual a escola está inserida, isto é, estar a par das inter- relações sociais, físicas, políticas, econômicas, culturais e históricas. Retomando a especificidade da discussão em relação ao currículo, ele é a seleção dos conteúdos produzidos por determinados grupos ou indivíduos baseada numa política educacional. Para Silva, O currículo é sempre o resultado de uma seleção: de um universo mais amplo de conhecimento e saberes seleciona-se aquela parte que vai construir, precisamente o currículo. As teorias do currículo tendo decidido quais conhecimentos devem ser selecionados, buscam justificar por que ‘esses conhecimentos’ e não ‘aqueles’ devem ser selecionados. (SILVA, 1999, p. 15). O professor tem sua importância na construção e no desenvolvimento do pensamento crítico do discente, por isso torna-se relevante compreender como esse currículo institucional, que muitas vezes vem sendo imposto precisa ser seguido, é apresentado na sala de aula, ou seja, é importante captar como o docente influencia na prática curricular. Segundo Malta, O currículo tem uma especificidade muito particular. É importante que, durante as aulas, haja problematização do mundo do trabalho e da própria produção social, de forma que os alunos tenham a possibilidade de refletir a partir das suas vivências cotidianas. Ainda recorrendo a Santos, A especificidade da EJA enquanto modalidade de ensino voltada para jovens e adultos é atendida quando se articula trabalho, seja em seu sentido social seja em sua dimensão histórica, à produção do espaço. Construir a ideia de que todos são responsáveis por essa produção, inclusive os trabalhadores, embora a sociedade de classes determine papéis e responsabilidades diferenciadas nesse processo, constitui- se tarefa da geografia escolar para todas as modalidades e níveis de ensino. Na EJA, entretanto, a proporção dessa premissa alcança graus bastante elevados, uma vez que muitos dos alunos já fazem parte do processo produtivo e já têm demarcado seu lugar na divisão social do trabalho, o que os torna ao mesmo tempo testemunhas e agentes de todo o processo estudado. (SANTOS, 2008, p. 279). Na Educação de Jovens e Adultos, ensinar os conteúdos a partir das vivências dos alunos facilita o processo de ensino-aprendizagem, já que suas práticas socioespaciais são ilustradas. Além disso, as discussões podem ser relevantes para o entendimento do cotidiano e da realidade social dos estudantes. Novamente evocando Santos, Pode-se dizer que o mundo do trabalho é a temática que mais imediatamente se relaciona com a Educação de Jovens e Adultos. Em uma proposta curricular voltada para essa modalidade, as questões e reflexões sobre o trabalho são praticamente imperativas se pretende atender o princípio através do qual a realidade vivida pelos estudantes deva ser considerada. (SANTOS, 2008, p. 184). Assim sendo, a problemática do mundo do trabalho e da experiência vivida pelos estudantes da EJA pode ser um elemento na construção de uma proposta curricular de Geografia que privilegie um pensamento autônomo e crítico, bem como das práticas socioespaciais. 2 O CURRÍCULO E A PRÁTICA DOCENTE EM GEOGRAFIA NA EDUCAÇÃO DE JOVENS E ADULTOS Todos os que dele participam e todos os que têm ingerência sobre o currículo, não o fazem de maneira neutra. Trata- se de uma área impregnada de valores, ideologias, forças, interesses e necessidades e exige, para uma definição mais exata, a explicitação de um quadro filosófica, histórica e política. (MALTA, 2013, p. 342). O currículo tem uma função social, uma vez que nele estão presentes os conteúdos a serem lecionados. Porém, como é feita as seleções desses conteúdos? Existe alguma intencionalidade? Refletindo sobre essas questões, compreende-se que o currículo é um documento de tamanha significância. Silva afirma que: Da perspectiva pós-estruturalista, podemos dizer que o currículo é também uma questão de poder e que as teorias do currículo, na medida em que buscam dizer o que o currículo deve ser, não podem deixar de estar envolvidas em questões de poder. Selecionar é uma operação de poder. Privilegiar um tipo de conhecimento é uma operação de poder. Destacar, entre as múltiplas possibilidades, uma identidade ou subjetividade como sendo a ideal é uma operação de poder. (SILVA, 1999, p. 16). SEÇÃO LIVRE SEÇÃO LIVRE 211 A organização curricular é baseada na concepção de alguns sujeitos ou de um determinado grupo, que através desse documento expressam a sua funcionalidade para o público da EJA. De acordo com Santos: Isso quer dizer que, ao nos referimos à EJA, devemos levar em conta uma série de complexas relações que envolvem a aquisição de saberes por parte de seu público. Mesmo em programas oficiais de ensino essa dimensão deve ser observada, sem a qual corremos o risco de abandonar toda trajetória histórica percorrida pela tentativa de possibilitar o acesso da classe trabalhadora a outras interpretações e visões de mundo para além daquelas construídas na luta pela sua sobrevivência. (SANTOS, 2008, p. 62). É importante que, durante as aulas, haja problematização do mundo do trabalho e da própria produção social, de forma que os alunos tenham a possibilidade de refletir a partir das suas vivências cotidianas. Ainda recorrendo a Santos, Desta forma, o discente conseguiria compreender de maneira mais simplificada a integração entre os conhecimentos escolares e os do cotidiano. De acordo com Santos, Desta forma, o discente conseguiria compreender de maneira mais simplificada a integração entre os conhecimentos escolares e os do cotidiano. De acordo com Santos, [...] a educação popular só faz sentido quando desenvolvida exatamente na fronteira das disciplinas, o que não quer dizer, obviamente, que os conhecimentos específicos destas não devam ser considerados. Assim, a escolha e o desenvolvimento do tema gerador a partir do universo temático dos alunos exigem conhecimentos espaciais que relacionem o local ao global, ou seja, impõem uma perspectiva que vê o mundo através da articulação das escalas de análise. (SANTOS, 2008, p. 146). Em relação à disciplina de Geografia, inúmeras foram as reformulações na ciência. Segundo Santos (2008, p. 141): “o objetivo de sintetizar o conteúdo da série escolar ocultava, na verdade, uma geografia tradicional, conservadora e que desprezava o espaço vivido e construído pelos próprios sujeitos participantes das classes supletivas”. A Geografia que se encontra presente na escola pode ser entendida a partir da abordagem histórica, sendo justificada por temáticas e questionamentos que são explicados como anacrônicos e ultrapassados. As transformações que ocorrem no ensino de Geografia na educação básica são consequências das políticas educacionais influenciadas pelas novas abordagens da ciência geográfica, que também está relacionada aos atuais contextos político, econômico e cultural. Diante disso, busca-se mostrar a importância que a Geografia Escolar tem no currículo da EJA, pois, a partir dessa ciência, é possível desenvolver em sala de aula reflexões sobre a vida do aluno trabalhador, auxiliando na criticidade da sua relação com a natureza e dos seus processos históricos, sociais, econômicos e culturais. Conforme Santos: [...] na seleção de conteúdos normalmente encontrada em livros didáticos e propostas curriculares para a Educação Básica, o mundo do trabalho sempre foi tratado como algo não muito propício a ser aprofundado nas aulas de geografia. É nesse sentido que ações pedagógicas na EJA podem contribuir para a centralidade dessa categoria na perspectiva geográfica. Em função disso, novos conteúdos passam a compor o temário geográfico escolar, dentre eles a maior atenção à geografia do trabalho. (SANTOS, 2011, p. 102). A disciplina de geografia no currículo escolar auxilia na construção de um pensamento autônomo, reflexivo e pode até mesmo proporcionar um raciocínio espacial do estudante da EJA a partir de suas vivências, sejam elas como discente ou como trabalhador. 3 A EDUCAÇÃO DE JOVENS E ADULTOS DO MUNICÍPIO DE DUQUE DE CAXIAS E SUA ESTRUTURA CURRICULAR EM GEOGRAFIA O currículo do ensino fundamental regular não deve ser comparado ao da EJA, mas revisto e apresentado de maneira distinta, afinal, o público alvo é diferenciado e com isso, torna- se inviável fazer qualquer tipo de comparação entre estes dois tipos de educando, pois cada faixa etária corresponde a diferentes fases. Tomando mais uma vez de empréstimo as palavras de Santos: Se é verdade que devemos respeitar as características desse público ao se pensar um conjunto de provas específicas para ele, é também fato que não devemos entender por isso que a EJA deve ser considerada uma modalidade menor, na qual os conhecimentos escolares devam ser menos prezados ou facilitados. O desafio é 212 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 justamente manter o nível de complexidade exigido no ensino dirigido para crianças e adolescentes, porém adaptado à realidade de vida dessas pessoas. (SANTOS, 2008, p. 150). Uma geografia assim concebida leva-nos fatalmente a considerar o aluno, em especial aquele oriundo das classes populares, como um ser neutro, sem vida, sem cultura, sem história – um ser que não trabalha, não produz a riqueza neste momento histórico e neste espaço geográfico determinado. O aluno não participa do espaço geográfico que ele estuda. Se o espaço não é encarado como algo em que o homem (aluno) está inserido, natureza que ele próprio ajuda a moldar, a verdade geográfica do indivíduo se perde e a Geografia se torna alheia para ele. (RESENDE, 1986, p. 18). Desta forma, o discente conseguiria compreender de maneira mais simplificada a integração entre os conhecimentos escolares e os do cotidiano. De acordo com Santos, Compreender a importância das diferentes linguagens (gravuras, músicas, literaturas, dados estatísticos, documentos de diferentes fontes, mapas, gráficos e tabelas) na leitura da paisagem, tornando-se capaz de interpretar, analisar e relacionar diversas informações sobre o espaço. Utilizar a linguagem gráfica para obter informações, compreendendo a espacialidade dos fenômenos geográficos. Fonte: Organizado pelos autores baseados na Secretaria Municipal de Educação de Duque de Caxias (2012). Tabela 1 - Objetivos Gerais da disciplina de Geografia do município de Duque de Caxias Objetivos Gerais de Geografia Redimensionar o entendimento de mundo a partir dos conceitos geográficos básicos, tais como: território, lugar, paisagem, espaço geográfico. Compreender a diversidade socioespacial, percebendo a importância de respeitá-la. Compreender a cidadania como parte das relações entre a sociedade e a natureza integrando-se como sujeito ativo, construtor/transformador de seu espaço histórico e cultural. Desenvolver a autonomia do aluno para que ele busque o conhecimento em diferentes tipos de linguagem. Compreender a importância das diferentes linguagens (gravuras, músicas, literaturas, dados estatísticos, documentos de diferentes fontes, mapas, gráficos e tabelas) na leitura da paisagem, tornando-se capaz de interpretar, analisar e relacionar diversas informações sobre o espaço. Utilizar a linguagem gráfica para obter informações, compreendendo a espacialidade dos fenômenos geográficos. Fonte: Organizado pelos autores baseados na Secretaria Municipal de Educação de Duque de Caxias (2012). p g Objetivos Gerais de Geografia O primeiro objetivo enfatiza a importância dos conceitos geográficos, sendo esses conceitos-chaves. Porém, é relevante incluir o conceito de região não citado no documento, pois esta parte da discussão sobre a disciplina permite compreender as diferentes organizações espaciais a partir de diversas formas de estruturação do território econômico, social, político e cultural, dentre outros âmbitos. O segundo objetivo, apesar de trazer a questão relacionada à “diversidade socioespacial”, não explica quais seriam os grupos ou o tipo de diversidade aos quais faz referência: quilombolas, ribeirinhos, indígenas, entre outros. Não é um objetivo direto, ficando aberto a diferentes interpretações para os professores. O segundo objetivo, apesar de trazer a questão relacionada à “diversidade socioespacial”, não explica quais seriam os grupos ou o tipo de diversidade aos quais faz referência: quilombolas, ribeirinhos, indígenas, entre outros. Não é um objetivo direto, ficando aberto a diferentes interpretações para os professores. O terceiro objetivo propõe o resgate da importância da cidadania na relação homem X natureza e do sujeito como ativo e transformador do espaço geográfico. Desta forma, o discente conseguiria compreender de maneira mais simplificada a integração entre os conhecimentos escolares e os do cotidiano. De acordo com Santos, Neste artigo, apresenta-se parte do currículo do ensino fundamental II, que está presente no Documento Oficial da Secretaria Municipal de Educação de Duque de Caxias (2012), para a EJA. Segundo o documento, a criação do currículo surgiu a partir de consultas realizadas junto à secretaria e aos professores de Geografia dos cursos de EJA. A intenção, portanto, é analisar esse currículo por meio dos objetivos gerais da disciplina de Geografia e dos objetivos específicos das etapas IV (sexto e sétimo anos) e V (oitavo e nono anos), saber como os conteúdos estão organizados e se há algum tipo de temática referente à cidade local ou se apenas seguem as orientações propostas pelo Ministério da Educação (MEC). Na tabela a seguir, estão os objetivos gerais do ciclo da disciplina de Geografia. SEÇÃO LIVRE 213 Tabela 1 - Objetivos Gerais da disciplina de Geografia do município de Duque de Caxias Objetivos Gerais de Geografia Redimensionar o entendimento de mundo a partir dos conceitos geográficos básicos, tais como: território, lugar, paisagem, espaço geográfico. Compreender a diversidade socioespacial, percebendo a importância de respeitá-la. Compreender a cidadania como parte das relações entre a sociedade e a natureza integrando-se como sujeito ativo, construtor/transformador de seu espaço histórico e cultural. Desenvolver a autonomia do aluno para que ele busque o conhecimento em diferentes tipos de linguagem. Compreender a importância das diferentes linguagens (gravuras, músicas, literaturas, dados estatísticos, documentos de diferentes fontes, mapas, gráficos e tabelas) na leitura da paisagem, tornando-se capaz de interpretar, analisar e relacionar diversas informações sobre o espaço. Utilizar a linguagem gráfica para obter informações, compreendendo a espacialidade dos fenômenos geográficos. Fonte: Organizado pelos autores baseados na Secretaria Municipal de Educação de Duque de Caxias (2012). Tabela 1 - Objetivos Gerais da disciplina de Geografia do município de Duque de Caxias Objetivos Gerais de Geografia Redimensionar o entendimento de mundo a partir dos conceitos geográficos básicos, tais como: território, lugar, paisagem, espaço geográfico. Compreender a diversidade socioespacial, percebendo a importância de respeitá-la. Compreender a cidadania como parte das relações entre a sociedade e a natureza integrando-se como sujeito ativo, construtor/transformador de seu espaço histórico e cultural. Desenvolver a autonomia do aluno para que ele busque o conhecimento em diferentes tipos de linguagem. NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Existe a necessidade de maior investimento na sistematização e na organização do currículo para a Educação de Jovens e Adultos. O trabalho pedagógico demanda comprometimento e uma produção própria de material didático, de maneira que os alunos sejam capazes de problematizar o que vivenciam, de criar o gosto por estar na sala de aula, de fazer relações e comparações para que o processo de aprendizagem seja contínuo. Nos objetivos específicos apresentados na tabela 2, serão apontadas explicações de como esse currículo poderia incluir conteúdos e temáticas que partilhassem a experiência e o cotidiano dos alunos. Tabela 2 - Objetivos Específicos da Etapa IV da disciplina de Geografia do município de Duque de Caxias Objetivos Específicos Conhecer os conceitos fundamentais que se relacionam aos temas específicos da geografia. Compreender os principais movimentos da Terra e suas influências sobre as atividades humanas. Desenvolver o uso da linguagem cartográfica. Compreender a dinâmica dos fatores internos e externos que afetam o relevo terrestre. Compreender as diferentes formas de regionalização do espaço geográfico brasileiro. Conhecer os diferentes setores da atividade econômica. Compreender a relação existente entre os processos de industrialização e urbanização. Reconhecer os indicadores da desigualdade social no Brasil decorrentes do processo de urbanização das grandes cidades. Compreender as especificidades dos espaços urbano e rural. Compreender os diferentes aspectos relacionados à dinâmica populacional brasileira. Fonte: Secretaria Municipal de Educação de Duque de Caxias, 2012, organizado pelos autores. Tabela 2 - Objetivos Específicos da Etapa IV da disciplina de Geografia do município de Duque de Caxias Objetivos Específicos Conhecer os conceitos fundamentais que se relacionam aos temas específicos da geografia. Compreender os principais movimentos da Terra e suas influências sobre as atividades humanas. Desenvolver o uso da linguagem cartográfica. Compreender a dinâmica dos fatores internos e externos que afetam o relevo terrestre. Compreender as diferentes formas de regionalização do espaço geográfico brasileiro. Conhecer os diferentes setores da atividade econômica. Compreender a relação existente entre os processos de industrialização e urbanização. Reconhecer os indicadores da desigualdade social no Brasil decorrentes do processo de urbanização das grandes cidades. Compreender as especificidades dos espaços urbano e rural. Compreender os diferentes aspectos relacionados à dinâmica populacional brasileira. Fonte: Secretaria Municipal de Educação de Duque de Caxias, 2012, organizado pelos autores. Compreender as especificidades dos espaços urbano e rural. Compreender as especificidades dos espaços urbano e rural. Compreender as especificidades dos espaços urbano e rural. p p p ç Compreender os diferentes aspectos relacionados à dinâmica populacional brasileira. Desta forma, o discente conseguiria compreender de maneira mais simplificada a integração entre os conhecimentos escolares e os do cotidiano. De acordo com Santos, Estudar cidadania na EJA é importante, pois o educador é capaz de auxiliar na criticidade sobre o direito de ir e vir, na construção do pensamento do que é viver dignamente com acesso à educação e à saúde de qualidade, à segurança, ao saneamento básico, à luz e à água encanada. Logo, essa discussão é relevante para o conhecimento do cidadão. O terceiro objetivo propõe o resgate da importância da cidadania na relação homem X natureza e do sujeito como ativo e transformador do espaço geográfico. Estudar cidadania na EJA é importante, pois o educador é capaz de auxiliar na criticidade sobre o direito de ir e vir, na construção do pensamento do que é viver dignamente com acesso à educação e à saúde de qualidade, à segurança, ao saneamento básico, à luz e à água encanada. Logo, essa discussão é relevante para o conhecimento do cidadão. p O quarto objetivo poderia ser substituído porque não destaca nenhum elemento geográfico coerente. E trabalhar com linguagens é parte do sistema operacional pedagógico. Os objetivos quinto e sexto fazem parte também do sistema operatório não apresentando conteúdo pertinente à disciplina de Geografia, pois eles integram as competências. Apesar de compreender a importância da análise das diferentes linguagens para ensinar Geografia, bem como dos diferentes documentos, entende-se que não só a paisagem pode ser analisada a partir dessas linguagens. O quarto objetivo poderia ser substituído porque não destaca nenhum elemento geográfico coerente. E trabalhar com linguagens é parte do sistema operacional pedagógico. Os objetivos quinto e sexto fazem parte também do sistema operatório não apresentando conteúdo pertinente à disciplina de Geografia, pois eles integram as competências. Apesar de compreender a importância da análise das diferentes linguagens para ensinar Geografia, bem como dos diferentes documentos, entende-se que não só a paisagem pode ser analisada a partir dessas linguagens. Os objetivos gerais presentes no currículo do município de Duque de Caxias são relevantes para a disciplina escolar. Entretanto, temáticas referentes ao cotidiano que privilegia as práticas socioespaciais dos estudantes da EJA poderiam estar incluídas no referido documento. O currículo seguido é o formal, que não tem de fato uma proposta para esse público. Para Resende: 214 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 a 2 - Objetivos Específicos da Etapa IV da disciplina de Geografia do município de Duque de Caxias Objetivos Específicos SEÇÃO LIVRE 215 SEÇÃO LIVRE espaço brasileiro, pois destacaram as diferentes regionalizações, não enfatizando qual tipo, ou seja, sem ressaltarem se era a subdivisão do IBGE, do Milton Santos, das Regiões Geoeconômicas, entre outras; mesmo apresentadas, essas regionalizações estão na categoria dos objetivos específicos. Por isso, retoma-se o discurso anterior: será que realmente esses conteúdos não poderiam ser trabalhados de outra maneira, levando em consideração, por exemplo, a contribuição das experiências do estudante, como o relato de um possível deslocamento migratório para o Sudeste? Por que não trabalhar o espaço brasileiro a partir da relação com o mundo do trabalho, fazendo uma abordagem espacial baseada nas cidades? É pautado nesse tipo de temática e conteúdo que o professor da EJA pode criar mecanismos para que o discente sinta-se motivado a participar, contribuir com suas experiências e se interessar pelo que está sendo desenvolvido. Para Resende, Acreditamos, isto sim, em assumir, dentro do sistema formal de ensino, o papel de agente de contradição, acumulando, através de uma prática ainda que só parcialmente inovadora e mesmo setorizada, forças que podem articular-se, ao nível de luta social concreta, com o avanço das reivindicações política dos trabalhadores por um outro sistema de ensino, por uma verdadeiramente nova, por uma nova sociedade. (RESENDE, 1986, p. 166). Acreditamos, isto sim, em assumir, dentro do sistema formal de ensino, o papel de agente de contradição, acumulando, através de uma prática ainda que só parcialmente inovadora e mesmo setorizada, forças que podem articular-se, ao nível de luta social concreta, com o avanço das reivindicações política dos trabalhadores por um outro sistema de ensino, por uma verdadeiramente nova, por uma nova sociedade. (RESENDE, 1986, p. 166). É necessário que a escola cumpra seu papel criando mecanismos para que a educação seja acessível ao público que a frequenta em diferentes turnos. É preciso que o conhecimento se torne acessível às camadas populares de forma a minimizar os impactos sofridos diariamente por sua realidade. Os demais objetivos apresentam conteúdos como: economia brasileira, industrialização, urbanização, desigualdades sociais, o urbano e o rural e dinâmicas populacionais. Desta forma, pode-se afirmar que, entre os conteúdos do 6º e 7º anos, há uma significativa parcela de temáticas voltadas para o espaço brasileiro em que o aluno terá a possibilidade de conhecer as relações existentes no seu país. p p p ç Compreender os diferentes aspectos relacionados à dinâmica populacional brasileira. p p p p Fonte: Secretaria Municipal de Educação de Duque de Caxias, 2012, organizado pelos autores. O primeiro objetivo aponta para a importância de conhecer os conceitos fundamentais da disciplina que já estão especificados nos objetivos gerais. O segundo, terceiro e quarto objetivos atendem aos conteúdos propostos para o 6º ano, embora ainda que estejam de forma bem resumida. Dentre uma gama de conteúdo, realizou-se uma seleção enfatizando os movimentos da terra, linguagens cartográficas e relevo terrestre. A linguagem cartográfica é fundamental para localização, orientação, interpretações de informações contidas em mapas, plantas, croquis, entre outros exemplos. Do mesmo modo, é necessário o conhecimento acerca do relevo, pois o aluno não precisa compreender o processo através da utilização de termos técnicos, como a orogênese, mas entender os tipos e formas para articular, por exemplo, com as inundações, com o formato de relevo encontrado na Baixada Fluminense e com suas características e especificidades. Esses conteúdos geográficos são relevantes para a disciplina, porém, nota-se que o mundo do trabalho não está inserido nos objetivos. Dessa maneira, não há reflexão sobre a vida do aluno trabalhador. Para Santos: A problemática e a análise do mundo do trabalho, portanto, devem se constituir em ações educativas fundamentais para uma proposta curricular que considere a ressignificação do saber, da experiência e a construção de um pensamento crítico e autônomo por parte dos alunos. Sem dúvida alguma, isso torna extremamente desafiador o trabalho docente e exige dos conhecimentos disciplinares a busca pela melhor forma de contribuir para esse processo, pois nem sempre a tradição seletiva do currículo das escolas para crianças contempla temas, abordagens e exemplos do mundo do trabalho. (SANTOS, 2011, p. 100). Por esse motivo, o papel da Geografia no âmbito escolar da EJA precisa ser repensado com o intento de trazer para a sala de aula conteúdos significativos daquilo que é ensinado, possibilitando a problematização e a experiência do mundo do trabalho que são instrumentos para a construção do pensamento geográfico. Como os demais objetivos dessa etapa são correspondentes ao 7º ano, entende-se que os mesmos foram bem específicos ao trabalharem o Tabela 3 - Objetivos Específicos da Etapa V da disciplina de Geografia do município de Duque de Caxias Objetivos Específicos Compreender a interação sociedade-natureza e as questões relacionadas aos problemas socioambientais. Compreender as diferentes formas de regionalização do mundo contemporâneo. Compreender os mais importantes aspectos do processo de globalização. Analisar a inserção do espaço Latino-Americano na lógica mundial do sistema capitalista. Analisar a posição da África no Mundo Contemporâneo e a influência de sua cultura na sociedade brasileira. Compreender questões relevantes no continente asiático: conflitos no Oriente Médio e o desenvolvimento da China. Analisar a crise do Estado de Bem-Estar Social e os conflitos sociais e étnico-religiosos no continente europeu. Fonte: Organizado pelos autores baseados na Secretaria Municipal de Educação de Duque de Caxias (2012). NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 NOVA REVISTA AMAZÔNICA - VOLUME IX - Nº 01 - MARÇO 2021 - ISSN: 2318-1346 Em relação aos objetivos da etapa V, partilham-se conteúdos do 8º e 9º anos. O primeiro objetivo ressalta a relação sociedade-natureza e os problemas socioambientais, porém, mais uma vez, não faz menção à realidade do aluno, sendo este um tema de suma relevância. Assim, observar-se a partir da análise da tabela 3 que a realidade socioambiental do município de Duque de Caxias não está contemplada no currículo. Nesse sentido, vale indagarmos: por que não apresentar aos discentes a relação da cidade local com os problemas socioambientais? Isto é, o Aterro Metropolitano que encerrou as atividades, a relação da Refinaria, a construção do Arco Metropolitano, por exemplo. Dessa forma, cabe ao professor fazer a articulação com o currículo, trazendo novas possibilidades a partir de diferentes escalas que podem partir do local- global ou vice-versa. g Os demais objetivos trazem os conteúdos a serem seguidos pelos Parâmetros Curriculares Nacionais (PCN) de Geografia (1998), como regionalização mundial, globalização e suas relações com os continentes americano, africano, asiático e europeu. É notória a preocupação em trazer as regionalizações mundiais nessa etapa, apresentando uma visão dos continentes de forma geral. O interessante é que não há a discussão do conceito de região no objetivo geral, como já foi mencionado anteriormente. Compreender a realidade dos outros continentes é importante para discutir e analisar questões sociais, econômicas, culturais, físicas, inclusive fazendo comparações com o espaço de vivência dos alunos, porém, se os alunos não têm o conhecimento da realidade local, não será possível fazer considerações em âmbito global. De acordo com Resende (1986, p. 156), se “o espaço vivido é sempre descrito e/ou julgado à luz da experiência concreta de trabalho e sobrevivência, à luz da posição que o sujeito ocupa nas relações sociais de produção [...]”, o saber geográfico pode estar “escondido” nas experiências que fazem parte da história real dos alunos. Diante disso, mostra-se relevante trazer uma concepção pedagógica que valorize as experiências do aluno e acredite na construção do conhecimento a partir das suas práticas socioespaciais. SEÇÃO LIVRE Todavia, não se ressaltou a questão da especificidade do Estado do Rio de Janeiro e de Duque de Caxias, por exemplo. Será que realmente os estudantes da Educação de Jovens Adultos conhecem minimamente a sua realidade, o seu bairro, a sua cidade ou o seu estado? De modo a obter resultados positivos no processo de ensino-aprendizagem, o educando precisa compreender que o seu cotidiano também faz parte desse dinamismo, sendo possível extrair dele conhecimentos que podem ser desenvolvidos na sala de aula. Nesse sentido, o professor pode mediar as práticas socioespaciais dos discentes, a geografia do trabalho, a geografia da percepção, voltando-se às temáticas trabalhadas na sala de aula para que o conhecimento seja uma troca, para que o saber científico seja construído coletivamente. 216 SEÇÃO LIVRE SEÇÃO LIVRE configura uma das instâncias formadoras da cidadania, que são as práticas cotidianas. A partir dessa concepção, a escola tem sua responsabilidade na discussão sobre o significado da cidadania que se faz presente na vida do aluno através da organização social e democrática e das lutas e incorporações dos direitos sociais. A EJA da Educação Básica está associada a um público que possui experiências próprias de vida, presentes no mundo do trabalho. Conforme Resende (1986, p. 20), “agir assim significaria, contudo, valorizar uma experiência de espaço do aluno, do aluno pobre – uma experiência de espaço que lhe é própria”. 4 CONSIDERAÇÕES FINAIS O professor como mediador do processo educativo, de forma didática, tem a possibilidade de inserir os conteúdos curriculares relacionando-os ao contexto do estudante. A compreensão do lugar e do cotidiano levam o aluno à reflexão e ao próprio entendimento do seu papel dentro daquela localidade. Trabalhar a realidade deve ser algo indispensável e mostra a preocupação do educador em querer que seu discente se (re) conheça dentro de sua realidade, possibilitando a aprendizagem significativa. A organização do currículo deve estar voltada para conteúdos que estimulem o desenvolvimento do raciocínio e da espacialidade, que valorizem o estudante como principal sujeito do processo de ensino-aprendizagem e que sejam construídos politicamente como um saber poderoso, crítico e reflexivo a ser passado, e não como um saber elitizado. O documento curricular do município de Duque de Caxias poderia destacar, na introdução do texto, informações sobre a disciplina de Geografia, sobre a relevância de se trabalhar com os conteúdos a partir das práticas socioespaciais dos estudantes. Desse modo, seria possível reforçar a necessidade de articulação entre a prática pedagógica do professor e a teoria e a prática. Então, cabe ao professor a responsabilidade de trazer determinados conteúdos que possibilitem aos educandos a compreensão da sua realidade, com exemplos da sua cidade e do seu espaço de vivência. 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Formação de professores dos anos iniciais do ensino fundamental: considerações sobre escola, conhecimento, linguagem e ensino de Geografia. Terra Livre, São Paulo, v. 1, n. 38, p. 99-120, 2012. Disponível em: https://www.agb.org.br/publicacoes/index.php/terralivre/article/view/462. Acesso em: 27 de jun de 2016 SILVA, Jorge Luiz Barcellos da. Formação de professores dos anos iniciais do ensino fundamental: considerações sobre escola, conhecimento, linguagem e ensino de Geografia. Terra Livre, São Paulo, v. 1, n. 38, p. 99-120, 2012. Disponível em: https://www.agb.org.br/publicacoes/index.php/terralivre/article/view/462. Acesso em: 27 de jun. de 2016. Terra Livre, São Paulo, v. 1, n. 38, p. 99-120, 2012. Disponível em: https://www.agb.org.br/publicacoes/index.php/terralivre/article/view/462. Acesso em: 27 de jun. de 2016. SILVA, Tomaz Tadeu da. Teoria do currículo. In: SILVA, Tomaz Tadeu da (org.). Documentos de identidade: uma introdução às teorias do currículo. Belo Horizonte: Autêntica, 1999. p. 11-17. SOUZA, Camila Vianna de. As práticas socioespaciais urbanas dos estudantes da EJA do município de Duque de Caxias - RJ. 2017. Dissertação (Mestrado em Geografia) - Programa de Pós-Graduação em Geografia, Faculdade de Formação de Professores da Universidade do Estado do Rio de Janeiro, São Gonçalo, 2017. TOMITA, Luzia Mitiko Saito. Ensino de geografia: aprendizagem significativa por meio de mapas conceituais. 2009. Tese (Doutorado em Geografia) – Programa de Pós-Graduação em Geografia Física, Departamento de Geografia, Faculdade de Filosofia, Letras e Ciências Humanas, Universidade de São Paulo, São Paulo, 2009.
https://openalex.org/W4289751828	https://zenodo.org/records/1317547/files/50.ARSLAN%20UQAILI.pdf	English	null	GAMMA-GLUTAMYL TRANSFERASE AS PREDICTOR OF EARLY VIROLOGIC RESPONSE IN SOFOSBUVIR TREATED CHRONIC HEPATITIS C GENOTYPE 3 PATIENTS	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	3,709	Abstract: Obj i Logistic regression analysis model for GGT as predictor of EVR shows 0.912 (91.2%) Area under curve (P 0 0001) ( ) Conclusion: The present study concludes low serum GGT level positively predicts an early virological response (EVR) in chronic hepatitis C patients. Key words: Gamma Glutamyl transferase, Chronic Hepatitis C, Early Virological Response ey wo ds: Gamma Glutamyl t ansfe ase, Ch onic epatitis C, a ly Vi ological esponse Corresponding Author: Burhan Memon, Medical Officer, Civil Hospital Matiari, Sindh, Pakistan, Email: burhanaslam.memon@hotmail.com, Contact no: +92-332-2605852 Please cite this article in press Burhan Memon et al., Gamma-Glutamyl Transferase as Predictor of Early Virologic Response in Sofosbuvir Treated Chronic Hepatitis C Genotype 3 Patients, Indo Am. J. P. Sci, 2018; 05(07). QR code Corresponding Author: Burhan Memon, Medical Officer, Civil Hospital Matiari, Sindh, Pakistan, Email: burhanaslam.memon@hotmail.com, Contact no: +92-332-2605852 Please cite this article in press Burhan Memon et al., Gamma-Glutamyl Transferase as Predictor of Early Virologic Response in Sofosbuvir Treated Chronic Hepatitis C Genotype 3 Patients, Indo Am. J. P. Sci, 2018; 05(07). QR code Corresponding Author: Burhan Memon, Medical Officer, Civil Hospital Matiari, Sindh, Pakistan, Email: burhanaslam.memon@hotmail.com, Contact no: +92-332-2605852 Please cite this article in press Burhan Memon et al., Gamma-Glutamyl Transferase as Predictor of Early Virologic Response in Sofosbuvir Treated Chronic Hepatitis C Genotype 3 Patients, Indo Am. J. P. Sci, 2018; 05(07). QR code Corresponding Author: Abstract: Obj i Abstract: Objective: Analyze the baseline Gamma-glutamyl transferase (GGT) as predictor of Early Virologic Response (EVR) in Sofosbuvir treated chronic hepatitis C genotype 3 patients. S d D i C i l d Study Design: Cross sectional study Study setting & Duration: Department of Medicine, Liaquat University Hospital Jamshoro/Hyderabad from August 2017 to March 2018 Subjects and Methods: A sample of 100 HCV- PCR positive; comprising of 63 male and 37 female was studied. Anti HCV- antibodies were detected by 3rd generation ELISA assay kit. Viral load HCV- RNA was detected by RT- PCR method on Cobas Amplicor (Roche Diagnostic Inc.). Hemoglobin, platelets, serum bilirubin, albumin, Prothrombin time (PT), alanine transaminase (ALT) and gamma glutamyl transferase (GGT) were detected. Data was analyzed on SPSS ver 22.0 by Student`s t- test and Chi- square test at 95% confidence interval (CI) (P < 0.05). Subjects and Methods: A sample of 100 HCV- PCR positive; comprising of 63 male and 37 female was studied. Anti HCV- antibodies were detected by 3rd generation ELISA assay kit. Viral load HCV- RNA was detected by RT- PCR method on Cobas Amplicor (Roche Diagnostic Inc.). Hemoglobin, platelets, serum bilirubin, albumin, Prothrombin time (PT), alanine transaminase (ALT) and gamma glutamyl transferase (GGT) were detected. Data was analyzed on SPSS ver 22.0 by Student`s t- test and Chi- square test at 95% confidence interval (CI) (P < 0.05). Results: Age (mean ± SD) of male and female was noted as 43.93±7.95 and 43.45±4.54 years respectively (P=0.032). Of 100 l 63% l d 37% f l (P 0 940) H l bi P th bi ti d GGT h i ifi t test and Chi- square test at 95% confidence interval (CI) (P < 0.05). Results: Age (mean ± SD) of male and female was noted as 43.93±7.95 and 43.45±4.54 years respectively (P=0.032). Of 100 sample, 63% were male and 37% were female (P=0.940). Hemoglobin, Prothrombin time and serum GGT shows significant differences between groups. Logistic regression analysis model for GGT as predictor of EVR shows 0.912 (91.2%) Area under curve (P=0.0001). q f ( ) ( ) Results: Age (mean ± SD) of male and female was noted as 43.93±7.95 and 43.45±4.54 years respectively (P=0.032). Of 100 sample, 63% were male and 37% were female (P=0.940). Hemoglobin, Prothrombin time and serum GGT shows significant differences between groups. IAJPS 2018, 05 (07), 6567-6571 IAJPS 2018, 05 (07), 6567-6571 Burhan Memon et al ISSN 2349-7750 Available online at: http://www.iajps.com Available online at: http://www.iajps.com GAMMA-GLUTAMYL TRANSFERASE AS PREDICTOR OF EARLY VIROLOGIC RESPONSE IN SOFOSBUVIR TREATED CHRONIC HEPATITIS C GENOTYPE 3 PATIENTS 1Burhan Memon, 2 Sanober Jamali, 3Hiba Jamali, 4 Pir Maroof Qureshi, 5 Zain Amar 1Medical Officer, Civil Hospital Matiari, Sindh, Pakistan, Email: burhanaslam.memon@hotmail.com, 2Medical Officer, Shafay Hospital Qasimabad, Hyderabad, Sindh Email: drsanober@yahoo.com, 3 Medical Student, Isra University Faculty of Medicine and Allied Medical Sciences, Hyderabad, Sindh, Email: jamali46@hotmail.com, 4 Medical Officer, Wali Bhai Rajputana Hospital, Hyderabad, Sindh Email: pir.maroof.qureshi.pmq@gmail.com, 5 Medical Officer, Jijal Maau Hospital, Hyderabad, Sindh, Pakistan Email: zain.amarsp@gmail.com, INTRODUCTION: were selected according to inclusion criteria. Age 40- 50 years, HCV- RNA PCR positive, genotype 3, drug naïve, without signs of liver fibrosis of both gender were included. Chronic hepatitis cases of relapse/recurrence with previous anti HCV therapy were excluded. Sofosbuvir 400mg daily and ribavirin (1000mg daily for body weight <75 kg and 1200mg for body weight >75 kg) were prescribed.6 Ribavirin was given in 2 doses. Anti HCV- antibodies were detected by 3rd generation ELISA assay kit. Viral load HCV- RNA was detected by reverse transcriptase polymerase chain reaction (RT- PCR) method. Cobas Amplicor (Roche Diagnostic Inc.) was used for RT- PCR analysis. HCV genotyping was performed by hybridization method. Lower limit of detection was 20 IU/ml. Serum HCV- RNA was detected as baseline and at 4 weeks, 12 weeks and at the end of therapy period. Rapid virological response (RVR), early virological response (EVR) and Sustained virological response (SVR) were defined as undetectable serum HCV-RNA at week 4, week 12 week and 24 weeks respectively.6 Study protocol was in accordance to the Helsinki`s declaration. Inclusion and exclusion were strictly exercised. They were informed that the information will not be shared to none and there will be no harm to them. Volunteers were interviewed to gain their confidence. Volunteers were requested for blood sampling for laboratory testing purpose. Volunteers were informed that they may withdraw at any time and this will not affect their medical therapy. Volunteers were asked to sign the consent form. Volunteers were examined clinically. Age, gender and blood pressure were noted. Venous blood was collected under strict aseptic conditions from prominent vein in the cubital fossa. Blood sample (2 ml) for hemoglobin and platelet estimation was collected in the EDTA tubes. 3 ml blood was centrifuged at x12, 000 g for ten minutes to separate the sera. Hemoglobin, platelets, serum bilirubin, albumin, Prothrombin time (PT), alanine transaminase (ALT) and gamma glutamyl transferase (GGT) were detected by standard laboratory methods. A proforma was designed for data collection. Confidentiality of patient’s data was secured. Study was approved by the institution`s ethical review committee (ERC). Data was analyzed on SPSS ver 22.0 by Student`s t-test and Chi- square test. Student`s t-test analyzed the continuous variables and results presented as mean +/- SD. Chi- square test was used for analysis of categorical variables and outcome presented as frequency and %. Burhan Memon, Page 6567 w w w . i a j p s . c o m IAJPS 2018, 05 (07), 6567-6571 IAJPS 2018, 05 (07), 6567-6571 ISSN 2349-7750 ISSN 2349-7750 Burhan Memon et al Burhan Memon et al INTRODUCTION: Data was analyzed at 95% confidence interval (CI) (P < 0.05). were selected according to inclusion criteria. Age 40- 50 years, HCV- RNA PCR positive, genotype 3, drug naïve, without signs of liver fibrosis of both gender were included. Chronic hepatitis cases of relapse/recurrence with previous anti HCV therapy were excluded. Sofosbuvir 400mg daily and ribavirin (1000mg daily for body weight <75 kg and 1200mg for body weight >75 kg) were prescribed.6 Ribavirin was given in 2 doses. Anti HCV- antibodies were detected by 3rd generation ELISA assay kit. Viral load HCV- RNA was detected by reverse transcriptase polymerase chain reaction (RT- PCR) method. Cobas Amplicor (Roche Diagnostic Inc.) was used for RT- PCR analysis. HCV genotyping was performed by hybridization method. Lower limit of detection was 20 IU/ml. Serum HCV- RNA was detected as baseline and at 4 weeks, 12 weeks and at the end of therapy period. Rapid virological response (RVR), early virological response (EVR) and Sustained virological response (SVR) were defined as undetectable serum HCV-RNA at week 4, week 12 week and 24 weeks respectively.6 Study protocol was in accordance to the Helsinki`s declaration. Inclusion and exclusion were strictly exercised. They were informed that the information will not be shared to none and there will be no harm to them. Volunteers were interviewed to gain their confidence. Volunteers were requested for blood sampling for laboratory testing purpose. Volunteers were informed that they may withdraw at any time and this will not affect their medical therapy. Volunteers were asked to sign the consent form. Volunteers were examined clinically. Age, gender and blood pressure were noted. Venous blood was collected under strict aseptic conditions from prominent vein in the cubital fossa. Blood sample (2 ml) for hemoglobin and platelet estimation was collected in the EDTA tubes. 3 ml blood was centrifuged at x12, 000 g for ten minutes to separate the sera. Hemoglobin, platelets, serum bilirubin, albumin, Prothrombin time (PT), alanine transaminase (ALT) and gamma glutamyl transferase (GGT) were detected by standard laboratory methods. A proforma was designed for data collection. Confidentiality of patient’s data was secured. Study was approved by the institution`s ethical review committee (ERC). Data was analyzed on SPSS ver 22.0 by Student`s t-test and Chi- square test. Student`s t-test analyzed the continuous variables and results presented as mean +/- SD. INTRODUCTION: Chi- square test was used for analysis of categorical variables and outcome presented as frequency and %. Data was analyzed at 95% fid i t l (CI) (P < 0 05) Globally hepatitis C viral (HCV) infection has emerged as a serious health problem. World incidence of HCV is estimated as 200 million (3.3%).1 Approximately 3-4 million people are infected by HCV throughout the World. Reported incidence of HCV ranges 0.2 – 40% and varies in different geography areas of World.2 An annual burden of 10 million chronic viral hepatitis C (CHC) has been reported in the Pakistan.3,4 Amongst highest HCV infection countries, the Pakistan ranks second with an approximate incident rate of 4.5% to 8%.1,5 HCV is a hepatotropic virus that disturbs the hepatocyte enzymes. Alanine transaminase (ALT) and gamma-glutamyl transferase (GGT) are liver enzymes of clinical significance.6 GGT has emerged as a biological indicator of viral response to drug therapy. GGT is expressed on the cell membranes of renal tubules, brain capillaries and biliary epithelium. GGT is a cell-surface heterodimeric glycoprotein that catalyzes the glutathione metabolism.7 GGT is used a clinical biomarker of hepatobiliary disorders, drug intake and alcohol consumption, etc. However, its diagnostic and prognostic worth is debatable.8 Few recent studies1,7,8 have highlighted its predictive, prognostic and diagnostic value. Raised GGT is reported as indicator of mortality in chronic liver disease, diabetes mellitus and neoplastic disorders.9 Serum GGT levels are raised in CHC patients.6-9 Previous studies have reported low baseline GGT level is a strong predictor of sustained virologic response (SVR) to anti viral drug therapy such as interferon.1,6-9 Serum GGT levels are reported to be predictor of hepatocellular carcinoma (HCC) in CHC patients. Its predictive value for the HCC development has been in non- cirrhotic CHC patients even after successful eradication of HCV.10-13 Whether serum GGT levels are associated or not with development of CHC are not well understood. A study from China reported serum GGT levels during various phases of CHC. This study reported GGT as a potential predictive clinical biomarker for hepatitis C in response to sofosbuvir therapy. The present prospective study analyzed the baseline Gamma- glutamyl transferase (GGT) as predictor of Early Virologic Response (EVR) in Sofosbuvir treated chronic hepatitis C patients in patients reporting at our tertiary care hospital SUBJECTS AND METHODS: SUBJECTS AND METHODS: Burhan Memon et al Burhan Memon et al ISSN 2349-7750 ISSN 2349-7750 SUBJECTS AND METHODS: A cross sectional study was conducted at the Department of Medicine, Liaquat University Hospital Jamshoro/Hyderabad from August 2017 to March 2018. A sample of 100 HCV- PCR positive; comprising of 63 male and 37 female Page 6568 w w w . i a j p s . c o m IAJPS 2018, 05 (07), 6567-6571 IAJPS 2018, 05 (07), 6567-6571 Burhan Memon et al DISCUSSION: corroborate the present study. In present study, the success rate was 100% EVR with sofosbuvir and low baseline GGT positively predicted the EVR. Lawaitz et al17 treated chronic HCV patients with moderate hepatic dysfunction with sofosbuvir and reported positive response of declining HCV RNA within 7 days of therapy. Mansoor et al22 studied 153 cases of chronic hepatitis C patients and reported Sofosbuvir was highly successful in eradication of HCV- RNA and achieved EVR irrespective of genotypes. They reported genotype 3 being the most common that supports the present study. There was no difference of sofosbuvir in achieving the EVR between male and female that is in agreement to above study.22 Sarwar et al23 found 83.1% of CHC patients achieving SVR with sofosbuvir/ribavirin therapy. The results of present study reports 100% results of EVR in CHC genotype 3 patients. The limitations of present study include a small sample size; however, the low baseline GGT as predictive marker of EVR is being reported for the first time. SCUSS O The present cross sectional study is the first time reporting on the baseline GGT as predictor of early virological response (EVR) in sofosbuvir treated chronic hepatitis C genotype 3 patients. Age (mean ± SD) of male and female was noted as 43.93±7.95 and 43.45±4.54 years respectively (P=0.032). Of 100 sample, 63% were male and 37% were female (P=0.940). These findings are in agreement with previous studies.1,14-15 Tahir et al14 reported age of 30- 59 years that is in agreement with present study. However, they reported increased incidence among female subjects that contradicts to the present study. In present study, male outnumbered to female that is in agreement to Sirhindi et al15 as they found increased among male patients. Siddique16 reported the mean age of 46 years that is in agreement to the present study with male dominancy. The present study included genotype 3 CHC patients that are in agreement to above studies. Pakistan has high prevalence of genotype 3 hepatic C virus. The prevalence is reported as 4.5-8.2%.1,14-16 Sofosbuvir is a newer oral anti viral drug that has yielded dramatic cure rates against chronic hepatitis C. Sofosbuvir specifically inhibits the NS5B polymerase enzyme of all genotypes of HCV. CONCLUSION: The present study concludes low serum GGT level positively predicts an early virological response (EVR) in chronic hepatitis C genotype 3 patients. The results of present study reports 100% results of EVR in CHC genotype 3 patients with sofosbuvir and ribavirin. REFERENCES: RESULTS: shown in table I. Results of present study reports 100% results of EVR in CHC genotype 3 patients and there was no response difference between male and female patients. Logistic regression analysis model for GGT as predictor of EVR shows AUC of 0.912 (91.2%) (P=0.0001) (Graph 1 and Table II). Age (mean ± SD) of male and female was noted as 43.93±7.95 and 43.45±4.54 years respectively (P=0.032). Of 100 sample, 63% were male and 37% were female (P=0.940) (Table I). Baseline hemoglobin, platelets, serum bilirubin, albumin, Prothrombin time (PT), alanine transaminase (ALT) and gamma glutamyl transferase (GGT) are Table 1. Demography and biochemical findings of study subjects (n= 100) Male Female P-value Gender 63% 37% 0.0001 Age (years) 43.93±7.95 43.45±4.54 0.940 Systolic BP (mmHg) 128.57±16.81 141.21±19.05 0.0001 Diastolic BP (mmHg) 72.76±9.62 78.35±13.47 0.001 Hemoglobin (g/dl) 13.37±2.27 11.90±4.26 0.027 Platelet (x106) 296.36±77.54 281.27±85.91 0.369 Bilirubin (mg/dl) 1.90±0.66 1.67±0.69 0.109 Albumin (g/dl) 3.83±0.97 3.65±0.76 0.345 PT (seconds) 20.68±6.96 13.81±5.15 0.0001 ALT (U/L) 82.79±18.98 90.40±28.32 0.345 GGT (U/L) 28.32±6.27 32.83±9.48 0.005 Graph 1. Logistic regression analysis model shows the area under curve of GGT as predictor of EVR aph 1. Logistic regression analysis model shows the area under curve of GGT as predictor of EVR Page 6569 w w w . i a j p s . c o m IAJPS 2018, 05 (07), 6567-6571 ISSN 2349-7750 Burhan Memon et al Table II. Area Under the Curve Test Result Variable(s): Predicted probability Area Std. Errora P-value.b Asymptotic 95% CI Lower Bound Upper Bound 0.912 0.026 0.0001 0.863 0.959 The test result variable(s): Predicted probability has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. a. Under the nonparametric assumption b. Null hypothesis: true area = 0.50 Table II. Area Under the Curve Test Result Variable(s): Predicted probability Area Std. Errora P-value.b Asymptotic 95% CI Lower Bound Upper Bound 0.912 0.026 0.0001 0.863 0.959 The test result variable(s): Predicted probability has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. a. Under the nonparametric assumption b. Null hypothesis: true area = 0.50 Table II. Area Under the Curve DISCUSSION: Hence sofosbuvir is pan- genotype nucleotide analogue effective against HCV.17,18 Genotype 3 HCV is predominantly found in the country and prevalence has been reported as around 78% in Pakistan.19,20 In present study, the logistic regression analysis model for GGT as predictor of EVR shows AUC of 0.912 (91.2%) (P=0.0001). Zebreska et al 21 reported poor response of genotype 3 HCV infection with elevated baseline GGT and low platelet counts treated with Pegylated interferon-α and ribavirin. The finding supports the observations of the presents study. They further added that the achieving a rapid (RVR) and early virological response (EVR) are associated with higher likelihood of a sustained virological response (SVR).21 Siddique et al16 treated 201 patients with sofosbuvir and ribavirin and reported excellent RVR and EVR at first month of therapy. The findings REFERENCES: 1. Saeed M, Iram S, Hussain S, Mobeen R, Ahmad M, Ashraf M. Hepatitis C virus infection; Frequency of a dumb murderer in blood donors’ community of Lahore. Professional Med J 2016; 23(5):546-552. 2. Akhtar AM, Khan MA, Ijaz T, Iqbal Z, Rana MY, Maqbool A, Rehman A. Seroprevalence and determinants of hepatitis-c virus infection in blood donors of Lahore, Pakistan. Pak J Zool 2013; 45:1–7. 3. Waheed Y, Shafi T, Safis Z, Qadri I. Hepatitis C virus in Pakistan: a systematic review of 3. Waheed Y, Shafi T, Safis Z, Qadri I. Hepatitis C virus in Pakistan: a systematic review of 3. Waheed Y, Shafi T, Safis Z, Qadri I. Hepatitis C virus in Pakistan: a systematic review of Page 6570 w w w . i a j p s . c o m IAJPS 2018, 05 (07), 6567-6571 Burhan Memon et al ISSN 2349-7750 prevalence, genotypes and risk factors. World J Gastroenterol 2009; 15:5647–5653. 14. Tahir M, Suhail AM, Tahir F, Hamza SA, Bukhari SAH. Detection and genotyping of HCV in patients of Sheikh Zayed Hospital, Lahore, Pakistan. J Uni Med Dent Coll 2016; 7(1):25-30. prevalence, genotypes and risk factors. World J Gastroenterol 2009; 15:5647–5653. 4. Raja Janjuaka NS. Epidemiology of hepatitis C virus infection in Pakistan. J Microbiolimmunol infect 2008; 41:4–8. 15. Sirhindi GA, Bajawa MA, Asif MJ, Khan LA, Alamgir MA. Frequency and different indications for anti-hcv screening. Pak J Med Health Sci 2012; 6(1):94-96. 5. Khattak MF, Salamat N, Bhatti FA, Qureshi TZ. Seroprevalence of hepatitis B, C and HIV in blood donors in Northern Pakistan. J Pak Med Assoc 2012; 52:398–402. 16. Siddique MS, Shoaib S, Saad A, Iqbal HJ, Durrani N. Rapid virological & End treatment response of patients treated with Sofosbuvir in Chronic Hepatitis C. Pak J Med Sci 2017; 33(4):813-817. 6. Huang R, Yang CC, Liu Y, Xia J, Su R, Xiong YL, Wang GY, Sun ZH, Yan XM, Lu S, Wu C. Association of serum gamma glutamyl transferase with treatment outcome in chronic hepatitis B patients. World J Gastroenterol 2015; 21(34): 9957-9965. 17. Lawitz E, Mangia A, Wyles D, Rodriguez-Torres M, Hassanein T, Gordon SC, et al. Sofosbuvir for previously untreated chronic hepatitis C infection. N Engl J Med 2013; 368:1878–87. 7. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009; 373: 582-592 [PMID: 19217993. 18. REFERENCES: Lawitz E, Poordad F, Brainard DM, Hyland RH, An D, Dvory-Sobol H, et al. Sofosbuvir with Peginterferon ribavirin for 12 weeks in previously treated patients with hepatitis C genotype 2 or 3 and cirrhosis. Hepatol 2015; 61:769–75. 8. Whitfield JB. Gamma glutamyl transferase. Crit Rev Clin Lab Sci 2001; 38: 263-355. 9. Everhart JE, Wright EC. Association of γ- glutamyl transferase (GGT) activity with treatment and clinical outcomes in chronic hepatitis C (HCV). Hepatol 2013; 57: 1725- 1733. 19. Steinbrenner N, Sprinzl MF, Zimmermann T, Wörns MA, Zimmerer T, Galle PR, et al. Early virological response may predict treatment response in sofosbuvir-based combination therapy of chronic hepatitis C in a multi-center “real-life” cohort.BMC Gastroenterology 2015; 15:97:2-8. 10. Ruhl CE, Everhart JE. Elevated serum alanine aminotransferase and gamma-glutamyl transferase and mortality in the United States population. Gastroenterol 2009; 136: 477-85.e11. 11. Güzelbulut F, Sezikli M, Cetinkaya ZA, Ozkara S, Gönen C, Ovünç AO. A lower serum gamma- glutamyl transferase level does not predict a sustained virological response in patients with chronic hepatitis C genotype 1. Gut Liver 2013; 7: 74-81. 20. Schneider MD, Sarrazin C. Antiviral therapy of hepatitis C in 2014: do we need resistance testing? Antiviral Res 2014; 105:64–71. 21. Güzelbulut F, Sezikli M, Cetinkaya ZA, Ozkara S, Gönen C, Ovünç AO. A lower serum gamma- glutamyltransferase level does not predict a sustained virological response in patients with chronic hepatitis C genotype 1. Gut Liver 2013; 7:74-81. 12. Huang CF, Yeh ML, Tsai PC, Hsieh MH, Yang HL, Hsieh MY, Yang JF, Lin ZY, Chen SC, Wang LY, Dai CY, Huang JF, Chuang WL, Yu ML. Baseline gamma-glutamyl transferase levels strongly correlate with hepatocellular carcinoma development in non-cirrhotic patients with successful hepatitis C virus eradication. J Hepatol 2014; 61: 67-74. 22. Mansoor VB, Ahmed U, Jahanzaib M, Ali Z, Haroon MA, Ahmed H, Munim A. End Treatment Response in Chronic Hepatitis C Patients to Sofosbuvir and Ribavirin. Ann Pak Inst Med Sci 2016; 12(3):127-130. 13. Dogan UB, Akin MS, Yalaki S. A low serum γ- glutamyl transferase level predicts a sustained virological response in patients with chronic hepatitis C genotype 1. Gut Liver 2014; 8: 113- 115. 23. Sarwar S, Khan AA. Sofosbuvir based therapy in Hepatitis C patients with and without cirrhosis: Is there difference? Pak J Med Sci 2017; 33(1):37-41. Page 6571 w w w . i a j p s . c o m
https://openalex.org/W2077827426	https://europepmc.org/articles/pmc2408563?pdf=render	English	null	Rotational stability of the AcrySof SA60TT toric intraocular lenses: A cohort study	BMC ophthalmology	2,008	cc-by	3,950	BioMed Central BioMed Central Received: 17 October 2007 Accepted: 6 May 2008 Received: 17 October 2007 Accepted: 6 May 2008 Received: 17 October 2007 Accepted: 6 May 2008 This article is available from: http://www.biomedcentral.com/1471-2415/8/8 © 2008 Zuberbuhler 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 Background: To evaluate the rotational stability of the three types of AcrySof SA60TT toric intraocular lenses (Alcon, Switzerland) in cataract surgery after the first postoperative week. Methods: A retrospective study of 44 eyes in 33 patients. All patients underwent similar uncomplicated phacoemulsification cataract surgery. Seven eyes with corneal astigmatism of less than 1.5 D were implanted with the AcrySof SA60T3 intraocular lens. Seventeen eyes with astigmatism between 1.5 D and 2.25 D received the SA60T4 intraocular lens, and 20 eyes with more than 2.25 D of corneal astigmatism received the SA60T5 intraocular lens. Intraoperatively, the axis of the toric lens was aligned to the steepest axis of the corneal astigmatism. Main outcome measure was the postoperative position of the lens, assessed at 1 week and 3 months, using a specially designed angle measuring eyepiece for the slit lamp. Results: There was no significant difference in the rotational stability of the three types of toric intraocular lenses. Overall, the postoperative rotation was within 5 degrees in 95% and within 2 degrees in 68% of eyes. The mean absolute rotation was 2.2 ± 2.2 degrees. No lens showed more than 9 degrees of rotation, and no lens required secondary repositioning. There was no trend for either clockwise or anti-clockwise rotation. The surgical procedure did not change the corneal astigmatism. Conclusion: Once placed to it's position, each of the three types of the AcrySof SA60TT toric intraocular lenses demonstrate rotational stability in the capsular bag. design as the AcrySof SA60AT monofocal IOL (Alcon, Switzerland). The toric IOL differs in that there is a toric component located on the posterior surface of the lens optic. The optic is marked with 3 peripheral dots that indi- cate the cylindrical axis of the lens and so enables its cor- rect alignment with the steepest axis of the corneal astigmatism during surgery. The AcrySof toric IOL is avail- Open Acce Research article Rotational stability of the AcrySof SA60TT toric intraocular lenses: A cohort study Bruno Zuberbuhler†1, Theo Signer†2, Richard Gale†3 and Eduard Haefliger†2 Address: 1Moorfields Eye Hospital NHS Foundation Trust, 162 City Road, London, EC1V 2PD, UK, 2Vista Klinik, Hauptstrasse 55, CH-4102 Open Access ddress: 1Moorfields Eye Hospital NHS Foundation Trust, 162 City Road, London, EC1V 2PD, UK, 2Vista Klinik, Hau nningen, Switzerland and 3St Thomas' Hospital, Ophthalmology Department, London, SE1 7EH, UK Corresponding author †Equal contributors Background g Corneal astigmatism above 1.5 D occurs in 19% of patients [1]. Implanting a toric intraocular lens (IOL) offers the possibility of correcting not only spherical equivalent refraction, but also astigmatism during phacoemulsification cataract surgery. The AcrySof SA60TT toric series of IOLs (Alcon, Switzerland) is made of hydro- phobic Acrylate and shares the same biconvex single-piece Page 1 of 5 (page number not for citation purposes) Page 1 of 5 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/8/8 BMC Ophthalmology 2008, 8:8 able in 3 types with different cylinder powers: 1.5 D (SA60T3), 2.25 D (SA60T4) and 3.0 D (SA60T5). incision, continuous curvilinear capsulorhexis, phacoe- mulsification with cleaver technique and bimanual irriga- tion/aspiration. The toric IOL was implanted into the capsular bag with a Monarch II injector and a B-Cartridge. Subsequently, the IOL was rotated with a second instru- ment, so that the cylindrical axis of the lens was aligned with the limbal marks of the corneal astigmatism. All patients were prescribed a combination of tobramicin and dexamethasone eye drops 4 times daily for 4 weeks. The success of a toric IOL can be judged not only by its ability to reduce refractive astigmatism immediately post- operatively, but also by its ability to maintain a stable position in the capsular bag in the longer term. However, many surgeons have stopped using toric IOLs because of reported positional instability [2]. The most frequent cause of IOL rotation following uncomplicated cataract surgery is because of capsular bag shrinkage due to fibrosis [3]. The majority of this occurs within the first three months after implantation [4]. Even a small rotational deviation of the toric IOL from its intended axis can result in large reduction of the astigmatic correction [5]. For example, a deviation of 10 degrees minimizes the poten- tial correction by 35%. At 1 week and 3 months postoperatively all patients underwent best-corrected distance visual acuity, subjec- tive refraction and slit lamp examination. The postopera- tive corneal astigmatism was assessed by Orbscan topography at 3 months. For the measurement of the axis of the toric lens the pupil was dilated to enable the peripheral dots on the optic to become visible. A specially designed eyepiece, replacing one of the 10× eyepieces, was inserted in the slit lamp. Background Looking through the eyepiece, a spirit level, a horizontal line, a black measurement line and a circular scale in the periphery with single degree steps could be seen. With the spirit level the horizontal line could be adjusted to the horizontal axis. A virtual reference line was used, formed by the line between the pupils of patient's eyes, to provide a patient's reference. This reference line was aligned with the horizontal line of the eyepiece at each follow-up to guarantee reproducible measuring conditions. The black measurement line then could be rotated into the axis of the toric IOL. The angle between the black measurement line and the horizontal line represented the angle of the toric IOL. The angle could be read on the peripheral scale in degrees. The aim of this study was to evaluate the postoperative rotational stability of the three types of AcrySof SA60TT toric IOLs in standard cataract surgery between the first postoperative week and the third month. Methods This is a retrospective cohort study of 44 eyes of 33 patients. The eyes all had corneal astigmatism of 1 to 5 D, and between July 2006 and March 2007 received an AcrySof toric intraocular lens during cataract surgery. Exclusion criteria were axial length of less than 22 mm, zonular weakness, a change in refractive astigmatism of more than 0.5 D within the 3 months prior to surgery, and coexisting maculopathy or glaucoma. Eyes with a corneal astigmatism (Orbscan, Carl Zeiss, Ger- many) of less than 1.5 D were implanted with the AcrySof SA60T3 IOL, eyes with a corneal astigmatism between 1.5 D and 2.25D were implanted with the SA60T4 IOL, and eyes showing a corneal astigmatism of more than 2.25 D were implanted with the SA60T5 IOL. A total of 44 eyes of 33 patients were recruited. Statistical analysis was performed using SPSS Software (SPSS Inc., Chicago, IL, USA). For comparative statistics the Wilcoxon matched-pairs test was used and for inde- pendent samples and the Mann-Whitney U-test was used. For association statistics the Spearman rank correlation test was applied. P-values less than 0.05 were considered statistically significant. Biometry was performed with the IOL Master (Carl Zeiss, Germany), using the Haigis formula for the IOL power calculation and the company's recommended A-constant of 118.4 for the toric IOL. The target postoperative spher- ical equivalent was aimed to emmetropia. Results Forty-four eyes of 33 patients underwent routine phacoe- mulsification cataract surgery with in-the-bag implanta- tion of a toric AcrySof IOLs. Seven eyes were implanted with the SA60T3 IOL, 17 eyes were implanted with the SA60T4 IOL, and 20 eyes were implanted with the SA60T5 lens (Table 1). There was no difference seen in patients' age, preoperative best-corrected visual acuity and preoper- ative spherical equivalent between the three types of IOLs. Preoperatively, the steepest axis of the corneal astigma- tism, determined by Orbscan topography was marked at the corneal limbus with a marker pen, using an angle measuring eyepiece on a BQ900 slit lamp (Haag Streit, Switzerland). Cataract surgery was performed by a single surgeon (EH) at the Vista Klinik, Binningen, Switzerland under topical local anesthesia. Each patient underwent the same technique with a 2.8 mm superior clear limbal There was no difference in rotational stability between the three IOL types (p > 0.18). Between week 1 and month 3 Page 2 of 5 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/8/8 BMC Ophthalmology 2008, 8:8 Table 1: Patient demographics, best-corrected distance visual acuity, refractive astigmatism and corneal astigmatism before and three months after implantation of a AcrySof SA60TT toric intraocular lens (SA60T3, SA60T4 or SA60T5 type). Results Parameters SA60T3 SA60T4 SA60T5 Summarized Eyes (n) 7 17 20 44 Patients (n) 7 14 17 33 Age (y) Mean ± SD 66 ± 9 71 ± 14 72 ± 13 71 ± 13 Range 52 to 76 35 to 88 35 to 86 35 to 88 Preoperative Parameters Mean BCDVA (LogMAR) ± SD 0.28 ± 0.22 0.27 ± 0.15 0.30 ± 0.17 0.28 ± 0.17 Mean SE (D) ± SD -1.90 ± 3.27 -1.60 ± 3.38 -1.50 ± 3.15 -1.60 ± 3.19 Mean refractive astigmatism (D) ± SD* 1.86 ± 0.83 2.24 ± 0.83 2.98 ± 1.07 - Mean corneal astigmatism (D) ± SD† 1.40 ± 0.55 1.95 ± 0.56 3.06 ± 0.74 - Postoperative Parameters Mean BCDVA (LogMAR) ± SD 0.00 ± 0.06 -0.02 ± 0.04 0.05 ± 0.15 0.01 ± 0.11 Mean SE (D) ± SD -0.75 ± 0.95 -0.80 ± 1.44 -0.38 ± 1.26 -0.61 ± 1.28 Mean refractive astigmatism (D) ± SD* 0.43 ± 0.59 0.22 ± 0.26 0.89 ± 1.29 - Mean corneal astigmatism (D) ± SD† 1.54 ± 0.51 1.85 ± 0.50 3.19 ± 0.93 - BCDVA = best-corrected distance visual acuity; SE = spherical equivalent The change in refractive astigmatism was statistically significant due to the lens implantation (SA60T3 p = 0.04; SA60T4 p < 0.001; SA60T5 p < 0.001). †The corneal astigmatism did not change significantly due to the surgery (SA60T3 p = 0.17; SA60T4 p = 0.98; SA60T5 p = 0.26). Table 1: Patient demographics, best-corrected distance visual acuity, refractive astigmatism and corneal astigmatism before and three months after implantation of a AcrySof SA60TT toric intraocular lens (SA60T3, SA60T4 or SA60T5 type). 0.001). †The corneal astigmatism did not change significantly due to the surgery (SA60T3 p = 0.17; SA60T4 p = 0.98; SA60T5 p = 0.26). 0.001). †The corneal astigmatism did not change significantly due to the surgery (SA60T3 p = 0.17; SA60T4 p = 0.98; SA60T p = 0.26 for SA60T5). The mean reduction in refractive astigmatism was 1.43 D for the SA60T3 IOL, 2.02D for the SA60T4 IOL, and 2.09D for the SA60T5 IOL. A reduction of at least 1 D was achieved in 71% of eyes receiving a SA60T3 IOL, a reduction of at least 1.5 D was achieved in 65% of eyes receiving a SA60T4 IOL, and a reduction of at least 2 D was achieved in 61% of eyes receiving a SA60T5 IOL. Results There was no correlation between the rotation of the lens and the amount or the change in refractive astigma- tism (p = 0.931). the overall mean absolute rotation of all 44 implanted IOLs was 2.2 ± 2.2 degrees. Sixteen (36%) IOLs rotated clockwise with a maximum rotation of 9 degrees, sixteen (36%) IOLs rotated anti-clockwise with a maximum rota- tion of 5 degrees, and twelve (28%) IOLs did not rotate (Figure 1). In 95% of eyes the IOL rotation was 5 degrees or less and in 68% of eyes it was 2 degrees or less. No cor- relation was seen between the amount of postoperative rotation and the topographic Sim K readings (p = 0.760), the axial length of the eye (p = 0.773) or the IOL power (p = 0.801). At three months, 90% of eyes were within 1.0 D and 81% of eyes within 0.5 D of the target spherical equivalent. Ninety-six percent of eyes gained one or more Snellen line of best-corrected distance visual acuity (BCDVA); 82% of eyes gained two or more Snellen lines of BCDVA. One eye lost one line of BCDVA. The corneal astigmatism did not significantly change fol- lowing surgery (p = 0.17 for SA60T3; p = 0.98 for SA60T4; Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months postoperative fol- low-up Figure 1 Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months post- operative follow-up. No complication was encountered during the intra- and postoperative course. None of the lenses required second- ary alignment. Discussion and Conclusion and direction of rotation of the AcrySof SA60TT toric intraocular lenses between the one week and three plantation in standard cataract surgery. Statistically, no difference in absolute rotation was noticed between the three types (p > 0.18). results of this study show a mean difference in refractive cylinder power between the two types of only 0.1 D. The- oretically, this could be due to errors in our refractive data. Further studies with a longer follow-up are necessary to analyze the refractive outcome of the toric IOLs. with Z-haptics (HumanOptics, Germany) that the mean rotation of this lens was 5.2 degrees and the maximum rotation was 15 degrees. Only 60% of the IOLs demon- strated five degrees or less of rotation. Sixty percent of the MicroSil toric IOLs rotated anti-clockwise [7]. Chang [8] demonstrated in a series of 50 STAAR TL toric IOLs (STAAR, USA) a maximum rotation of 20 degrees and 72% of the IOLs were within 5 degrees of the intended axis. A smaller diameter version of this STAAR IOL (STAAR TF toric IOL) demonstrated rotation of up to 80 degrees and required subsequent repositioning in 50% of cases [8]. Other currently used toric IOLs include the T- flex 573T and T-flex 623T (Rayner, United Kingdom), and the Acri.LISA Toric 466TD and Acri.Comfort 646TLC (Acri.Tec, Germany). Different methods can be used to accurately determine the position of a toric IOL. Weinand et al. [9] and Becker et al. [10] analyzed digital and conventional photographs, taken preoperative and postoperative through the slit lamp and operating microscope. This method with photo- graphs in retrograde illumination has become the 'gold standard' for evaluating the centration and axial position- ing of a toric intraocular lens. Viestenz et al. [11,12] and Quentin et al. [13] used the method of simultaneous slide projection to evaluate the rotation of the toric lens. This method has shown superior accuracy in calculating the rotational stability, because it respects the autorotation of the eye which has shown to be up to 11.5 degrees (mean of 2.3 degrees) [12,14]. The negative side of the methods with photographs or slides is the increased time consume and demand for specific equipment. In this study, we choose the eyepiece measuring device because it was only a postoperative analysis of the toric lens axis. Discussion and Conclusion This is the first study to report the rotational stability of the AcrySof SA60TT toric IOL in a series of 44 cases 3 months after implantation in cataract surgery. The rotational stability of the SA60TT toric IOLs compares favorably with other current-generation toric IOLs. The SA60TT showed a mean absolute rotation of 2.2 degrees, a maximum rotation of 9 degrees and 95% of IOLs showed 5 degrees or less of rotation (Table 2). De Silva et al. [6] showed in a series of 21 MicroSil 6116TU toric IOLs Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months postoperative fol- low-up Figure 1 Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months post- operative follow-up. Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months postoperative fol- low-up Figure 1 Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months post- operative follow-up. Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months postoperative fol low up Figure 1 Rotation of the AcrySof SA60TT toric intraocular lens between the one week and three months post- operative follow-up. Page 3 of 5 (page number not for citation purposes) BMC Ophthalmology 2008, 8:8 http://www.biomedcentral.com/1471-2415/8/8 http://www.biomedcentral.com/1471-2415/8/8 BMC Ophthalmology 2008, 8:8 Table 2: Absolute rotation and direction of rotation of the AcrySof SA60TT toric intraocular lenses between the one week and three months follow-up after implantation in standard cataract surgery. Parameters SA60T3 SA60T4 SA60T5 Summarized Eyes (n) 7 17 20 44 Absolute rotation (degrees) Mean ± SD 1.6 ± 1.0* 3.3 ± 2.4* 1.5 ± 2.0* 2.2 ± 2.2 Range 0 to 3 0 to 9 0 to 7 0 to 9 Direction of rotation, n (%) Clockwise 4 (57%) 7 (41%) 5 (25%) 16 (36%) Anti-clockwise 2 (29%) 7 (41%) 7 (35%) 16 (36%) No rotation 1 (14%) 3 (18%) 8 (40%) 12 (28%) *Statistically, no difference in absolute rotation was noticed between the three types (p > 0.18). Table 2: Absolute rotation and direction of rotation of the AcrySof SA60TT toric intraocular lenses between the one week and three months follow-up after implantation in standard cataract surgery. e 2: Absolute rotation and direction of rotation of the AcrySof SA60TT toric intraocular lenses between the hs follow-up after implantation in standard cataract surgery. Competing interests The authors declare that they have no competing interests. Discussion and Conclusion The device was easy and fast to handle and allowed the ophthalmol- ogist to perform axis measurements at any follow-up, even in busy clinics. It also showed good reproducibility when the patient's head was correctly aligned with the eyepiece and the slit lamp. Because the eyepiece provided data with one degree precision, this unit was used for the study, too. The precision of one degree steps, of course is on the optimistic side, and needs to be set in relation to the errors occurring from autorotation and head disloca- tion. For comparisons of misalignments of the toric lens, analyzing preoperative and postoperative axis, respecting parallaxes and cyclorotation, we recommend to use the superior technique of simultaneous slide projection. In this series, the largest rotation of the toric IOL was 9 degrees, seen in one eye only. No specific cause was iden- tified for this large deviation and the patient did not require secondary repositioning of the lens because of vis- ual satisfaction. One eye lost a single line of BCDVA because of a disadvantageous change in the regularity of the corneal astigmatism. No reason could be found for this anomaly. This patient was managed conservatively. A toric lens has to be implanted using an astigmatically neutral procedure: a technique that will have no effect on the preoperative corneal astigmatism [5]. This study dem- onstrated no significant change in corneal astigmatism from the preoperative assessment to the 3 month postop- erative assessment. Caution should be exercised while implanting and rotating the toric lens in the capsular bag so as not to cause capsular damage. Very small eyes, eyes with zonular instability or floppy capsules (post-vitrec- tomy eyes) may be considered a relative contraindication. Except for the SA60T5, the AcrySof SA60TT lenses reduced refractive astigmatism as expected. The manufacturer's data claim that the cylinder power of the SA60T5 IOL is 0.75 diopters higher at the IOL plane, and 0.51 D higher at the corneal plane, than the SA60T4. However, the In conclusion, the three types of AcrySof SA60TT toric IOLs demonstrated no significant rotation three months after implantation. The surgical technique used for Page 4 of 5 (page number not for citation purposes) http://www.biomedcentral.com/1471-2415/8/8 BMC Ophthalmology 2008, 8:8 http://www.biomedcentral.com/1471-2415/8/8/prepub http://www.biomedcentral.com/1471-2415/8/8/prepub implantation did not significantly change the axis of cor- neal astigmatism. implantation did not significantly change the axis of cor- neal astigmatism. Authors' contributions BZ conceived of the study, participated in its design and coordination, collected data and wrote the article. RG per- formed the statistical analysis and contributed to writing the article. TS and EH conceived of the study, participated in its design and contributed to writing the article. All authors read and approved the final manuscript. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 5 of 5 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge f g 13. Quentin CD, Genée D: Rotationsstabilität der Silikon-HKL mit C-Haptik versus Z-Haptik im Kapselsack und mit einer C- Haptik im Sulcus ciliaris. 17. Kongress der Deutschsprachigen Ges- ellschaft für Intraokularlinsen-Implantation und refraktiven Chirurgie :175-180. 14. Viestenz A, Walter S, Viestenz A, Behrens-Baumann W, Langen- bucher A: Torische Intraokularlinse und Astigmatismuskor- rektur. Ophthalmologe 2007, 104:620-627. References 1. Hoffer KJ: Biometry of 7,500 cataractous eyes. Am J Ophthalmol 1980, 90:360-368. correction, 890 1. Hoffer KJ: Biometry of 7,500 cataractous eyes. Am J Ophthalmol 1980, 90:360-368. correction, 890 2. Horn JD: Status of toric intraocular lenses. Curr Opin Ophthalmol 2007, 18(1):58-61. 2. Horn JD: Status of toric intraocular lenses. Curr Opin Ophthalmol 2007, 18(1):58-61. 3. Ohmi S: Decentration associated with asymmetric capsular shrinkage and intraocular lens size. J Cataract Refract Surg 1993, 19:640-643. 4. Strenn K, Menapace R, Vass C: Capsular bag shrinkage after implantation of an open-loop silicone lens and a poly(methyl methacrylate) capsule tension ring. J Cataract Refract Surg 1997, 23:1543-1547. 5. Sanders DR, Grabow HB, Shepherd J: The toric IOL. In Sutureless Cataract Surgery; An Evolution Toward Minimally Invasive Technique Edited by: Gills JP, Martin RG, Sanders DR. Thorofare, NJ, Slack; 1992:183-197. 6. De Silva DJ, Ramkissoon YD, Bloom PA: Evaluation of a toric intraocular lens with Z-haptic. J Cataract Refract Surg 2006, 32:1492-1498. 7. Warlo I, Krummenauer F, Dick HB: Rotational stability in intraocular lenses with C-haptics versus Z-haptics in cataract surgery. A prospective randomised comparison. Ophthalmol- oge 2005, 102:987-992. g 8. Chang DF: Early rotational stability of the longer Staar toric intraocular lens: fifty consecutive cases. J Cataract Refract Surg 2003, 29:935-940. 9. Weinand F, Jung A, Stein A, Pfotzner A, Becker R, Pavlovic S: Rota- tional stability of a single-piece hydrophobic acrylic intraoc- ular lens: new method for high-precision rotation control. J Cataract Refract Surg 2007, 33:800-803. f g 10. Becker KA, Auffarth GU, Völcker HE: Measurement method for the determination of rotation and decentration of intraocu- lar lenses. Ophthalmologe 2004, 101:600-603. p g 11. Viestenz A, Langenbucher A, Seitz B: Impact of the eye's cycloro- tation on axial orientation analysis of toric intraocular lenses: recommendations for an optimized evaluation of rotational stability of toric IOLs. Klin Monatsbl Augenheilkd 2006, 223:134-140. 12. Viestenz A, Seitz B, Langenbucher A: Evaluating the eye's rota- tional stability during standard photography. Effect on deter- mining the axial orientation of toric intraocular lenses. J Cataract Refract Surg 2005, 31:557-561. Pre-publication history The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here:
https://openalex.org/W1973489762	https://scholarscompass.vcu.edu/cgi/viewcontent.cgi?article=1023&context=obgyn_pubs	English	null	Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences?	PloS one	2,014	cc-by	8,422	Part of the Obstetrics and Gynecology Commons Copyright: © 2014 Chang 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. Virginia Commonwealth University VCU Scholars Compass Obstetrics and Gynecology Publications Dept. of Obstetrics and Gynecology 2014 Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? Jen Jen Chang Saint Louis University Jerome F. Strauss Virginia Commonwealth University, jfstrauss@vcu.edu Jonathan P. DeShazo Virginia Commonwealth University, jpdeshazo@vcu.edu See next page for additional authors Follow this and additional works at: http://scholarscompass.vcu.edu/obgyn_pubs Part of the Obstetrics and Gynecology Commons Copyright: © 2014 Chang 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. This Article is brought to you for free and open access by the Dept. of Obstetrics and Gynecology at VCU Scholars Compass. It has been accepted for inclusion in Obstetrics and Gynecology Publications by an authorized administrator of VCU Scholars Compass For more information, please contact Recommended Citation Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? Chang JJ, Strauss JF III, Deshazo JP, Rigby FB, Chelmow DP, et al. (2014) Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? PLoS ONE 9(10): e106446. doi: 10.1371/journal.pone.0106446 Virginia Commonwealth University VCU Scholars Compass Dept. of Obstetrics and Gynecology Obstetrics and Gynecology Publications Follow this and additional works at: http://scholarscompass.vcu.edu/obgyn_pubs Follow this and additional works at: http://scholarscompass.vcu.edu/obgyn_pubs Part of the Obstetrics and Gynecology Commons Part of the Obstetrics and Gynecology Commons Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? Jonathan P. DeShazo Virginia Commonwealth University, jpdeshazo@vcu.edu Authors Jen Jen Chang, Jerome F. Strauss, Jonathan P. DeShazo, Fidelma B. Rigby, David P. Chelmow, and George A. Macones This article is available at VCU Scholars Compass: http://scholarscompass.vcu.edu/obgyn_pubs/24 Abstract Objective: To investigate the association between cigarette use during pregnancy and pregnancy-induced hypertension/ preeclampsia/eclampsia (PIH) by maternal race/ethnicity and age. Methods: This retrospective cohort study was based on the U.S. 2010 natality data. Our study sample included U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation without major fetal anomalies in 2010 (n = 3,113,164). Multivariate logistic regression models were fit to estimate crude and adjusted odds ratios and the corresponding 95% confidence intervals. Results: We observed that the association between maternal smoking and PIH varied by maternal race/ethnicity and age. Compared with non-smokers, reduced odds of PIH among pregnant smokers was only evident for non-Hispanic white and non-Hispanic American Indian women aged less than 35 years. Non-Hispanic Asian/Pacific Islander women who smoked during pregnancy had increased odds of PIH regardless of maternal age. Non-Hispanic white and non-Hispanic black women 35 years or older who smoked during pregnancy also had increased odds of PIH. Conclusion: Our study findings suggest important differences by maternal race/ethnicity and age in the association between cigarette use during pregnancy and PIH. More research is needed to establish the biologic and social mechanisms that might explain the variations with maternal age and race/ethnicity that were observed in our study. Citation: Chang JJ, Strauss JF III, Deshazo JP, Rigby FB, Chelmow DP, et al. (2014) Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? PLoS ONE 9(10): e106446. doi:10.1371/journal.pone.0106446 Editor: Ana Claudia Zenclussen, Medical Faculty, Otto-von-Guericke University Magdeburg, Medical Faculty, Germany Received March 16, 2014; Accepted August 5, 2014; Published October 22, 2014 Received March 16, 2014; Accepted August 5, 2014; Published October 22, 2014 ang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. pyright: 2014 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Chang 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. 1, Jerome F. Strauss III2, Jon P. Deshazo3, Fidelma B. Rigby4, David P. Chelmow4, 5 1 Department of Epidemiology, College for Public Health and Social Justice, Saint Louis University, St. Louis, Missouri, United States of America, 2 VCU Medical Center School of Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America, 3 Department of Health Administration, School of Allied Health Professions, Virginia Commonwealth University, Richmond, Virginia, United States of America, 4 Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, Virginia, United States of America, 5 Department of Obstetrics and Gynecology, Washington University, St. Louis, Missouri, United States of America Abstract Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The primary data utilized in our analyses are deposited in public databases. The 2010 public-use U.S. natality file from Center for Disease Control and Prevention’s National Center for Health Statistics is publicly available at http://www.cdc.gov/nchs/data_access/Vitalstatsonline.htm The U.S. National Inpatient Sample (NIS) database is publicly available at http:// www.hcup-us.ahrq.gov/nisoverview.jsp. Funding: This work was supported by the National Institute of Health (Grant P60 MD002256). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Email: jjchang@slu.edu ogen activation [7], apoptosis [8,9], reactive oxygen species formation [10], and sFlt-1, an antiangiogenic factor [11,12]. Recommended Citation Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? Chang JJ, Strauss JF III, Deshazo JP, Rigby FB, Chelmow DP, et al. (2014) Reassessing the Impact of Smoking on Preeclampsia/Eclampsia: Are There Age and Racial Differences? PLoS ONE 9(10): e106446. doi: 10.1371/journal.pone.0106446 This Article is brought to you for free and open access by the Dept. of Obstetrics and Gynecology at VCU Scholars Compass. It has been accepted for inclusion in Obstetrics and Gynecology Publications by an authorized administrator of VCU Scholars Compass. For more information, please contact libcompass@vcu.edu. libcompass@vcu.edu. October 2014 \| Volume 9 \| Issue 10 \| e106446 Materials and Methods This population-based, retrospective cohort study was based on the 2010 public-use U.S. natality file from Center for Disease Control and Prevention’s National Center for Health Statistics. We used data based on the 1989 (unrevised) version of the birth certificate for this analysis because the 2003 release has substantial missing data for some key variables for the present study. The natality file includes data on parental demographics, medical and obstetrical characteristics and complications, and neonatal status at birth. Our study sample consisted of U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation in 2010. Pregnancies complicated by major fetal anomalies were excluded. These exclusions resulted in a study sample of 3,113,164 pregnancies. U.S. natality data is a publicly available data set and qualified for exemption from Institutional Review Board approval. The exposure, cigarette use during pregnancy, was indicated with a yes/no binary variable based on maternal self-report on the birth certificate. The outcome was PIH and included all women who had the condition ‘‘pregnancy-associated hypertension (i.e., PIH/preeclampsia)’’ or ‘‘eclampsia’’ checked on the 1989 version of the birth certificate [26,27]. Pregnancy-associated hypertension was defined as pregnancy- induced hypertension after the 20th week of gestation that resulted in an increase in blood pressure of at least 30 mm Hg systolic or 15 mm Hg diastolic on two measurements taken 6 hours apart [27]. Eclampsia, defined as the occurrence of convulsions and/or coma unrelated to other cerebral conditions in women with signs and symptoms of preeclampsia [27], was included in the preeclampsia group due to the small number of women affected within each ethnic group after stratification by maternal race/ethnicity and age. Mother’s race and ethnicity were self-reported and categorized as non- Hispanic white, non-Hispanic black, Hispanic, non-Hispanic Asian/Pacific Islander (non-Hispanic Asian), and non-Hispanic American Indians/Alaskan natives (non-Hispanic American Indi- ans), and Hispanics. This population-based, retrospective cohort study was based on the 2010 public-use U.S. natality file from Center for Disease Control and Prevention’s National Center for Health Statistics. We used data based on the 1989 (unrevised) version of the birth certificate for this analysis because the 2003 release has substantial missing data for some key variables for the present study. The natality file includes data on parental demographics, medical and obstetrical characteristics and complications, and neonatal status at birth. Our study sample consisted of U.S. women who delivered singleton pregnancies between 20 and 44 weeks of gestation in 2010. Materials and Methods Pregnancies complicated by major fetal anomalies were excluded. These exclusions resulted in a study sample of 3,113,164 pregnancies. U.S. natality data is a publicly available data set and qualified for exemption from Institutional Review Board approval. Smoking during Pregnancy and Preeclampsia Smoking during Pregnancy and Preeclampsia chronic hypertension, diabetes, and kidney disease [24]. For instance, it has been shown that white women are more likely to smoke heavily during pregnancy ($20 cigarette daily) than women of other race and ethnicity [23,25]. birth using Diagnosis Related Groups codes (765, 766, 767,768, 774, 775) assigned to inpatient visits from years 2006, 2008, and 2010. NIS data from 2006 and 2008 were included to increase the sample size due to the small number of eclampsia cases available. Multiple births were excluded (as defined by ICD9 codes V31– V37). This resulted in a NIS data sample of 12,326,151 pregnancies. We used ICD-9-CM codes to identify the exposure (i.e. cigarette use during pregnancy) and outcome (PIH) as well as maternal medical conditions as follows: tobacco use (305.1, V. 158.2), preeclampsia and pregnancy-induced hypertension (642.46, 642.56, 642.36), and eclampsia (642.61, 642.62, 642.63, 642.64), diabetes (ICD9 250), and chronic hypertension (642.2, 401). Despite the acknowledged disparity in hypertensive disorders during pregnancy by maternal age and race/ethnicity, few prior studies have examined the impact of their joint interactive effect with other known risk factors. The objective of this study was to examine the association between cigarette use during pregnancy and PIH by maternal race/ethnicity and age. Statistical analysis Mother’s race and ethnicity were self-reported and categorized as non- Hispanic white, non-Hispanic black, Hispanic, non-Hispanic Asian/Pacific Islander (non-Hispanic Asian), and non-Hispanic American Indians/Alaskan natives (non-Hispanic American Indi- ans), and Hispanics. Statistical analysis y Differences in sample characteristics by race/ethnicity were assessed by using the Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. Multivariable binary logistic regression models were used to estimate the crude and adjusted odds ratios (aOR) and the corresponding 95% confidence interval (95% CI). To reduce the bias in the parameter estimation, potential confounders were included in the multivariate analysis, including maternal age, marital status, parity, Kotelchuck prenatal care index, gestational weight gain, and maternal medical risk factors (i.e., diabetes and chronic hypertension). To evaluate if maternal race/ethnicity and age are effect modifiers, the Wald test was used to test if the regression coefficient of the product term of cigarette use during pregnancy, maternal race/ethnicity, and maternal age was statistically different from zero. Advanced maternal age, defined as 35 years or older, has been associated with adverse pregnancy [32]. Therefore, we dichotomized maternal age using a cutoff value of 35. We detected a significant interaction effect (interaction term P- value,0.01) for the product term of cigarette use during pregnancy, maternal race/ethnicity, and maternal age. Therefore, we stratified by race/ethnicity and age in the multivariable analysis. To generate nationally repre- sentative estimates, the analyses based on the NIS sample data were weighted and adjusted for clustering data structure using the survey commands. All tests were 2 tailed and P,0.05 was considered significant. All statistical analyses were performed with STATA (version 10.0, STATA Corp, College Station, TX). q p pp The exposure, cigarette use during pregnancy, was indicated with a yes/no binary variable based on maternal self-report on the birth certificate. The outcome was PIH and included all women who had the condition ‘‘pregnancy-associated hypertension (i.e., PIH/preeclampsia)’’ or ‘‘eclampsia’’ checked on the 1989 version of the birth certificate [26,27]. Pregnancy-associated hypertension was defined as pregnancy- induced hypertension after the 20th week of gestation that resulted in an increase in blood pressure of at least 30 mm Hg systolic or 15 mm Hg diastolic on two measurements taken 6 hours apart [27]. Eclampsia, defined as the occurrence of convulsions and/or coma unrelated to other cerebral conditions in women with signs and symptoms of preeclampsia [27], was included in the preeclampsia group due to the small number of women affected within each ethnic group after stratification by maternal race/ethnicity and age. Introduction Preeclampsia is associated with significant pregnancy-related morbidity and mortality, [1]. The etiology of preeclampsia is still not well understood, but several risk factors have been identified. These include genetic factors [2,3], nulliparity, multifetal gesta- tions, maternal race and age, and pre-existing conditions such as preeclampsia in a prior pregnancy, chronic hypertension, kidney disease, diabetes mellitus, and obesity [1]. Advanced maternal age and race/ethnicity have been well documented to be significant risk factors for a number of adverse pregnancy outcomes. Gregory and Korst observed that older pregnant women experienced increased risk of a number of maternal, fetal, and placental conditions including hypertension [13]. Rates of hypertensive disorders and preeclampsia appear to vary by race and ethnicity, as do the presentation and course of the disease [13–23]. In the U.S., preeclampsia risk is higher in ethnic minority women compared with non-Hispanic white women, with African-American women having the highest rate [18,22,23]. The causal mechanisms explaining the racial and ethnic differences in hypertensive disorders during pregnancy are largely unknown. These disparities might be related to a number of risk factors that are associated with race and ethnicity for preeclampsia, including Numerous studies have documented an inverse association between cigarette smoking during pregnancy and preeclampsia in different populations with a reduction of risk by up to 50% [4]. The underlying mechanism for this association is still not well understood with several hypothesized pathways, including carbon monoxide-mediated inhibition of inflammation [5], enhanced vasodilation [6], suppression of platelet aggregation [7], plasmin- October 2014 \| Volume 9 \| Issue 10 \| e106446 October 2014 \| Volume 9 \| Issue 10 \| e106446 1 PLOS ONE \| www.plosone.org Results Characteristic Natality data NIS samples p-value* (n = 3,113,164) (n = 12,326,151) % % Mother’s age Mean (SD) 26.6 (6.1) 27.5 (13.6) ,0.0001 Mother’s age ,20 9.4 10.1 ,0.0001 20–34 76.5 75.6 35+ 14.1 14.3 Tobacco use 9.2 2.1 ,0.0001 PIH/Preeclampsia 4.1 6.7 ,0.0001 Eclampsia 0.27 0.08 ,0.0001 Chronic hypertension 1.3 0.7 ,0.0001 Diabetes 5.0 0.8 ,0.0001 Marital status 40.1 na Not married Parity na 1 59.0 $2 41.0 Prenatal care adequacy Inadequate 14.7 na Intermediate 10.1 Adequate 42.1 Adequate+ 33.1 Weight gain 0–19 lbs 24.8 na 20–39 lbs 51.2 40+ lbs 24.0 Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension. p value from Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. doi:10.1371/journal.pone.0106446.t001 Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension. p value from Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. doi:10.1371/journal.pone.0106446.t001 Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension. p value from Pearson Chi-square test (x2) for categorical variables and t-test for continuous variables. doi:10.1371/journal.pone.0106446.t001 Table 4 shows that the inverse relationship between maternal smoking and PIH was age dependent. The odds of PIH increased with age in all ethnic groups after adjusting for potential confounders. The reduced odds of PIH was only evident for non-Hispanic white and non-Hispanic American Indian women young than 35 years old who smoked during pregnancy based on the natality data. This decreased odds of PIH conferred by maternal smoking was similarly observed in non-Hispanic white women younger than 35 years old based on NIS sample, albeit with a borderline significant and weaker strength of association. It is noteworthy that the increased odds of PIH among non-Hispanic Asian women who smoked during pregnancy persisted regardless of maternal age, with stronger association among older women within this ethnic group (Table 4). Furthermore, data from both the natality and the NIS sample indicated increased odds of PIH among non-Hispanic white women 35 years or older who smoked during pregnancy compared with those who did not, with stronger association based on the NIS data (Table 4). Interestingly, non- Hispanic white women also had the lowest aOR in the older age group, suggesting that there may still be a mitigating effect of smoking within this ethnic group. care compared to women of other ethnic origins. Results In this study, data from the birth certificates served as the primary source because they provide more demographic and lifestyle variables than the NIS data as potential confounders for the multivariable analysis. As expected by the differing data sources, there were statistically significant differences in selected maternal characteristics between the natality and the NIS data samples (Table 1). Women in the NIS data were slightly older than those from the natality data. Prevalence of pregnancy-induced hypertension was greater in the NIS sample than in the natality data. The prevalence of cigarette use during pregnancy, eclampsia, chronic hypertension, and diabetes were lower in the NIS data than in the natality data (Table 1). Factors that may be associated with maternal smoking and PIH were evaluated as potential confounders. Data for the following demographic and lifestyle variables were obtained from the birth certificate: maternal age (,20, 20 to 34, and $35 years), marital status (single or married), parity, Kotelchuck prenatal care index [28], gestational weight gain (,20 lbs, 20 to 39 lbs, and $40 lbs), and maternal medical risk factors including diabetes and chronic hypertension. Some studies had suggested potential misclassifications of medical conditions with data from birth certificates [29,30]. To address this issue, we attempted to validate our study findings from the natality data by replicating the same analysis as in the birth certificate data in a nationally representative sample of maternal hospital discharges from the U.S. National Inpatient Sample (NIS) database, the largest all-payer, publicly available inpatient database in the U.S. [31]. We identified in NIS records representing hospital stays for women who were pregnant or gave Table 2 shows the maternal characteristics in different ethnic groups from the natality data. All variables showed significant differences by race/ethnicity. The mean (standard deviation) maternal age of non-Hispanic Asian [30.6 years (5.3)] was higher (P,0.01) than that of other ethnic groups. Non-Hispanic American Indian women had the highest proportions of teenage births (16%), tobacco use during pregnancy (18.6%), PIH/ preeclampsia (5.3%), eclampsia (0.6%), and inadequate prenatal October 2014 \| Volume 9 \| Issue 10 \| e106446 PLOS ONE \| www.plosone.org 2 Smoking during Pregnancy and Preeclampsia Table 1. Study Sample Characteristics. October 2014 \| Volume 9 \| Issue 10 \| e106446 Results Relative to women in other ethnic groups, non-Hispanic black women had the greatest proportions of chronic hypertension (2.8%) and being single (72.5%) whereas non-Hispanic Asian had the largest percentage of women with diabetes (8.7%). Using the natality data, we found decreased odds of PIH among non-Hispanic white and non-Hispanic American Indian women who smoked during pregnancy (aOR = 0.90, 95% CI: 0.88, 0.92 and aOR = 0.80, 95% CI: 0.70, 0.91, respectively, Table 3) compared to those who did not smoke during pregnancy, after controlling for confounders that included maternal age, marital status, parity, Kotelchuck prenatal care index, gestational weight gain, chronic hypertension, and diabetes. On the contrary, non- Hispanic black, non-Hispanic Asian, and Hispanic women who smoked during pregnancy had increased likelihood of PIH, after adjusting for confounders (Table 3). The increased odds of PIH for non-Hispanic Asian women who smoked during pregnancy was also observed in the NIS sample (aOR = 1.53, 95% CI: 1.14, 2.04) after controlling for maternal age, chronic hypertension, and diabetes (Table 3). Some covariates including marital status, parity, Kotelchuck prenatal care index, gestational weight gain were not available in NIS dataset and thus could not be included in the multivariate analysis for the NIS sample. There may be potential overlap between the 2010 US natality and the NIS sample, which includes data from year 2006, 2008, and 2010. Therefore, we conducted a subsample analysis for the October 2014 \| Volume 9 \| Issue 10 \| e106446 October 2014 \| Volume 9 \| Issue 10 \| e106446 PLOS ONE \| www.plosone.org 3 Smoking during Pregnancy and Preeclampsia Table 2. Maternal Characteristics by Race/Ethnicity, US 2010 Natality file (n = 3,113,164). Results Characteristic NH White NH Black NH American Indian NH Asian/Pacific Islander Hispanic P Value (n = 1,718,634) (n = 402,472) (n = 34,348) (n = 192,141) (n = 765,569) n(%) n(%) n(%) n(%) n(%) Mother’s age Mean (SD) 28.2 (5.8) 25.9 (6.2) 26.4 (5.8) 30.6 (5.3) 30.2 (5.4) ,0.01 Mother’s age ,0.01 ,20 117,061 (6.8) 62,348 (15.5) 5,503(16.0) 4,155(2.2) 104,003 (13.6) 20–34 1,347,959(78.4) 298,208 (74.1) 26,092 (76.0) 141,816 (73.8) 567,453 (74.1) 35+ 253,614 (14.8) 41,916 (10.4) 2,753(8.0) 46,170 (24.0) 94,113 (12.3) Tobacco use 227,997 (13.3) 33,300 (8.3) 6,389 (18.6) 2,800(1.5) 15,931(2.1) ,0.01 PIH/Preeclampsia 77,812 (4.5) 20,616 (5.1) 1,835 (5.3) 4,558 (2.4) 23,505(3.1) ,0.01 Eclampsia 4,554 (0.3) 1,720 (0.4) 195(0.6) 404(0.2) 1,635(0.2) ,0.01 Chronic hypertension 21,610 (1.3) 11,330 (2.8) 601 (1.8) 1,535 (0.8) 5,363(0.7) ,0.01 Diabetes 79,167 (4.6) 17,915 (4.5) 2,397 (7.0) 16,656(8.7) 40,005 (5.2) ,0.01 Marital status ,0.01 Married 1,224,022(71.2) 110,690 (27.5) 11,519 (33.5) 162,467 (84.6) 356,277 (46.5) Not married 494,612 (28. 8) 291,782 (72.5) 22,829 (66.5) 29,674 (15.4) 409,292 (53.5) Parity ,0.01 1 741,778 (43.2) 164,284 (40.8) 12,524 (36.5) 88,947 (46.3) 269,956 (35.3) $2 976,856 (56.8) 238,188 (59.2) 21,824 (63.5) 103,194 (53.7) 495,613 (64.7) Prenatal care Adequacy ,0.01 Inadequate 184,037 (10.7) 88,795 (22.1) 8,826 (25.7) 23,195 (12.1) 152,607 (19.3) Intermediate 161,565 (9.4) 43,267 (10.8) 4,719 (13.7) 21,244 (11.1) 84,207 (11.0) Adequate 769,893 (44.8) 144,370 (35.9) 12,315 (35.9) 84,611 (44.0) 298,586 (39.0) Adequate+ 603,139 (35.1) 126,040 (31.3) 8,488 (24.7) 63,091 (32.8) 230,169 (30.1) Weight gain ,0.01 0–19 lbs 356,997 (20.8) 128,957 (32.0) 10,390 (30.3) 43,571 (22.7) 232,815 (30.4) 20–39 lbs 899,157 (52.3) 177,754 (44.2) 15,565 (45.3) 113,758 (59.2) 388,071 (50.7) 40+ lbs 462,480 (26.9) 95,761 (23.8) 8,393 (24.4) 34,812 (18.1) 144,683 (18.9) Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi Square test Table 3. Odds Ratios for the Effect of Smoking on PIH among Ethnic Groups. djusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabete djusted for maternal age, chronic hypertension, and diabetes. ; , p , , g y yp maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi-Square tes p g y yp p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (contin (categorical variables). Abbreviation: SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension bb e at o : SD, sta da d de at o ; , o spa c, , eg a cy duced ype te s o p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi-Square test Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. + adjusted for maternal age, chronic hypertension, and diabetes. doi:10 1371/journal pone 0106446 t003 reviation: OR, odds ratio, 95% CI, 95% confidence interval. : SD, standard deviation; NH, non-Hispanic, IH, Pregnancy induced hypertension , ; , p , , g y yp p value for the association of maternal race/ethnicity with all maternal characteristics in the study based on analysis of variance (continuous variable) or Chi-Square test (categorical variables) Results adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. +adjusted for maternal age, chronic hypertension, and diabetes. doi:10.1371/journal.pone.0106446.t004 on the natality data and conferred for the older age group by the NIS data. 2010 US natality data set among primiparous women and the NIS sample from only 2006 and 2008, separately. In this subsample analysis, we obtained results that are largely consistent with the analysis of the US natality and the multi-year NIS sample. We observed that the association between maternal cigarette use and hypertensive disorders of pregnancy varied by maternal race/ ethnicity and age (Table S1). Specifically, the decreased odds of PIH among women who smoked during pregnancy was only apparent in non-Hispanic white and American Indian women younger than 35 years old who smoked during pregnancy, based on the natality data and among non-Hispanic white and blacks only based on the NIS sample. We also observed that maternal cigarette use during pregnancy was associated with increased odds of PIH for non-Hispanic Asians younger than 35 years old based on the natality data and the increased odds was also conferred by the NIS data regardless of maternal age (Table S2). In general, we observed an association in the same direction with cigarette use during pregnancy and PIH between the two data sets, albeit with differences in the strength of association with a weaker association from the NIS data for women younger than 35 years of age, but a stronger association for mothers with age greater than or equal to 35 (Table 4). This discrepancy in findings between the two data sets may be explained by the different sources of information between the two data sets. In the NIS data, measures of interest were defined based on ICD-9 discharge diagnosis codes, whereas in the natality data smoking is self- reported by the patient and hypertensive disorders were coded by chart extractors. Hence, compared to the natality data, prevalence of maternal smoking during pregnancy in the NIS data was much lower, whereas, the prevalence of the outcome was higher (Table 1). Geller and colleagues [34] evaluated the accuracy of the ICD-9 revision codes for preeclampsia and eclampsia and observed variation in accuracy of diagnosis with a positive predictive value for severe preeclampsia of 84.8%, 45.3% for mild preeclampsia, and 41.7% for eclampsia. Results The potential misclassification in NIS data for exposure and the outcome were likely non-differential, however, which would bias the point estimate toward the null value and may explain the weaker strength of association observed in the younger women in the NIS data. Results Natality data (n = 3,113,164) NIS data (n = 12,326,151) Crude OR 95 % CI Adjusted OR* 95 % CI Adjusted OR+ 95 % CI NH White 0.89 0.87, 0.91 0.90 0.88, 0.92 0.99 0.94–1.03 NH Black 0.99 0.94, 1.04 1.07 1.02, 1.13 0.95 0.85, 1.06 NH American Indian 0.74 0.65, 0.84 0.80 0.70, 0.91 0.97 0.65, 1.43 NH Asian/Pacific Islander 1.73 1.44, 2.09 1.64 1.36, 1.99 1.53 1.14, 2.04 Hispanic 1.11 1.02, 1.21 1.09 1.01, 1.19 0.98 0.84, 1.14 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. + adjusted for maternal age, chronic hypertension, and diabetes. doi:10 1371/journal pone 0106446 t003 * adjusted for maternal age, marital status, parity, kotelchuck pren + adjusted for maternal age, chronic hypertension, and diabetes. October 2014 \| Volume 9 \| Issue 10 \| e106446 October 2014 \| Volume 9 \| Issue 10 \| e106446 PLOS ONE \| www.plosone.org 4 Smoking during Pregnancy and Preeclampsia Table 4. Odds Ratios for the Effect of Smoking on PIH Among Ethnic Groups by Maternal Age. Natality data NIS data (n = 12,326,151) (n = 3,113,164) Women ,35 yrs Adjusted OR* 95 % CI Adjusted OR+ 95 % CI NH White 0.89 0.86, 0.91 0.95 0.91, 1.00 NH Black 1.05 1.00, 1.09 0.91 0.81, 1.03 NH American Indian 0.76 0.66, 0.87 0.96 0.64, 1.45 NH Asian/Pacific Islander 1.66 1.35, 2.05 1.36 0.96, 1.93 Hispanic 1.09 1.00, 1.20 0.99 0.84, 1.17 Natality data NIS data (n = 12,326,151) (n = 3,113,164) Women $35 yrs Adjusted OR* 95 % CI Adjusted OR+ 95 % CI NH White 1.17 1.09, 1.26 1.29 1.13, 1.47 NH Black 1.18 1.01, 1.37 1.30 0.97, 1.62 NH American Indian 1.29 0.88, 1.89 1.07 0.25, 4.63 NH Asian/Pacific Islander 1.71 1.07, 2.72 2.32 1.35, 3.99 Hispanic 1.18 0.91, 1.54 0.91 0.57, 1.46 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. +adjusted for maternal age, chronic hypertension, and diabetes. doi:10.1371/journal.pone.0106446.t004 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. * adjusted for maternal age, marital status, parity, kotelchuck prenatal care index, gestational weight gain, chronic hypertension, diabetes. +adjusted for maternal age, chronic hypertension, and diabetes. doi:10.1371/journal.pone.0106446.t004 Abbreviation: OR, odds ratio, 95% CI, 95% confidence interval. October 2014 \| Volume 9 \| Issue 10 \| e106446 Smoking during Pregnancy and Preeclampsia Nevertheless, this measurement error of exposure was likely non-differential, which we expect would result in the attenuation of our point estimate toward the null value [36],which may explain the weaker association in our findings. In addition, the different rates of maternal smoking and preeclampsia across ethnic groups observed by previous studies may have contributed to this discrepancy. Our observation of the increased odds of PIH among non- Hispanic Asian women was unexpected and of particular interest. In the non-pregnant population, it has been observed that Asian and Indian adults have an increased risk of diabetes, hypertension, and dyslipidemia at lower BMI levels than European adults [37]. Additionally, in a study of ethnic variations in HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome among pregnant women with preexisting hypertension, Williams et al. observed that Caucasian and Chinese women were more likely than East Indians to develop HELLP syndrome [38]. Asian women may also differ from women of other ethnic origins in socioeconomic status, stress, physical activities, diet, and other social or life style factors that could contribute to this differential risk in hypertensive disorders of pregnancy. p p Our study findings are also limited by the self-reported information on tobacco use status during pregnancy. However, the prevalence of cigarette use during pregnancy in natality data from the present study is consistent with the population-based estimate from the 2010 Pregnancy Risk Assessment and Monitor- ing System data from 27 states conducted by the U.S. Centers for Disease Control and Prevention [52]. England et al. reported that 24% of smokers during pregnancy were misclassified as quitters in a large multicenter randomized study of nulliparous women [53]. Llurba and associates observed a 90% concordance between self- reported smoking and cotinine levels among 125 healthy Spanish pregnant women at 24 weeks of gestation in a case control study [54]. To estimate the bias introduced by the potential misclassi- fication of maternal smoking status in our study based on natality data file, we conducted a sensitivity analysis. Assuming a 76% sensitivity and 100% specificity of our exposure measurement, our parameter estimates remained largely the same for results shown in Table 3 and 4 after correcting for the measurement error [55]. Our study is further limited by the lack of information on the timing, intensity, and frequency of maternal smoking, which could potentially vary by race/ethnicity. In addition, our analysis used broad categorizations of ethnicity (e.g. Smoking during Pregnancy and Preeclampsia non-Hispanic Asian and Hispanic), which may obscure an association between maternal smoking and PIH within ethnic subgroups. When analyzing different ethnic and racial groups in the present study based on a U.S. population, it is difficult to ascertain whether the categori- zation of maternal race/ethnicity summarizes genetics or envi- ronment. While racial group implies a specific genetic inheritance, ethnicity reflects culture and is therefore changeable. In this study no attempt has been made to distinguish between the two potential effects in the role of maternal ethnic origin as an effect modifier. p g y In general, race/ethnicity may influence health outcomes due to differences in socio-economic status, life style (i.e., diet and physical activity), access to medical care, and medical conditions among ethnic groups [19]. For instance, blacks have higher prevalence of hypertension [39] and obesity [40] than whites. Cigarette use during pregnancy was less prevalent among Asians and blacks compared to whites [35,41,42]. Knuistet al suggested that risk factors for preeclampsia differed in prevalence among women of different ethnic groups such that the diastolic blood pressure was the strongest predictor for preeclampsia in white women whereas high maternal age was a better predictor for preeclampsia in blacks [23]. Previous studies also suggested that the underlying pathophysiology of hypertensive disorders may be different in blacks than in whites [43,44]. The varying rates of preeclampsia by race/ethnicity could also be attributed to genetic predisposition to developing diseases [45,46]. Prasmusinto et al. demonstrated ethnic differences in the association of factor V Leiden mutation and the C677T methylenetetrahydrofolate reductase gene polymorphism with preeclampsia among white and Indonesian mothers [47]. Variations in the distribution in the risk factors of preeclampsia including maternal smoking among ethnic groups combined with ethnic variation in genetic polymor- phisms predisposing to preeclampsia may explain the observed ethnic differences in the association between maternal smoking and preeclampsia. Further studies are needed to elucidate the biologic mechanisms underlying the ethnic differences in this relationship. The strength of this study rests in its use of a large population- based sample of U.S. women and the availability of information on many potential confounders that may affect the risk of PIH from the natality data. The large sample size provided adequate statistical power to detect significant associations, increased precision in the risk estimates, and the ability to evaluate the potential effect modifying role of maternal race/ethnicity and age. Smoking during Pregnancy and Preeclampsia data [35]. Knuist et al. observed an inverse association between maternal smoking and preeclampsia in both white [adjusted relative risk: 0.8, 95% CI: 0.3, 1.7] and black women (adjusted relative risk: 0.5, 95% CI: 0.1, 4.4), but the results were not statistically significant. Findings from Knuist et al. may be limited by the lack of sufficient statistical power for a stratified analysis by maternal race [23]. discharge data from New York City, Engel et al. found that the reduced risk of preeclampsia among women who smoked during pregnancy was limited to women aged 30 years or younger and that more advanced maternal age may be associated with greater risk of hypertension with preeclampsia [50]. Our findings are in general agreement with this observation. data [35]. Knuist et al. observed an inverse association between maternal smoking and preeclampsia in both white [adjusted relative risk: 0.8, 95% CI: 0.3, 1.7] and black women (adjusted relative risk: 0.5, 95% CI: 0.1, 4.4), but the results were not statistically significant. Findings from Knuist et al. may be limited by the lack of sufficient statistical power for a stratified analysis by maternal race [23]. Some methodological limitations of this study need to be considered in interpreting our study findings. They include the potential for inaccurate reporting, residual confounding by socioeconomic and other unmeasured maternal characteristics (e.g., stress, physical activities, nutrition), the lack of information regarding the diagnosis, timing and severity of preeclampsia, and misclassification of medical and obstetrical conditions. A prior validation study has indicated that the reporting rate of preeclampsia on birth certificates with a check-box format is fairly good, ranging from 85% to 97% when compared with risks based on hospital discharge data [51]. In our study, we used pregnancy induced hypertension to approximate preeclampsia as our outcome which may results in measurement error. It is unclear whether pregnancy induced hypertension and preeclampsia are two distinct disorders that share a similar symptom (i.e., hypertension) or if pregnancy induced hypertension is a precursor of preeclampsia [16]. The weaker association between maternal smoking and PIH from our study may be attributed to potential misclassification of the exposure, maternal smoking, due to recall bias or under- reporting for both natality and the NIS data. Discussion Numerous studies have observed a decreased risk of both preeclampsia and gestational hypertension among women who smoked during pregnancy with an average aOR of 0.7 [4,33]. However, to our knowledge, no prior studies have examined the joint impact of maternal race/ethnicity and age on the relationship between smoking and hypertensive disorders of pregnancy. The present study found that the association between maternal cigarette use and hypertensive disorders of pregnancy varied by maternal race/ethnicity and age. Specifically, the decreased odds of PIH among women who smoked during pregnancy was only apparent in non-Hispanic white and American Indian women younger than 35 years old who smoked during pregnancy, based on the natality data and among non-Hispanic white only based on the NIS sample. Interestingly, we also observed that maternal cigarette use during pregnancy was associated with increased odds of PIH for non-Hispanic Asians regardless of maternal age, based The inverse association between maternal smoking and PIH among non-Hispanic white and American Indian women in our study appears to be weaker compared to findings from prior research [23,33,35]. Misra et al. found that maternal smoking was associated with reduced odds of gestational hypertension in white women (aOR: 0.17, 95% CI: 0.12 to 0.24) but not in black women (aOR: 0.35, 95% CI: 0.09–1.37) [35]. However, results from this study may have been affected by selection bias as more than half of the eligible subjects were excluded from the study due to missing October 2014 \| Volume 9 \| Issue 10 \| e106446 October 2014 \| Volume 9 \| Issue 10 \| e106446 PLOS ONE \| www.plosone.org 5 October 2014 \| Volume 9 \| Issue 10 \| e106446 References 20. Janakiraman V, Gantz M, Maynard S, El-Mohandes A (2009) Association of cotinine levels and preeclampsia among African-American women. Nicotine Tob Res 11: 679–684. 1. Ananth CV, Vintzileos AM (2011) Ischemic placental disease: epidemiology and risk factors. Eur J Obstet Gynecol Reprod Biol 159: 77–82. y p 2. Nejatizadeh A, Stobdan T, Malhotra N, Pasha MA (2008) The genetic aspects of pre-eclampsia: achievements and limitations. Biochem Genet 46: 451–479. 21. Anderson NH, Sadler LC, Stewart AW, Fyfe EM, McCowan LM (2012) Ethnicity, body mass index and risk of pre-eclampsia in a multiethnic New Zealand population. Aust N Z J Obstet Gynaecol 52: 552–558. 3. Palei AC, Spradley FT, Warrington JP, George EM, Granger JP (2013) Pathophysiology of hypertension in pre-eclampsia: a lesson in integrative physiology. Acta Physiol (Oxf) 208: 224–233. 22. Caughey AB, Stotland NE, Washington AE, Escobar GJ (2005) Maternal ethnicity, paternal ethnicity, and parental ethnic discordance: predictors of preeclampsia. Obstet Gynecol 106: 156–161. 4. England L, Zhang J (2007) Smoking and risk of preeclampsia: a systematic review. Frontiers in Bioscience 12: 2471–2483. 23. Knuist M, Bonsel GJ, Zondervan HA, Treffers PE (1998) Risk factors for preeclampsia in nulliparous women in distinct ethnic groups: a prospective cohort study. Obstet Gynecol 92: 174–178. 5. Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, et al. (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 6: 422–428. 24. Roberts J (2009) Maternal-Fetal Medicine: principles and practice. In: Creasy R, Resnik R, Iams JD, editors. Pregnancy-related hypertension. Philadelphia, PA: Saunders Elsevier; . pp. 650–688. p y 6. Zhang F, Kaide JI, Rodriguez-Mulero F, Abraham NG, Nasjletti A (2001) Vasoregulatory function of the heme-heme oxygenase-carbon monoxide system. Am J Hypertens 14: 62S–67S. 25. Perreira KM, Cortes KE (2006) Race/ethnicity and nativity differences in alcohol and tobacco use during pregnancy. Am J Public Health 96: 1629–1636. 7. Fujita T, Toda K, Karimova A, Yan SF, Naka Y, et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat Med 7: 598–604. 26. (1996) ACOG technical bulletin. Hypertension in pregnancy. Number 219– January 1996 (replaces no. 91, February 1986). Committee on Technical Bulletins of the American College of Obstetricians and Gynecologists. Int J Gynaecol Obstet 53: 175–183. 8. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, et al. References (2000) Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 192: 1015–1026. 27. (n.d.) Division of Vital Statistics. Technical appendix from vital statistics of the United States, Natality, March, 2001 Hyattsville, Maryland: National Center for Health Statistics. 9. Liu XM, Chapman GB, Peyton KJ, Schafer AI, Durante W (2002) Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc Res 55: 396–405. 28. Bloch JR, Dawley K, Suplee PD (2009) Application of the Kessner and Kotelchuck prenatal care adequacy indices in a preterm birth population. Public Health Nurs 26: 449–459. 10. Wang X, Wang Y, Kim HP, Nakahira K, Ryter SW, et al. (2007) Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation. J Biol Chem 282: 1718–1726. 29. Watkins ML, Edmonds L, McClearn A, Mullins L, Mulinare J, et al. (1996) The surveillance of birth defects: the usefulness of the revised US standard birth certificate. Am J Public Health 86: 731–734. 11. Powers RW, Roberts JM, Cooper KM, Gallaher MJ, Frank MP, et al. (2005) Maternal serum soluble fms-like tyrosine kinase 1 concentrations are not increased in early pregnancy and decrease more slowly postpartum in women who develop preeclampsia. Am J Obstet Gynecol 193: 185–191. 30. Allen AM, Dietz PM, Tong VT, England L, Prince CB (2008) Prenatal smoking prevalence ascertained from two population-based data sources: birth certificates and PRAMS questionnaires, 2004. Public Health Rep 123: 586–592. 12. Cudmore M, Ahmad S, Al-Ani B, Fujisawa T, Coxall H, et al. (2007) Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation 115: 1789–1797. 31. Health care Cost and Utilization Project (HCUP) (2011) Introduction to the NIS:2009, HCUP NIS related reports. Rockville, MD: Agency for Healthcare Research and Quality. 13. Gregory KD, Korst LM (2003) Age and racial/ethnic differences in maternal, fetal, and placental conditions in laboring patients. Am J Obstet Gynecol 188: 1602–1606; discussion 1606–1608. 32. Waldenstrom U, Aasheim V, Nilsen AB, Rasmussen S, Pettersson HJ, et al. (2014) Adverse pregnancy outcomes related to advanced maternal age compared with smoking and being overweight. Obstet Gynecol 123: 104–112. ; 14. Goodwin AA, Mercer BM (2005) Does maternal race or ethnicity affect the expression of severe preeclampsia? Am J Obstet Gynecol 193: 973–978. 33. Conde-Agudelo A, Althabe F, Belizan JM, Kafury-Goeta AC (1999) Cigarette smoking during pregnancy and risk of preeclampsia: a systematic review. Acknowledgments This work was supported by the National Institute of Health (Grant P60 MD002256). We thank Dr. Emmanuel A. Anum for his contributions in data analysis to the early phases of this work. This work was supported by the National Institute of Health (Grant P60 MD002256). We thank Dr. Emmanuel A. Anum for his contributions in data analysis to the early phases of this work. Smoking during Pregnancy and Preeclampsia In addition, our study used two independent data sources and observed similar findings albeit with variations in the strength of association for the effect of interest. Using the NIS data from Few studies have sufficient sample size to examine the effect modifying role of maternal age in the association between maternal smoking and preeclampsia. Advanced maternal age, especially after age 35, is associated with increased risk of preeclampsia [48] and other adverse pregnancy outcomes [49]. In a large study based on birth records linked with hospital October 2014 \| Volume 9 \| Issue 10 \| e106446 PLOS ONE \| www.plosone.org 6 Smoking during Pregnancy and Preeclampsia hospital inpatient records, which is superior to birth certificate check boxes further supports and enhances the validity of our study findings. Author Contributions Conceived and designed the experiments: JS DC JJC GM. Performed the experiments: JD JJC DC. Analyzed the data: JD JJC. Contributed reagents/materials/analysis tools: JJC JS JD FR DC GM. Wrote the paper: JJC JS JD FR DC GM. Supporting Information Table S1 Odds Ratios for the Effect of Smoking on PIH Among Ethnic Groups. (DOCX) Our study findings suggested important differences by maternal race/ethnicity and age in the association between maternal smoking and PIH. How race/ethnicity modifies this relationship is not clearly understood. It may be explained by a combination of social, behavioral, and genetic polymorphisms and disease susceptibility. While this disparity needs to be confirmed in future studies, our study results may help health professional identify specific subgroups of women who are at higher risk for PIH. Although the pathophysiologic pathways of preeclampsia are largely unknown, separation of women into etiologically homoge- neous groups in future studies of preeclampsia may improve our understanding and prediction of the disease. It is plausible that women of different racial and ethnic origins may have different clinical presentations and clinical courses of preeclampsia. More research is needed to establish the biologic and social mechanisms that might explain the variations by maternal age and race/ ethnicity that were observed in our study. Table S2 Odds Ratios for the Effect of Smoking on PIH Among Ethnic Groups by Maternal Age. (DOCX) Smoking during Pregnancy and Preeclampsia 38. Williams KP, Wilson S (1997) Ethnic variation in the incidence of HELLP syndrome in a hypertensive pregnant population. J Perinat Med 25: 498–501. trahydrofolate reductase gene polymorphism with preeclampsia. Eur J Obstet Gynecol Reprod Biol 112: 162–169. 39. Gillum RF (1996) Epidemiology of hypertension in African American women. Am Heart J 131: 385–395. 48. Duckitt K, Harrington D (2005) Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies. BMJ 330: 565. 40. Wattigney WA, Webber LS, Srinivasan SR, Berenson GS (1995) The emergence of clinically abnormal levels of cardiovascular disease risk factor variables among young adults: the Bogalusa Heart Study. Prev Med 24: 617–626. 49. Khalil A, Syngelaki A, Maiz N, Zinevich Y, Nicolaides KH (2013) Maternal age and adverse pregnancy outcomes: a cohort study. Ultrasound Obstet Gynecol. 50. Engel SM, Janevic TM, Stein CR, Savitz DA (2009) Maternal smoking, preeclampsia, and infant health outcomes in New York City, 1995–2003. Am J Epidemiol 169: 33–40. y g g y 41. Tuck SM, Cardozo LD, Studd JW, Gibb DM, Cooper DJ (1983) Obstetric characteristics in different racial groups. Br J Obstet Gynaecol 90: 892–897. 41. Tuck SM, Cardozo LD, Studd JW, Gibb DM, Cooper DJ (19 characteristics in different racial groups. Br J Obstet Gynaecol 90 g p y 42. Perry IJ, Beevers DG, Whincup PH, Bareford D (1995) Predictors of ratio of placental weight to fetal weight in multiethnic community. BMJ 310: 436–439. 42. Perry IJ, Beevers DG, Whincup PH, Bareford D (1995) Predictors of ratio of l t l i ht t f t l i ht i lti th i it BMJ 310 436 439 42. Perry IJ, Beevers DG, Whincup PH, Bareford D (1995) Predict 51. Frost F, Starzyk P, George S, McLaughlin JF (1984) Birth complication reporting: the effect of birth certificate design. Am J Public Health 74: 505–506. placental weight to fetal weight in multiethnic community. BMJ 310: 436–439. 43. Falkner B (1990) Differences in blacks and whites with essential hypertension: biochemistry and endocrine. State of the art lecture. Hypertension 15: 681–686. 43. Falkner B (1990) Differences in blacks and whites with essential hypertension: biochemistry and endocrine. State of the art lecture. Hypertension 15: 681–686. 52. Division of Reproductive Health, U.S. Centers for Disease Control and Prevention. (n.d.) Tobacco use and pregnancy. 44. Frohlich ED (1990) Hemodynamic differences between black patients and white patients with essential hypertension. References Am J Obstet Gynecol 181: 1026–1035. 15. Marshall NE, Guild C, Cheng YW, Caughey AB, Halloran DR (2013) Racial disparities in pregnancy outcomes in obese women. J Matern Fetal Neonatal Med. 34. Geller SE, Ahmed S, Brown ML, Cox SM, Rosenberg D, et al. (2004) International Classification of Diseases-9th revision coding for preeclampsia: how accurate is it? Am J Obstet Gynecol 190: 1629–1633; discussion 1633– 1624. 16. Wolf M, Shah A, Jimenez-Kimble R, Sauk J, Ecker JL, et al. (2004) Differential risk of hypertensive disorders of pregnancy among Hispanic women. J Am Soc Nephrol 15: 1330–1338. 17. Bouthoorn SH, Gaillard R, Steegers EA, Hofman A, Jaddoe VW, et al. (2012) Ethnic differences in blood pressure and hypertensive complications during pregnancy: the Generation R study. Hypertension 60: 198–205. 35. Misra DP, Kiely JL (1995) The effect of smoking on the risk of gestational hypertension. Early Hum Dev 40: 95–107. 36. Dosemeci M, Wacholder S, Lubin JH (1990) Does nondifferential misclassifi- cation of exposure always bias a true effect toward the null value? Am J Epidemiol 132: 746–748. 18. Tanaka M, Jaamaa G, Kaiser M, Hills E, Soim A, et al. (2007) Racial disparity in hypertensive disorders of pregnancy in New York State: a 10-year longitudinal population-based study. Am J Public Health 97: 163–170. 37. (2004) World Health Organization expert consultation. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet: 157–163. 19. Gong J, Savitz DA, Stein CR, Engel SM (2012) Maternal ethnicity and pre- eclampsia in New York City, 1995–2003. Paediatr Perinat Epidemiol 26: 45–52. October 2014 \| Volume 9 \| Issue 10 \| e106446 October 2014 \| Volume 9 \| Issue 10 \| e106446 7 PLOS ONE \| www.plosone.org trahydrofolate reductase gene polymorphism with preeclampsia. Eur J Obstet Gynecol Reprod Biol 112: 162–169. Smoking during Pregnancy and Preeclampsia State of the art lecture. Hypertension 15: 675–680. 53. England LJ, Grauman A, Qian C, Wilkins DG, Schisterman EF, et al. (2007) Misclassification of maternal smoking status and its effects on an epidemiologic study of pregnancy outcomes. Nicotine & Tobacco Research 9: 1005–1013. 45. El Sahly HM, Reich RA, Dou SJ, Musser JM, Graviss EA (2004) The effect of mannose binding lectin gene polymorphisms on susceptibility to tuberculosis in different ethnic groups. Scand J Infect Dis 36: 106–108. 54. Llurba E, Sanchez O, Dominguez C, Soro G, Goya M, et al. (2013) Smoking during pregnancy: changes in mid-gestation angiogenic factors in women at risk of developing preeclampsia according to uterine artery Doppler findings. Hypertens Pregnancy 32: 50–59. g p J 46. Pegoraro RJ, Hira B, Rom L, Moodley J (2003) Plasminogen activator inhibitor type 1 (PAI1) and platelet glycoprotein IIIa (PGIIIa) polymorphisms in Black South Africans with pre-eclampsia. Acta Obstet Gynecol Scand 82: 313–317. 55. Fink AK, Lash TL (2003) A null association between smoking during pregnancy and breast cancer using Massachusetts registry data (United States). Cancer Causes Control 14: 497–503. p p y 47. Prasmusinto D, Skrablin S, Fimmers R, van der Ven K (2004) Ethnic differences in the association of factor V Leiden mutation and the C677T methylenete- October 2014 \| Volume 9 \| Issue 10 \| e106446 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8
https://openalex.org/W2312459518	https://zenodo.org/records/2379161/files/article.pdf	English	null	Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of 1917–1918	The Boston medical and surgical journal/Boston medical and surgical journal	1,918	public-domain	3,333	ered us all winter, and that is really one of the most interesting and one of the most im- portant problems that has arisen during the winter's work among the new recruits. The cream is made by adding the plaster little by little to a basin holding about a quart of cold water, the water being gently but contin- uously agitated with the hand as the plaster is added. When the consistency is such that the withdrawn hand appears to be covered with a white cotton glove, the correct combination has been made. The form is immediately plunged in the plaster bouillon as described above. Many ingenious forms are used, especially the Delbet appareil de marche for.fractures of the leg. Upon the completion of all operative work and the healing of all wounds the patient is sent to a physiotherapeutic center. Here he receives massage, mechano-therapy, electricity, heat and gymnastic training. Following this, if unable to return to the Army, he enters a re- educational school where, with the assistance of many ingenious types of apparatus, he is taught to adapt himself to his physical incapac- ity, and after from three to six months enters once more into civil life. Our problems are quite different from those encountered in the camps in 1898, a difference largely due to the elimination of typhoid fever and the so-called "typhoid malaria." The ab- sence of these diseases is due chiefly to the triple inoculation with typhoid and para- typhoid vaccines; but the splendid work of the Medical Corps of the Army in improving camp sanitation, in the careful selection of camp sites, with reference to their natural soils and drainage, and eliminating the sources of ma- laria and other insect-borne diseases, has been an important contributing factor. p g In spite of all possible care, the herding to- gether in barrack life of large numbers of men, especially from the country districts, has fa- vored the spread of certain other infections. To a lesser degree we have scarlet fever and diphtheria; to a greater degree mumps and cerebrospinal meningitis, although we have been very fortunate in having little of the lat- ter in New England. And to a still greater degree we have had measles and pneumonia, which, have taken the place to a considerable extent of typhoid and its allied diseases, as the most common and the most dangerous diseases incident to camp life. They are always present in our base hospitals in larger proportion than one sees in our civil hospitals; and at periods of great activity, as after the arrival of large numbers of new recruits, constitute a clinic the value of which cannot be appreciated by those not in the service. The clinic thus con- stituted is an exceedingly valuable post-grad- uate course for the general practitioner. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. g p y The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. DISCUSSION. Dr. David Cheever, Boston : I was very much interested in what Dr. Clark had to say, especially about the guillotine amputations. That was the method used in the British army in 1915-1916, but I note that lately in the Brit- ish surgical literature there have been many protests against this method. They necessi- tated secondary amputations, which resulted in marked additional shortening of the limb, and in a certain number of cases secondary tetanus appeared. Of course that could, no doubt, be handled by additional prophylactic injections of tetanus serum, so that the latent and encap- sulated organisms freed by the secondary op- eration would be destroyed. It would seem that flap amputations, with the tissues held widely apart by appropriate packing with gauze and protective tissue, would be as effec- tive in preventing gas bacillus infection and give better stumps. g From a surgical point of view, the most in- teresting thing in connection with the epidemic of pneumonia and measles has been the study of the empyema with which they have been so frequently complicated. In our experience about twenty per cent, of all cases of pneu- monia have been followed by empyema, fifteen per cent, of the straight lobar pneumonias, and twenty-one per cent, of the post-measles pneu- monias. The empyema met with has been very different from the type which we have been accustomed to see in civil practice and in our local hospitals. Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. The Boston Medical and Surgical Journal as published by Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. It is of rapid development; does not wait for the cri- sis; in some instances fluid being present with- in three or four days after the symptoms of the pneumonia. More rarely it appears with- out the preceding pneumonia. In eighty-one cases of pneumonia with empyema, fluid was found, in sixty per cent, within the first two weeks, and in most of these within the first week. In other words, it has usually appeared before the pneumonia has cleared up, thus adding very greatly to the danger and diffi- culties of surgical treatment. Then again the fluid has collected very rapidly and suddenly, often reaching large amounts within twenty- four hours. y We believe that the empyema was simply a local manifestation of a blood infection, that is to say, it was but an expression of a general constitutional infection. We felt that only upon some such basis could we explain the presence of the infection in both pleurae at the same time, in the pericardium, and even in the peritoneum. To these we occasionally added joint infections so that we really had a poly- serositis. Although the pericardium was often involved, the endocardium was seldom infected. I remember but one instance, a case that was apparently convalescent, but which terminated fatally. The patient was sitting up in bed about ten days after thoracotomy, talking with his father, apparently very well, when he was seized with a sudden dyspnea and cyanosis, and died within five minutes, as if from pulmonary embolus. Autopsy showed an organized clot on the side of the right auricle, partly detached, and blocking the auriculo-ventricular opening. A noticeable feature of the autopsy was the presence of multiple pulmonary abscesses, some of them very minute, some holding two or three ce of pus. When these were at the periphery of the lung, it seemed possible that their rup- ture might have given rise to a direct contami- nation of the pleural cavity, perhaps even with the pneumothorax that was occasionally met with. Another feature that has distinguished these cases from those ordinarily met with has been the character of the fluid. In very few cases have we found frank pus. We get instead a cloudy, serous fluid with much fibrin, showing many leucocytes microscopically. The fluid is often so clear that it seems hardly necessary to drain. Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. In some, where the mortality has been very low, the streptococcus has been absent, but apparently it has been quite uni- versal. We undertook one or two interesting experiments to see how prevalent it was among the officers. From the medical officers at the Base Hospital cultures from the throats showed that seventy-two per cent, carried the hemo- lvtic streptococcus in their throats. Then we tried it among the nurses, and found that sixty per cent, of the nurses carried it. Then, as a check, we took the camp surgeons, including the regimental surgeons, and only five per cent, of them were streptococcus carriers. Yet, of course, the cases of pneumonia were all coming from the camp. There were no cases of the streptococcus pneumonia among the per- sonnel of the hospital, in spite of the fact that seventy per cent, of us were carriers. We believe that the empyema was simply a local manifestation of a blood infection, that is to say, it was but an expression of a general constitutional infection. We felt that only upon some such basis could we explain the presence of the infection in both pleurae at the same time, in the pericardium, and even in the peritoneum. To these we occasionally added joint infections so that we really had a poly- serositis. Although the pericardium was often involved, the endocardium was seldom infected. I remember but one instance, a case that was apparently convalescent, but which terminated fatally. The patient was sitting up in bed about ten days after thoracotomy, talking with his father, apparently very well, when he was seized with a sudden dyspnea and cyanosis, and died within five minutes, as if from pulmonary embolus. Autopsy showed an organized clot on the side of the right auricle, partly detached, and blocking the auriculo-ventricular opening. A noticeable feature of the autopsy was the presence of multiple pulmonary abscesses, some of them very minute, some holding two or three ce of pus. When these were at the periphery of the lung, it seemed possible that their rup- ture might have given rise to a direct contami- nation of the pleural cavity, perhaps even with the pneumothorax that was occasionally met with. One other interesting point disclosed by g p Now the empyema of camp life during the past winter has been very different. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. g p y The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine Downloaded from nejm org at UNIVERSITY OF VIRGINIA on July 1 2016 Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. BY Major Homer Gage, M.R.C., Worcester, Mass. BY Major Homer Gage, M.R.C., Worcester, Mass. In response to the invitation of your com- mittee, I am very glad to say a few words about one of the camp problems that has both- p Ordinarily, if, after the crisis of the pneu- shown hemolytic streptococcus, both in smear and culture ; and this has been true even of the lobar pneumonia. Often a Type I or Type II pneumonia would be accompanied by a strep- tococcus empyema. In some, where the mortality has been very low, the streptococcus has been absent, but apparently it has been quite uni- versal. We undertook one or two interesting experiments to see how prevalent it was among the officers. From the medical officers at the Base Hospital cultures from the throats showed that seventy-two per cent, carried the hemo- lvtic streptococcus in their throats. Then we tried it among the nurses, and found that sixty per cent, of the nurses carried it. Then, as a check, we took the camp surgeons, including the regimental surgeons, and only five per cent, of them were streptococcus carriers. Yet, of course, the cases of pneumonia were all coming from the camp. There were no cases of the streptococcus pneumonia among the per- sonnel of the hospital, in spite of the fact that seventy per cent, of us were carriers. monia, we have a subsequent rise in temper- ature, or when the temperature remains up after the crisis should have occurred, we begin to look out for the physical signs of empyema, and when this seems sufficiently evident we aspi- rate, and if we find thick, purulent fluid, the case is turned over to the surgeon. Except in older, neglected cases it is not often regarded as an emergency, but is operated on, as the fluid reaccumulates, often under general anesthesia, and usually with the resection of f rib. If the case is neglected or unsuspected, we get a pic- ture of general sepsis with marked dyspnea from lung compression shown hemolytic streptococcus, both in smear and culture ; and this has been true even of the lobar pneumonia. Often a Type I or Type II pneumonia would be accompanied by a strep- tococcus empyema. The Boston Medical and Surgical Journal as published by Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. y The clinical picture differs from ordinary empyema, chiefly in its early appearance as a complication of the pneumonic process. The patients are, therefore, as a rule, much sicker than we have been accustomed to see them. If they live long enough for resolution to take place, they present a picture not unlike that commonly seen in civil practice. y p act ce In establishing the diagnosis, aside from the ordinary physical signs, the x-ray is of great value, often indicating and locating the pneu- monia even before the physical signs are clearly defined, and revealing the presence and prog- ress of a pleural effusion. Chief reliance as to the presence of fluid must, however, be placed upon repeated tapping of the chest with a good-sized needle. Some of the patients have to be tapped many times. As soon as you suspect the fluid, tap, and then tap again, and keep it up until you are thoroughly satisfied. It ap- parently does not disturb the patients much, if carefully and properly done, and it leads to a much earlier diagnosis than you get in any other way. y p To summarize : I think our experience illus- trates very well the absolute importance and necessity of operation. Its value seems to have been clearly brought out in our group of cases, and my experience and observation of the re- sults in other camps leads me to stick strongly to my opinion about the value of simple thor- acotomy with local anesthesia, as against the excision of rib, believing that the less you do to a patient so sick, in getting relief from the immediate difficulty, the better the chance you give him to get well. The only change that I should make in a future series of cases would be to put them immediately upon irrigation with Carrel-Dakin solution. y As to the interesting problem of treatment, of course we have the alternatives of frequent repeated aspirations and of drainage, and drainage may be obtained with or without rib resection. Repeated aspiration in some camps has turned out pretty well. In a few of the camps too extensive operation seems to have raised the mortality. Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. We aspirated frequently, but in presence of a streptococcus infection, as proved by smear and culture, or in the pres- ence of frank pus, we operated, and we oper- ated as early as either condition was estab- lished. As to the form of operation we felt, and still feel, that the patients were so very sickf that it was unwise to add any greater discom- fort or shock than was absolutely necessary, and in all instances we did a simple thoracot- omy under local anesthesia. In the adult, one can get a large drainage tube between the ribs, and secure such good drainage that rib resec- tion would not seem to be called for. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. Discussion on Empyema and Its Treatment in Military Cantonments during the Winter of1917-1918. I do not know what is the mortality of em- pyema in civil life, although I believe it to be higher than has been generally realized. How- ever, I do not think it has been comparable to the mortality from empyema as experienced during the last winter in the different camps. In one it was eighty-four per cent., in others fifty-three per cent., fifty-seven per cent., and sixty-five per cent. In twenty-five different camps there was an average mortality of over thirty per cent. It is clear, therefore, that pneumonia and empyema have been very im- portant factors in the mortality as well as the morbidity statistics of the army camps. One other interesting point disclosed by One other interesting point disclosed by o b d ty y Bacteriologically, most of these cases have As for irrigation, we did not irrigate except in cases where there was a foul odor to the fluid (and that happened only once), and in eases where there were large fibrin clots, which we could not remove without irrigation. We had twenty-one cases in which we had pneumocoecus infection alone. Of these, seven were operated on without any deaths, and of the fourteen not operated, eleven died. There were forty-one cases with the streptococcus alone, twenty-si? of which were operated on, with, up to the present time, eight deaths, and fifteen not operated on with eleven deaths. The infection was a mixed streptococcus and pneumocoecus in five operative cases without a death, and in eight cases not operated on, of which four died. Of the cases in which the infection was unknown, or not recorded there were four, all operated on, and one of them died. Out of twenty-four operative cases, there has been up to the present time a mor- tality of eighteen per cent., and of thirty-seven non-operative- cases there was a mortality of seventy per cent. autopsy was the presence of a large collection of fluid, in a few of our cases, between the left lung and the pericardium. It was really a walled-off abscess lying between two layers of the pleura. In one instance we found three or four hundred ec. in this locality. If we could have located it ante mortem, it couicl have been drained, but we were unable to detect it, e1 en : with the assistance of the x-ray. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF VIRGINIA on July 1, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. DISCUSSION. S. J. MrxTER, Maj. M.R.C. : These cases of pneumonia and empyema have been most in- teresting as well as most trying. I have had a chance to see them in the base hospitals, and also to go over the statistics and the figures as they come in from week to week in the office at Washington. S. J. MrxTER, Maj. M.R.C. : These cases of pneumonia and empyema have been most in- teresting as well as most trying. I have had a chance to see them in the base hospitals, and also to go over the statistics and the figures as they come in from week to week in the office at Washington. g The first thing which strikes one is the ap- parent difference in the virulence of empyema in different localities. I do not think the differ- ence in the mortality in different places is en- tirely due to differences in operations, al- though it has been found that the more exten-
https://openalex.org/W2887369361	https://europepmc.org/articles/pmc6307003?pdf=render	English	null	The association between periodontal disease and adverse pregnancy outcomes in Northern Tanzania: a cross-sectional study	African health sciences	2,018	cc-by	7,755	Abstract Background: For the past two decades, studies have investigated the relationship between periodontal disease and adverse preg- nancy outcomes such as pre-eclampsia, preterm birth, low birth weight and preterm premature rupture of membranes. Background: For the past two decades, studies have investigated the relationship between periodontal disease and adverse preg- nancy outcomes such as pre-eclampsia, preterm birth, low birth weight and preterm premature rupture of membranes. Objectives: To determine the prevalence of periodontal disease and associated adverse pregnancy outcomes among women delivering at the Kilimanjaro Christian Medical Centre (KCMC).i Objectives: To determine the prevalence of periodontal disease and associated adverse pregnancy outcomes among women delivering at the Kilimanjaro Christian Medical Centre (KCMC).i Methods: This cross-sectional study was based on the use of patients’ files, clinical examinations and oral interviews with moth- ers who delivered at the KCMC. Pregnant women with singleton babies (N=1117) who delivered at the KCMC were recruited for the study. Intra-oral examination was performed within five days of birth. The Community Periodontal Index was used to assess periodontal diseasei Results: The prevalence of periodontal disease was 14.2%. Periodontal disease was significantly associated with higher odds of pre-eclampsia [adjusted Odds Ratio 95% Confidence Interval (aOR=4.12;95%CI:2.20-7.90)], low birth weight (aOR=2.41;95%- CI:1.34-4.33) and preterm birth (aOR=2.32;95%CI:1.33-4.27). There was no significant association between periodontal disease and preterm premature rupture of membranes (aORs 1.83;95%CI:0.75-4.21) and eclampsia (3.71;95%CI:0.80-17.13). Conclusion: Maternal periodontal disease is a potential independent risk indicator for pre-eclampsia, low birth weight, and preterm birth. Periodontal assessment and therapy should form part of the preventive antenatal care provided to women in developing countries. p g Keywords: Preterm birth; low birth weight; pre-eclampsia; eclampsia; preterm premature rupture of membranes; periodontal disease; periodontitis; cross-sectional studies. DOI: https://dx.doi.org/10.4314/ahs.v18i3.18 p g Cite as: Gesase N, Miranda-Rius J, Brunet-Llobet L, Lahor-Soler E, Mahande MJ, Masenga G. The association between periodontal disease and adverse pregnancy outcomes in Northern Tanzania: a cross-sectional study. Afri Health Sci. 2018;18(3): 601-611. https://dx.doi.org/10.4314/ h 18i3 18 Cite as: Gesase N, Miranda-Rius J, Brunet-Llobet L, Lahor-Soler E, Mahande MJ, Masenga G. The association between periodontal disease and adverse pregnancy outcomes in Northern Tanzania: a cross-sectional study. Afri Health Sci. 2018;18(3): 601-611. https://dx.doi.org/10.4314/ ahs.v18i3.18 Nyobasi Gesase1, Jaume Miranda-Rius2,3, Lluís Brunet-Llobet3,4, Eduard Lahor-Soler2,3, Michael J Mahande5, Gileard Masenga1 1. Department of Obstetrics and Gynaecology, Kilimanjaro Christian Medical Centre (KCMC)/Kilimanjaro Christian Medical University College (KCMU College), Moshi, Tanzania. 1. Department of Obstetrics and Gynaecology, Kilimanjaro Christian Medical Centre (KCMC)/Kilimanjaro Christian University College (KCMU College), Moshi, Tanzania. 2. Department of Odontostomatology. Faculty of Medicine and Health Sciences. Universitat de Barcelona, Barcelona, 3. Hospital Dentistry & Periodontal Medicine Research Group, Institut de Recerca Sant Joan de Déu (IRSJD), Funda Joan de Déu, Barcelona, Spain. 4. Division of Pediatric Dentistry. Hospital Universitari Sant Joan de Déu. Universitat de Barcelona, Barcelona, Spain. 5. Department of Epidemiology and Biostatistics. Institute of Public Health. Kilimanjaro Christian Medical University (KCMU College), Moshi, Tanzania. 2. Department of Odontostomatology. Faculty of Medicine and Health Sciences. Universitat de Barcelona, Barcelona, Spain. 3. Hospital Dentistry & Periodontal Medicine Research Group, Institut de Recerca Sant Joan de Déu (IRSJD), Fundació Sant Joan de Déu, Barcelona, Spain. J p 4. Division of Pediatric Dentistry. Hospital Universitari Sant Joan de Déu. Universitat de Barcelona, Barcelona, Spain. ric Dentistry. Hospital Universitari Sant Joan de Déu. Universitat de Barcelona, Barcelona, Spa 4. Division of Pediatric Dentistry. Hospital Universitari Sant Joan de Déu. Universitat de Barcelona, Barcelona, Spain. 5. Department of Epidemiology and Biostatistics. Institute of Public Health. Kilimanjaro Christian Medical University College (KCMU College), Moshi, Tanzania. 5. Department of Epidemiology and Biostatistics. Institute of Public Health. Kilimanjaro Christian Medical University College (KCMU College), Moshi, Tanzania. African Health Sciences © 2018 Gesase et al. Licensee African Health Sciences. 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. The association between periodontal disease and adverse pregnancy outcomes in Northern Tanzania: a cross-sectional study Nyobasi Gesase1, Jaume Miranda-Rius2,3, Lluís Brunet-Llobet3,4, Eduard Lahor-Soler2,3, Michael J Mahande5, Gileard Masenga1 Nyobasi Gesase1, Jaume Miranda-Rius2,3, Lluís Brunet-Llobet3,4, Eduard Lahor-Soler2,3, Michael J Mahande5, Gileard Masenga1 Introduction Adverse pregnancy outcomes such as pre-eclampsia (PE), eclampsia, preterm premature rupture of membranes (pPROM), preterm birth (PTB) and low birth weight (LBW) are all associated with maternal and neonatal mor- bidity and mortality. These outcomes also represent a se- rious public health problem. Maternal periodontal disease can adversely affect pregnancy, by causing bacteria and inflammatory mediators to spread from the oral cavity to the fetal placental unit via the blood1. African Health Sciences © 2018 Gesase et al. Licensee African Health Sciences. 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. African Health Sciences Vol 18 Issue 3, September, 2018 African Health Sciences Vol 18 Issue 3, September, 2018 601 In periodontitis, periodontal pathogens or by-products may reach the placenta and spread to the fetal circulation and amniotic fluid. Their presence in the fetal-placental compartment can stimulate a fetal immune/inflamma- tory response characterized by the production of IgM antibodies against the pathogens and the secretion of el- evated levels of inflammatory mediators, which in turn may cause miscarriage or premature birth. Moreover, infection/inflammation may cause placental structural changes leading to pre-eclampsia and impaired nutrient support, thus reducing birth weight. Fetal exposure may also result in tissue damage and increase the risk of peri- natal morbidity and mortality14. The American Academy of Periodontology defines peri- odontal disease as an inflammatory disease that affects the soft and hard structures supporting the teeth2. In the early stage of the disease, which is known as gingivitis, the gums become swollen and red due to inflammation, which is the body’s natural response to the presence of harmful bacteria. In the more serious form of periodon- tal disease, called periodontitis, the gums pull away from the tooth and the supporting gum tissues are destroyed2. Periodontitis is principally caused by Gram-negative an- aerobic bacteria, which raise local and systemic levels of pro-inflammatory mediators3. The prevalence of periodontal disease varies depending on the population studied and the definition used. For example, in industrialized areas it affects between 20% and 50% of pregnant women4,5. The World Bank Coun- try classification defines the United Republic of Tanza- nia as a low–income economy6. A systematic review and meta regression of the global burden of severe periodon- titis calculated its prevalence in Tanzania to be 20%7. Introduction In Africa, and specifically in Tanzania, data on periodontal health in pregnancy are limited. However, one study in a rural population in Uganda reported 67% prevalence of periodontal disease among pregnant women8. The international definition of low birth weight is a weight “below 2500g” regardless of gestational age. Weights be- low 1500g are considered very low (VLBW) and weights below 1000g as extremely low (ELBW)15. In Tanzania, a birth weight less than 1000g is considered as an abortion, as the fetus is non-viable.i Preterm or premature birth (PTB) is usually defined as a gestational age of less than 37 completed weeks. Preterm premature rupture of membranes (pPROM) is defined as rupture of the fetal membranes before the onset of labour and prior to 37 weeks of gestation10. From the epidemiological perspective, the incidence of PTB re- ported in the literature varies, because it is a multifactorial problem influenced by geographical and socio-economic factors, racial characteristics, age, and quality of prenatal care. PTB occurs in approximately 8-10% of pregnan- cies in developed countries but this figure may be as high as 43% in parts of the developing world. Unfortunately, preterm birth and low birth weight rates are also high elsewhere: Europe 4 to 12%, Asia 15%, Africa 10 to 12%, Australia 6% and North America 7%16. At the 1996 World Workshop in Periodontics, the term “periodontal medicine” was introduced to define a disci- pline focused on the evaluation of the two-way relation- ship between the fields. Periodontal disease can have a major impact on individual systemic health, and in turn systemic diseases may influence periodontal health as well9. Gingivitis and periodontitis usually involve active chronic infections which may increase the risk of adverse preg- nancy outcomes such as PTB, LBW, pPROM and PE. The presence of a large ulcerated epithelium surface in periodontal pockets allows bacteria and their products to reach other parts of the body, causing lesions at different levels. Certain bacteria such as Porphyromona gingivalis and Aggregatibacter actinomycetemcomitans may even invade cells and tissues directly10-12. Periodontal bacteria in pregnant women with periodontitis may induce the cascade of im- muno-inflammatory mediators such as PGE2, IL-6, IL-1, and TNF-α which may be implicated in adverse pregnan- cy outcomes4,13. Pre-eclampsia is a multi-factorial inflammatory disorder that is a major cause of maternal and perinatal morbidity and mortality. African Health Sciences Vol 18 Issue 3, September, 2018 Measurements In this study the outcome variables were pPROM, PE/ eclampsia, PTB and LBW, while periodontal disease (PD) was the main exposure of interest. Other variables such as age, marital status, level of education, height, weight, body mass index, cigarette smoking, alcohol consump- tion, previous history of pPROM, LBW, PE, PTB, HIV status, urinalysis, VDRL, blood group and Rh factor, haemoglobin level, blood slide for malaria parasite/ma- laria rapid diagnostic test gestation age at delivery, birth weight, mode of delivery, number of teeth, and tooth mobility were also assessed as co-variates. Study subjects This study included all pregnant women (n=1117) deliv- ering at the KCMC labour ward, aged from 18 to 46 years, with singleton intrauterine fetuses of a gestational age be- tween 28 and 42 weeks. Multiple gestations, women with any systemic infection apart from periodontitis, and those who lacked the number of teeth necessary to register the Community Periodontal Index (CPI) were excluded. Study participants were recruited at the time of admis- sion to the labour and delivery area17,18. They all provided signed consent and their information was gathered to- gether using a structured questionnaire, where their so- ciodemographic characteristics, previous obstetric histo- ry and the index pregnancy information were recorded. Periodontal examination was performed within five days of delivery by the same obstetrician (NG), who had re- ceived training in oral examination from a senior research- er in periodontics (JMR). The intraexaminer calibration was followed by assessments of the clinician’s ability over a three-month period prior to the start of the study19. This study included all pregnant women (n=1117) deliv- ering at the KCMC labour ward, aged from 18 to 46 years, with singleton intrauterine fetuses of a gestational age be- tween 28 and 42 weeks. Multiple gestations, women with any systemic infection apart from periodontitis, and those who lacked the number of teeth necessary to register the Community Periodontal Index (CPI) were excluded. Study participants were recruited at the time of admis- sion to the labour and delivery area17,18. They all provided signed consent and their information was gathered to- gether using a structured questionnaire, where their so- ciodemographic characteristics, previous obstetric histo- ry and the index pregnancy information were recorded.i Statistical analysis Data were analysed using SPSS version 20. Descriptive statistics were summarized using mean and SD for con- tinuous variables while frequency and proportions were used for categorical variables. Odds ratios (OR) with 95% Confidence Intervals (CI) for the adverse pregnant out- comes associated with periodontal disease were estimat- ed using a multivariable logistic model. A p-value of less than 0.05 (2-tailed) was considered statistically significant. Periodontal examination was performed within five days of delivery by the same obstetrician (NG), who had re- ceived training in oral examination from a senior research- er in periodontics (JMR). The intraexaminer calibration was followed by assessments of the clinician’s ability over a three-month period prior to the start of the study19. Study design g We performed a cross-sectional study of all pregnant women delivering at the Kilimanjaro Christian Medical Center (KCMC) in Moshi, Northern Tanzania,between September 2015 and April 2016. The KCMC is a zonal consultant and university teaching hospital serving the local community and referred cases from six regions in Northern Tanzania. These include Kilimanjaro, Arusha, Manyara, Tanga, Singida and Dodoma with approximate- ly 15 million inhabitants. The Department of Obstetrics and Gynaecology (KCMC Birth Medical Registry 2012) provides birth care services to pregnant women from the nearby communities as well as referral cases from other regions. It has an average of 3,300 deliveries per year. Introduction The syndrome is characterised by inappro- priate inflammatory and abnormal vascular response to placentation which causes endothelial dysfunction, result- ing in maternal hypertension during pregnancy3. Several studies have explored the relationship between periodontal health status and adverse pregnancy out- comes such as PTB, LBW, PE and pPROM. However most of these studies have focused almost exclusively on African Health Sciences Vol 18 Issue 3, September, 2018 602 assessed (17, 16, 11, 26, 27, 47, 46, 31, 36, 37) using a standard periodontal probe (Michigan 8/11, Hu-Friedy, and Chicago, IL, USA). The probing depth involved three measurements in mesial-medial-distal for buccal surfaces and lingual-palatal surfaces respectively. The CPI scoring criteria were defined as follows: 0 = no periodontal dis- ease; 1 = bleeding on probing; probing depth ≤ 3mm; 2 = calculus with plaque seen or felt by probing; 3 = patho- logical pocket > 3mm and < 6mm, and 4 = pathological pocket ≥ 6mm; thus, a score of 0 meant no periodontal disease, 1 - 2 gingivitis and 3 - 4 periodontitis. Assessment of gingival recession included only one measurement in medial for buccal and lingual-palatal surfaces20. Dental mobility was classified as grade I, II, or III. Other param- eters such as gingival enlargement21, were also considered. PTB and LBW in relation to systemic maternal infections. The aim of our study was to determine the prevalence of periodontal disease among pregnant women in North- ern Tanzania, and to analyse its possible association with these adverse pregnancy outcomes. African Health Sciences Vol 18 Issue 3, September, 2018 African Health Sciences Vol 18 Issue 3, September, 2018 Results A total of 1,117 participants were eligible and were en- rolled in the study. Their demographic characteristics are shown in Table 1. Mean age was 28.5 years (SD ± 5.9), 568 (50.9%) were aged between 26 and 35 years, 435 (38.9%) attained post-secondary school education, and 1005 (90%) were married. The vast majority 967 (86.6%) were between parity 1 and parity 3, 150 (13.4%) had par- Clinical periodontal examination was done using the ep- idemiological section of the Community Periodontal In- dex of Treatment Needs known as the Community Peri- odontal Index (CPI)17. In each participant, ten teeth were African Health Sciences Vol 18 Issue 3, September, 2018 603 ity >3, and mean parity was 2 (SD ± 1). One thousand and four (89.9%) had between one and three living chil- dren; 63.1% gave birth via vaginal delivery, 35.3% by cesarean section, 1.1% by vacuum and 0.5% by assisted breech. Mean gestation age at birth was 39.0 weeks (SD ± 2.3, 28.0-44.0), with preterm delivery accounting for 9.8% (n=110); 897 (80.3%) were term deliveries, and 110 (9.8%) were post-term; 123 (11.0%) were LBW, 84.2% were normal birth weight (>2.5kg to <4kg) and 4.7% were overweight (>4kg). Results Table 1: Characteristics of study participants (N = 1117) Variable n (%) Age (years): 18 - 25 383 (34.3) 26 - 35 568 (50.9) 36 - 46 166 (14.9) Mean (±SD) 28.5 (±5.9) Level of education: No formal education 15 (1.3) Primary 416 (37.2) Secondary 251 (22.5) Post-secondary 435 (38.9) Marital status: Never married 112 (10.0) Ever married 1005 (90.0) Parity: 1 – 3 967 (86.6) More than 3 150 (13.4) Mean (±SD) 2 (±1) Number of living children: 1 – 3 1004 (89.9) More than 3 113 (10.1) Mean (±SD) 2 (±1, 0-11) Body Mass Index (kg/m2): Underweight 43 (3.8) Normal 605 (54.2) Overweight 320 (28.6) Obese 149 (13.3) Mean (±SD) 24.9 (±4.4) Blood pressure (mmHg): Normal 1014 (90.8) High 103 (9.2) First antenatal visit (weeks) (n=1110): Up to 14 281 (25.3) More than 14 829 (74.7) Mean (±SD) 17.9 (±5.3) Mode of delivery: Spontaneous vaginal 705 (63.1) Caesarian section 394 (35.3) Vacuum extraction 12 (1.1) Assisted breach 6 (0.5) Alcohol consumption: Yes 208 (18.6) No 909 (81.4) Table 1: Characteristics of study participants (N = 1117) Table 1: Characteristics of stu Variable Age (years): 18 - 25 26 - 35 36 - 46 Mean (±SD) Level of education: No formal education Primary Secondary Post-secondary Marital status: Never married Ever married Parity: 1 – 3 More than 3 Mean (±SD) Number of living children: 1 – 3 More than 3 Mean (±SD) Body Mass Index (kg/m2): Underweight Normal Overweight Obese Mean (±SD) Blood pressure (mmHg): Normal High First antenatal visit (weeks) ( Up to 14 More than 14 Mean (±SD) Mode of delivery: Spontaneous vaginal Caesarian section Vacuum extraction Assisted breach Alcohol consumption: Yes No African Health Sciences Vol 18 Issue 3, September, 2018 604 [Odds Ratio 95% Confidence Interval (OR = 2.1; 95% CI: 1.30-3.40)] compared with the youngest group (18 to 25 years). When parity was compared, the odds of having periodontal disease in the 1 - 3 parity group was half that recorded in women with higher parity (OR = 0.5; 95% CI: 0.30-0.70). Other variables such as level of education, marital status, HIV status, body mass index and alcohol consumption did not influence the occurrence of peri- odontal disease (Table 2). The prevalence of periodontal disease was 14.2% (n=159); however, severe periodontitis (grade 4) was found in only 5.0% of this group. Results The characteristics of study participants and the presence of periodontal dis- ease are shown in Table 2. With reference to the distribution of periodontal disease, adjusted for the age of the sample, women aged 36 to 46 years had a 2.1-times higher odds of periodontal disease Table 2: Characteristics of postpartum mothers and presence of periodontal disease (n = 111 Variable Total Presence of periodontal disease OR (95% CI) p-value Present Absent n (%) n (%) Age (years): MD (95% CI) Mean (SD) 30.0 (6.6) 28.2 (5.7) 1.8 (0.8-2.7) 0.001 18 - 25 383 50 (13.1) 333 (86.9) 1.0 26 - 35 568 69 (12.1) 499 (87.9) 0.9 (0.6-1.4) 0.679 36 - 46 168 40 (24.1) 126 (75.9) 2.1 (1.3-3.4) 0.001 Level of education: No formal education 15 4 (26.7) 11 (73.3) 1.0 Primary 416 74 (17.8) 342 (82.2) 0.6 (0.2-1.9) 0.491* Secondary 251 34 (13.5) 217 (86.5) 0.4 (0.1-1.4) 0.243* Post-secondary 435 47 (10.8) 388 (89.2) 0.3 (0.1-1.1) 0.078* Marital status: Never married 112 13 (11.6) 99 (88.4) Ever married 1005 146 (14.5) 859 (85.5) 0.8 (0.4-1.4) 0.401 Body mass index (kg/m2): Underweight 43 6 (14.0) 37 (86.0) 1.0 Normal 605 86 (14.2) 519 (85.8) 1.0 (0.4-2.5) 0.962 Overweight 320 45 (14.1) 275 (85.9) 1.0 (0.4-2.5) 0.985 Obese 149 22 (14.8) 127 (85.2) 1.1 (0.4-2.8) 0.895 Parity: 1 – 3 967 123 (12.7) 844 (87.3) 0.5 (0.3-0.7) <0.001 More than 3 150 36 (24.0) 114 (76.0) 1.0 Known HIV status (n=1114) Positive 29 6 (20.7) 23 (79.3) Negative 1085 151 (13.9) 934 (86.1) 1.6 (0.6-4.0) 0.301 Alcohol consumption: Yes 208 30 (14.4) 178 (85.6) 1.0 No 909 129 (14.2) 780 (85.8) 1.0 (0.7-1.6) 0.931 MD=Mean Difference; =Fisher Exact p-value Characteristics of postpartum mothers and presence of periodontal disease (n = 1117) African Health Sciences Vol 18 Issue 3, September, 2018 605 Adverse maternal outcomes and their associations with PD are summarized in Table 3. Postpartum women with PD at delivery had a 3.4 higher odds of pre-eclampsia than women without (OR = 3.4; 95% CI: 2.20-5.40). Results In contrast, there was no statistically significant association between PD and eclampsia, preterm premature rupture of membranes or term rupture Table 3: Adverse maternal outcome associated with periodontal disease (N = 1117) Outcome Total Periodontal disease OR (95% CI) p-value Present Absent n (%) n (%) Pre-eclampsia Yes 101 33 (32.7) 68 (67.3) No 1016 126 (12.4) 890 (87.6) 3.4 (2.2-5.4) <0.001 Eclampsia Yes 16 5 (31.2) 11 (68.8) No 1101 154 (14.0) 947 (86.0) 2.8 (1.0-8.2) 0.050 Severity of pre- eclampsia (n=101): Mild 39 9 (23.1) 30 (76.9) Severe 62 24 (38.7) 38 (61.3) 0.5 (0.2-1.2) 0.103 Pre-term premature rupture of membranes (n=110): Yes 26 9 (34.6) 17 (65.4) No 84 22 (26.2) 62 (73.8) 1.5 (0.6 -3.8) 0.404 Table 3: Adverse maternal outcome associated with periodontal disease (N = 1117) Adverse fetal outcomes and their association with PD are summarized in Table 4. Women shown to have had PD at delivery had a 2.6 higher odds of delivering children with LBW (<2.5 kg) than women without PD (OR = 2.6; 95% CI: 1.70-4.00). When gestation age at delivery was analysed, mothers with PD were significantly (2.7 times) more likely to deliver before term (<37 weeks gestation) than mothers without PD (OR = 2.7; 95% CI: 1.70-4.20). Crude analysis for the main variables was performed us- ing logistic regression with cut off points for maternal/ neonatal characteristics, and the significant associations were entered in the final model (Table 5). African Health Sciences Vol 18 Issue 3, September, 20 Table 4: Adverse fetal outcome associated with periodontal disease (N = 1117) Outcomes Total Periodontal disease OR (95% CI) p-value Present Absent n (%) n (%) Gestational age at delivery (weeks): Term/ postdate 1007 128 (12.7) 879 (87.3) Preterm 110 31 (28.2) 79 (71.8) 2.7 (1.7-4.2) <0.001 Birth-weight (kg): Mean (SD) 1117 2.9 (0.7) 3.1 (0.6) -0.2 [(-0.3 – (-0.1)] <0.001 Normal 941 122 (13.0) 819 (87.0) 1.0 Low birthweight 123 34 (27.6) 89 (72.4) 2.6 (1.7-4.0) <0.001 Overweight 53 3 (5.7) 50 (94.3) 0.4 (0.1-1.3) 0.138 *=Fisher Exact p-value Table 4: Adverse fetal outcome associated with periodontal disease (N = 1117) VARIABLE As regards the relationship between PD and adverse ma- ternal outcomes PE/eclampsia and pPROM, after ad- justment for age, parity, and previous history of these events the association with PE was significantly higher in women with PD adjusted Odds Ratio 95% Confidence Interval (aOR = 4.12; 95% CI: 2.20-7.90), but it was not statistically significant for pPROM and eclampsia which presented aORs of 1.83; 95% CI: 0.75-4.21) and (3.71; 95% CI: 0.80-17.13)respectively. As for the association between PD and adverse fetal outcomes, after adjustment for age, parity, and previous history the aORs were (2.41; 95% CI: 1.34-4.33) for LBW and (2.32; 95% CI: 1.33- 4.27) for PTB and were statistically significant in both cases. duce hyper-irritability of the smooth muscle of the uter- us, enhancing contractility, cervical thinning and cervical dilatation, and may thus trigger preterm labour4,23,24. For its part, maternal periodontal disease can adversely affect pregnancy by causing bacteria and inflammatory media- tors to spread from the oral cavity to the fetal placental unit via the blood.1 In Tanzania, there is no epidemiological data regard- ing periodontal disease in pregnant women. One of the strengths of our study is the sample size: more than 1100 pregnant women were included, of whom 14.2% present- ed with periodontal disease. The prevalence was strongly influenced by older age and parity above 3. These results are in agreement with a study in Brazil which found a prevalence of 11%25, but other authors have reported higher rates of PD among pregnant women3,12,26,27. The associations found in our study between lower rates of PD, age under 35 and parity of 1 – 3 are probably due to the higher levels of education in the younger popula- tion and the study setting. Our study was carried out at a tertiary teaching hospital in an area where most of the community lives in an urban setting, a fact which may have influenced the results of the study: well-educated individuals in urban environments are likely to have a bet- ter understanding of the need for good oral hygiene and have a lower risk of developing periodontal disease. Oth- er studies have associated the prevalence of periodontal disease with low educational level, residence in rural en- In Tanzania, there is no epidemiological data regard- ing periodontal disease in pregnant women. VARIABLE One of the strengths of our study is the sample size: more than 1100 pregnant women were included, of whom 14.2% present- ed with periodontal disease. The prevalence was strongly influenced by older age and parity above 3. These results are in agreement with a study in Brazil which found a prevalence of 11%25, but other authors have reported higher rates of PD among pregnant women3,12,26,27. Table 4: Adverse fetal outcome associated with periodontal disease (N = 1117) African Health Sciences Vol 18 Issue 3, September, 2018 606 Table 5: Adjusted odds ratio of maternal and neonatal characteristics and presence of periodontal disease (N = 1117) VARIABLE COR (95%CI) AOR (95%CI) Pre-eclampsia at delivery 1,2 3.43 (2.23-5.41) 4.12(2.20-7.90) Preterm premature rupture of membranes 1,2 2.62 (1.45-5.01) 1.83 (0.75-4.21) Eclampsia 1,2 2.85 (1.05-8.25) 3.71 (0.80-17.13) Low birthweight (<2500g) 1,2,3 2.69 (1.74-4.27) 2.41 (1.34-4.33) Pre-term birth (gestation age < 37 weeks)1,2,4 2.69 (1.71-4.25) 2.32 (1.33-4.27) COR: Crude Odds Ratio; AOR: Adjusted Odds Ratio; (95%CI): 95% Confidence Interval; 1: adjusted for age, parity 2: adjusted for the same adverse fetal outcome 3: adjusted for previous low birth weight and pre-eclampsia 4: adjusted for previous preterm birth and pre-eclampsia VARIABLE COR (95%CI) AOR (95%CI) Pre-eclampsia at delivery 1,2 3.43 (2.23-5.41) 4.12(2.20-7.90) Preterm premature rupture of membranes 1,2 2.62 (1.45-5.01) 1.83 (0.75-4.21) Eclampsia 1,2 2.85 (1.05-8.25) 3.71 (0.80-17.13) Low birthweight (<2500g) 1,2,3 2.69 (1.74-4.27) 2.41 (1.34-4.33) Pre-term birth (gestation age < 37 weeks)1,2,4 2.69 (1.71-4.25) 2.32 (1.33-4.27) African Health Sciences Vol 18 Issue 3, September, 2018 African Health Sciences Vol 18 Issue 3, September, 2018 Discussion Periodontal disease has been associated with certain ad- verse pregnancy outcomes such as LBW, PTB, pPROM and PE. Our principal findings indicate that maternal periodontal disease is a potential independent risk indica- tor for pre-eclampsia, low birth weight and preterm birth. Modelled estimates for Tanzania suggest that 23% of newborn deaths are due to complications of PTB. Eighty- six per cent of newborn deaths are also LBW, many of which are PTB22. Maternal systemic infections can elicit an inflammatory response that results in inflammation of the maternal-fetal-placental unit including the uterus, chorioamniotic membranes, placenta, amniotic fluid, fe- tal lungs and circulation. These inflammatory stimuli in- The associations found in our study between lower rates of PD, age under 35 and parity of 1 – 3 are probably due to the higher levels of education in the younger popula- tion and the study setting. Our study was carried out at a tertiary teaching hospital in an area where most of the community lives in an urban setting, a fact which may have influenced the results of the study: well-educated individuals in urban environments are likely to have a bet- ter understanding of the need for good oral hygiene and have a lower risk of developing periodontal disease. Oth- er studies have associated the prevalence of periodontal disease with low educational level, residence in rural en- African Health Sciences Vol 18 Issue 3, September, 2018 607 in the development and progression of atherosclerosis, leading to coronary vascular disease. Periodontal disease may cause a chronic burden of endotoxin and inflam- matory cytokines which serve to initiate and exacerbate athrogenesis and thrombogenesis. It is possible that the placenta may be similarly burdened in pregnant wom- en who develop pre-eclampsia37,38,39. Women with active periodontal disease during pregnancy may have transient translocation of oral organisms to the uteroplacental unit, causing placental inflammation or oxidative stress early in pregnancy which ultimately produces placental damage and the clinical manifestation of pre-eclampsia. Studying umbilical cord serum, Madianos et al assessed the pres- ence of fetal immunoglobulin M against oral pathogens, and identified the production of fetal IgM against Por- phyromonas gingivalis; this finding indicates a fetal humoral response to organisms distant from the intrauterine envi- ronment and suggests the possibility of translocation of oral pathogens to the uteroplacental unit14. Discussion Other authors have detected a molecular increase of oral bacteria such as Fusobacterium nucleatum in the placenta tissues of preg- nant periodontitis patients40. vironments, poor oral hygiene and cigarette smoking8,28. Cigarette smoking is not a common habit among Tan- zanian women and indeed none of our population were smokers. The prevalence of PD varies depending on the popula- tion investigated and the definition and the recording in- dex used. One of the weaknesses of most studies is the inconsistency in the recording of prevalence and severity of PD, a drawback that influences the results and lim- its the possibility of making valid comparisons between studies. In this study the CPI scoring system was the method used for measuring periodontal status29.i The prevalence of PD varies depending on the popula- tion investigated and the definition and the recording in- dex used. One of the weaknesses of most studies is the inconsistency in the recording of prevalence and severity of PD, a drawback that influences the results and lim- its the possibility of making valid comparisons between studies. In this study the CPI scoring system was the method used for measuring periodontal status29. This study found a significant association between PD and PE [aOR 4.12 (95% CI: 2.20-7.90)] indicating that PD may influence adverse pregnancy outcomes in Northern Tanzania. Parity more than 3, age 36 - 45 and history of previous PE were strongly associated with development of PE in women with PD, in agreement with previous re- ports in the US30, Iran31, Italy32, and Brazil33 which found significant associations between pre-eclampsia and PD. In a meta-analysis including 13 observational case control studies and two cohort studies, Wei and colleagues also found a significant association between PD and pre-ec- lampsia34. This study found a significant association between PD and PE [aOR 4.12 (95% CI: 2.20-7.90)] indicating that PD may influence adverse pregnancy outcomes in Northern Tanzania. Parity more than 3, age 36 - 45 and history of previous PE were strongly associated with development of PE in women with PD, in agreement with previous re- ports in the US30, Iran31, Italy32, and Brazil33 which found significant associations between pre-eclampsia and PD. In a meta-analysis including 13 observational case control studies and two cohort studies, Wei and colleagues also found a significant association between PD and pre-ec- lampsia34. African Health Sciences Vol 18 Issue 3, September, 2018 Ethical considerationi 6. World Bank Country and Lending Groups. The World Bank Group – worldbank.org. 2017 https://datahelp- desk.worldbank.org/knowledgebase/articles/906519- world-bank-country-and-lending-groups (Last accessed Nov 2017). Ethical clearance certificate No. 822 was obtained from the Kilimanjaro Christian Medical University College Re- search and Ethics Committee. Study participants signed an informed consent form before data collection and all the information was treated confidentially. Participants who did not provide consent received the same care as those who did. 7. Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJ, Marcenes W. Global burden of severe peri- odontitis in 1990-2010: a systematic review and meta-re- gression. J Dent Res. 2014; 93(11): 1045-1053. PubMed. DOI:10.1177/0022034514552491. Discussion PTB or LBW newborns are a major cause of infant mortality and morbidity, and those who survive suffer a higher risk of developing neurodevelopmental, respirato- ry, cardiovascular and metabolic abnormalities as well as learning disabilities. It is clear that the causes of PTB and LBW are complex and multifactorial, but the mechanisms involved may present common pathways. Infection is an important risk factor for preterm low birth weight and so periodontal disease may be linked to this process41. Due to the high morbidity and mortality rates, PTB represents a significant public health problem in Tanzania, which is among the countries with the highest perinatal and child mortality indices22. However, studies conducted by Nabet et al.3 and Patta- nashetti et al.35 found a strong association between PD and induced PTB due to preeclampsia. Our findings are at odds with those of studies performed in Iran and Italy by Abati et al.1 and Yaghini et al.36, who did not report this association. Certain similarities have been reported between pre- eclampsia and atherosclerosis. Like preeclampsia, athero- sclerosis is associated with endothelial dysfunction, which may be caused by oxidative stress and subsequent lipid peroxidation, hyperlipidemia or homocysteinemia37. Epi- demiological factors such as obesity, history of hyperten- sion and African ancestry render the person susceptible to pre-eclampsia and atherosclerosis. One of the reasons for abnormalities in endothelial function is the presence of severe inflammatory responses. Periodontal disease, a chronic oral Gram-negative infection, has been asso- ciated with atherosclerosis, thromboembolic events and hypercholesterinemia. Oral pathogens have been detect- ed in atherosclerotic plaque where they may play a role y This study also found associations between PD and PTB and LBW. After adjusting for participant’s age, parity, presence of pre-eclampsia and history of previous LBW and PTB, the relationship was statistically significant in all cases. These findings corroborate those of studies else- where in Africa and in other parts of the world, which have recorded significant associations between PD, PTB and LBW26,42-47. However, two studies conducted in Ita- ly1,48 and one in Taiwan49 found no association between PTB and PD, and an earlier study in Tanzania conducted African Health Sciences Vol 18 Issue 3, September, 2018 608 Conflict of interest The authors declare that they have no conflict of interest. by Mumghamba and Manji50 failed to demonstrate the as- sociation between PD and preterm LBW. References 1. Abati S, Villa A, Cetin I, Dessole S, Lugliè PF, Strohmenger L, et al. Lack of association between mater- nal periodontal status and adverse pregnancy outcomes: a multicentric epidemiologic study. J Matern Fetal Neonatal Med. 2013; 26 (4): 369–372. DOI:10.3109/14767058.201 2.733776. 1. Abati S, Villa A, Cetin I, Dessole S, Lugliè PF, Strohmenger L, et al. Lack of association between mater- nal periodontal status and adverse pregnancy outcomes: a multicentric epidemiologic study. J Matern Fetal Neonatal Med. 2013; 26 (4): 369–372. DOI:10.3109/14767058.201 2.733776. Future studies should aim to identify possible correlations between the maternal subgingival microbiota and the pla- cental microbioma, and analyse common inflammatory factors at both these sites. In any case, new policies are needed to provide systematic periodontal treatment for all pregnant patients with periodontal disease and thus control one of the main risk factors for adverse pregnan- cy outcomes. 2. Periodontal Disease Fact Sheet. American Acade- my of Periodontology - Perio.org. IL. Chicago 2017. https://www.perio.org/newsroom/periodontal-dis- ease-fact-sheet (Last accessed Feb 2017). 3. Nabet C, Lelong N, Colombier M-L, Sixou M, Mus- set A-M, Goffinet F, et al. Maternal periodontitis and the causes of preterm birth: the case-control Epipap study. J Clin Periodontol. 2010; 37 (1): 37–45. DOI:10.1111/j.1600- 051X.2009.01503.x. 2. Periodontal Disease Fact Sheet. American Acade- my of Periodontology - Perio.org. IL. Chicago 2017. https://www.perio.org/newsroom/periodontal-dis- ease-fact-sheet (Last accessed Feb 2017). 3. Nabet C, Lelong N, Colombier M-L, Sixou M, Mus- set A-M, Goffinet F, et al. Maternal periodontitis and the causes of preterm birth: the case-control Epipap study. J Clin Periodontol. 2010; 37 (1): 37–45. DOI:10.1111/j.1600- 051X.2009.01503.x. Conclusion I In summary, maternal periodontal disease is a potential independent risk indicator for pre-eclampsia, low birth weight and preterm birth. Periodontal examination and treatment for women with pre-existing periodontal dis- ease can reduce the risk of recurrence or deterioration, and should form part of the preventive antenatal care provided in developing countries. Taken together with other studies, our findings suggest that the disparate re- sults regarding the association between periodontal dis- ease and adverse outcomes are partly due to the defini- tions used and the populations studied. 4. 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African Health Sciences Vol 18 Issue 3, September, 2018 Discussion Our study did not find a significant statistical association between PD and pPROM or eclampsia, even though the findings were clinically significant. Similarly, Abati et al. in Italy found no association between PD and adverse pregnancy outcomes including pPROM1. Our study did not find a significant statistical association between PD and pPROM or eclampsia, even though the findings were clinically significant. Similarly, Abati et al. in Italy found no association between PD and adverse pregnancy outcomes including pPROM1. Funding information The study was self- funded by the authors. Acknowledgements Clínics Associates, a well-established Professional Part- nership in Terrassa (Barcelona), deserves a special men- tion for leading and supporting KiliBarnaDental project at Soweto General Hospital in Arusha, United Repub- lic of Tanzania, East Africa. 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DOI:10.1111/ j.1600-0757.2010.00358.x. 48. Calabrese N, Calabrese A, Nibali L, Rosati A, Fiengo S, Di Renzo GC. Is there any association be- tween periodontitis and preterm low birth weight? J Matern Fetal Neonatal Med. 2010; 23 (11): 1288–1293. DOI:10.3109/14767051003615467. 40. Blanc V, O’Valle F, Pozo E, Puertas A, León R, Mesa F. Oral bacteria in placental tissues: increased molecular detection in pregnant periodontitis patients. Oral Dis. 2015; 21 (7): 905-912. DOI:10.1111/odi.12364. 49. Wang YL, Liou JD, Pan WL. Association between ma- ternal periodontal disease and preterm delivery and low birth weight. Taiwan J Obstet Gynecol. 2013; 52 (1): 71-76. DOI: 10.1016/j.tjog.2013.01.011. 49. Wang YL, Liou JD, Pan WL. Association between ma- ternal periodontal disease and preterm delivery and low birth weight. Taiwan J Obstet Gynecol. 2013; 52 (1): 71-76. DOI: 10.1016/j.tjog.2013.01.011. 50. Mumghamba EGS, Manji KP. Maternal oral health status and preterm low birth weight at Muhimbili Na- tional Hospital, Tanzania: a case-control study. BMC Oral Health 2007; Jun 26 (7):8. DOI:10.1186/1472-6831-7-8. detection in pregnant periodontitis patients. Oral Dis. 2015; 21 (7): 905-912. DOI:10.1111/odi.12364. 41. Vergnes J-N, Sixou M. Preterm low birth weight and maternal periodontal status: a meta-analysis. Am J Obstet Gynecol. 2007; 196 (2) 135: e1–7.DOI:10.1016/j. ajog.2006.09.028. /j j g 50. Acknowledgements Mumghamba EGS, Manji KP. Maternal oral health status and preterm low birth weight at Muhimbili Na- tional Hospital, Tanzania: a case-control study. BMC Oral Health 2007; Jun 26 (7):8. DOI:10.1186/1472-6831-7-8. 42. Jacob PS, Nath S. Periodontitis among poor rural Indi- 611 African Health Sciences Vol 18 Issue 3, September, 2018
https://openalex.org/W4252006552	https://newprairiepress.org/cgi/viewcontent.cgi?article=6356&context=kaesrr	English	null	Comparison of conventional and low-inhibitor soybeans with different heat treatments and lysine concentrations in diets for finishing pigs	Kansas Agricultural Experiment Station research reports	1,991	cc-by	296	Comparison of conventional and low-inhibitor soybeans with Comparison of conventional and low-inhibitor soybeans with different heat treatments and lysine concentrations in diets for different heat treatments and lysine concentrations in diets for finishing pigs (1991) finishing pigs (1991) Follow this and additional works at: https://newprairiepress.org/kaesrr Part of the Other Animal Sciences Commons ansas Agricultural Experiment Station Research Reports ansas Agricultural Experiment Station Research Reports ansas Agricultural Experiment Station Research Reports ansas Agricultural Experiment Station Research Reports Volume 0 Issue 10 Swine Day (1968-2014) Article 516 This research report is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/kaesrr/vol0/iss10/516 Recommended Citation Recommended Citation Giesemann, M A.; Healy, B J.; Lewis, A J.; and Hancock, Joe D. (1991) "Comparison of conventional and low-inhibitor soybeans with different heat treatments and lysine concentrations in diets for finishing pigs (1991)," Kansas Agricultural Experiment Station Research Reports: Vol. 0: Iss. 10. https://doi.org/10.4148/ 2378-5977.6356 This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1991 the Author(s). Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. Authors Authors Authors Authors M A. Giesemann, B J. Healy, A J. Lewis, and Joe D. Hancock Authors Authors M A. Giesemann, B J. Healy, A J. Lewis, and Joe D. Hancock M A. Giesemann, B J. Healy, A J. Lewis, and Joe D. Hancock This research report is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/kaesrr/vol0/iss10/516
https://openalex.org/W2768976121	https://e-journal.unair.ac.id/MKG/article/download/5874/4021	English	null	The Effect of a combination of 12% spirulina and 20% chitosan on macrophage, PMN, and lymphocyte cell expressions in post extraction wound	Dental Journal	2,017	cc-by-sa	4,456	abstract Background: Tooth extraction is the ultimate treatment option for defective teeth followed by the need for dentures. Inflammation is one phase of the healing process that should be minimized in order to preserve alveolar bone for denture support. Macrophage, PMN and lymphocyte cells are indicators of acute inflammation. Spirulina and chitosan are natural compounds with the potential to be anti-inflammatory agents. Purpose: This study aimed to determine macrophage, PMN and lymphocyte cells of animal models treated with a combination of 12% spirulina and 20% chitosan on the 1st, 2nd and 3rd post-extraction day. Methods: Animal models were randomly divided into control (K) and treatment (P) groups. Each group was further divided into three subgroups (KI, KII, KIII and PI, PII, PIII). The post-extraction sockets of the control group animals were then filled with CMC Na 3%. Meanwhile, the post-extraction sockets of the treatment group members were filled with a combination of 12% spirulina and 20% chitosan. Subsequently, the number of PMN, macrophage and lymphocyte cells was analyzed by means of HE analysis on the 1st, 2nd and 3 rd days. Statistical analysis was then performed using a T-test. Results: There was a decrease in PMN cells and an increase in macrophage and lymphocyte cells on Days 1, 2 and 3. Conclusion: It can be concluded that a combination of 12% spirulina and 20% chitosan can not only decrease PMN cells, but can also increase macrophage and lymphocyte cells on day 1, 2 and 3 after tooth extraction. Keywords: spirulina; chitosan; inflammation; PMN; macrophage; lymphocyte Correspondence: Nike Hendrijantini, Department of Prosthodontics, Faculty of Dental Medicine, Universitas Airlangga. Jl. Mayjend. Prof. Dr. Moestopo no. 47 Surabaya 60132, Indonesia. Email: nike-h@fkg.unair.ac.id bone while promoting prosthetic restoration, biomaterial is required to eliminate the inflammatory process and accelerate the post-extraction healing process. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v50.i2.p106–110 106 106 Dental Journal (Majalah Kedokteran Gigi) 2017 June; 50(2): 106–110 The Effect of a combination of 12% spirulina and 20% chitosan on macrophage, PMN, and lymphocyte cell expressions in post extraction wound Nike Hendrijantini, Rostiny, Mefina Kuntjoro, Kevin Young, Bunga Shafira, and Yunita Pratiwi Department of Prosthodontics Faculty of Dental Medicine, Universitas Airlangga Surabaya- Indonesia Nike Hendrijantini, Rostiny, Mefina Kuntjoro, Kevin Young, Bunga Shafira, and Yunita Pratiwi Department of Prosthodontics Nike Hendrijantini, Rostiny, Mefina Kuntjoro, Kevin Young, Bunga Shafira, and Yunita Pratiwi Department of Prosthodontics Faculty of Dental Medicine, Universitas Airlangga Surabaya- Indonesia introduction Tooth loss is one of dental health problem. Its prevalence in Indonesia, according to RISKESDAS data in 2013, was 14.51% within the 45-54 years age group, 25.02% in the 55- 64 years age group, and 43.79% in the >65 years age group.1 Tooth loss can occur due to extraction which can increase injury to dental tissues resulting in an acute inflammatory reaction as well as inflammatory cell infiltration, such as polymorphonuclear (PMN), macrophages, and lymphocytes.2 Clinical inflammatory reaction is usually indicated by edema, redness, and pain.3 The inflammatory phase commonly lasts from the point of injury to Day 6 after its occurrence.4 Thus, in order to maintain the alveolar Recently, the use of natural materials in accelerating the wound healing process has been widely studied since they are safer than synthetic materials. One of the natural ingredients that have many benefits for the wound healing process is spirulina - a greenish-green algae containing C-phycocyanin, B-carotenoids, vitamin E, zinc, and other components useful to the human body. C-phycocianin is even considered to have an anti-inflammatory and antioxidant components.5,6 Another natural ingredient in the healing process that is commonly studied is chitosan, a polymer of deacetylated 107 107 Hendrijantini, et al./Dent. J. (Majalah Kedokteran Gigi) 2017 June; 50(2): 106–110 chitin. Chitin is a copolymer of N-acetyl-d-glucosamine and D-glucosamine bound by ß- (1-4) glycosidic bonds. Chitin and chitosan can both be found in aquatic and terrestrial organisms. Chitosan can currently be obtained via the food industry through the processing of waste derived from shrimps, lobsters, crabs, and squids.7 Chitosan is widely used as one of ingredients for drug delivery systems, wound healing processes, and orthopedic implants, while also being tknown to increase the activities of immune cells, inflammatory cells and angioendothelial cells. Chitosan oligosaccharides have anti-inflammatory properties since chitosan can inhibit the production of tumor necrotizing factor-α (TNF-α) in inflammation stimulated by lipopolysaccharide (LPS).6 Other studies have also shown that chitosan can reduce the inflammation associated with allergic responses by inhibiting the secretion of interleukin-8 (IL-8) and TNF-α.7 The animal specimens were subsequently divided into two groups, namely; control and treatment. The control (K) and treatment (P) groups were each sub-divided into three sub-groups referred to as KI, KII, KIII, PI, PII, and PIII. The roman numerals I, II and III represent day 1, 2, and 3 after the specimens had been terminated. introduction p Thereafter, the mandibular incisors of the members of all groups were extracted under ketamine anastesi (Ketalar, PT Pfizer, Jakarta, Indonesia) at a dose of 40 mg/kgBW. After the extraction, the sockets of the KI, KII, and KIII control groups were filled with 3% sodium-carboxymethyl cellulose natrium (CMC Na). Meanwhile, the sockets of the treatment groups, PI, PII, and PIII, were filled with a combination of 3% CMC Na, 12% spirulina and 20% chitosan using a 0.1 cc syringe. The sockets were stitched with 3/0-size silk threat. After the treatment, these animals were returned to their cages. On the first day, members of the KI and PI groups were decapitated, a process repeated for the KII and PII groups on day 2, as well as the KIII and PIII groups on day 3. Mandible samples were then taken and fixed. ( ) Some cases in the field of prosthodontics, such as immediate denture and ovate pontik installation, also require a more rapid wound healing process. Consequently, a shorter treatment that can lead to optimal results for patient comfort is necessary. Considerable previous research into the effects of spirulina and chitosan induction on collagen, osteoblast, and osteoclast cells in animal models has been conducted. The results of past investigations have shown that a combination of 12% spirulina and 20% chitosan can not only increase collagen and osteoblast expressions, but can also decrease osteoclast expression. As a result, it can be said that spirulina and chitosan has the potential to promote the bone healing process.8 The effects of spirulina and chitosan in this regard have actually already been studied, unlike the effects of a combination of spirulina and chitosan on inflammatory cells. Therefore, this research aimed to reveal the effects of a combination of 12% spirulina and 20% chitosan on PMN, macrophage and lymphocyte cells in animal models on day 1, 2 and 3 after extraction. At that point, the mandibles were with 2.5% nitric acid for 2 days. Thereafter, the sagittal incisive socket area of the specimens was cut and soaked in a 10% buffered formalin for 24 hours. Mixed preparations were then performed by using eosin haematoxylin (HE) before PMN, macrophages, and lymphocytes on one-third of the sockets’ area were observed using a light microscope (Nikon H600L®, Tokyo, Japan) at a magnification of 400x. results The results of the observation of PMN, macrophage, and lymphocyte cells in the control and treatment groups can be seen in Table 1. Moreover, the results of HE staining in both groups are contained in Figure 1. The results of HE staining illustrate that PMN possessed a large cell picture, a nucleus featuring lobes (2-5 lobes), chromatin in a condensed nucleus and visible organelle in cytoplasm. They also depicted macrophages as having a large cell image and generally kidney-shaped, erratically located cell nuclei. Furthermore, the cytoplasm cells were solid and appeared to be composed of a pink and purple granule, introduction The data obtained was then analyzed by means of a Saphiro Wilk test to analyze the data distribution, followed by an Independent t-test to identify the differences between the groups. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v50.i2.p106–110 materials and methodS This investigation reported here constituted laboratory- based experimental research incorporating a post test- only control group design and using male Cavia cobaya specimens (n = 42) weighing 300-350 grams and aged 3-3.5 months. The research passed an ethical test performed by the Faculty of Dental Medicine, Universitas Airlangga (no. 110/KKEPK.FKG/VII/2016). Table 1. Mean and standard deviation of PMN, macrophage, and lymphocyte expressions No Groups n PMN Macrophages Lymphocytes 1 KI 7 41.85±5.87 1.43±0.53 3.00±0.82 2 PI 7 32.00±3.26 5.57±1.51 12.00±2.00 3 KII 7 35.29±2.81 2.00±1.15 8.28±2.28 4 PII 7 11.71±1.60 19.71±1.98 21.43±2.37 5 KIII 7 20.57±3.69 15.57±1.72 19.14±2.41 6 PIII 7 15.14±2.41 20.57±2.70 22.43±3.31 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v50.i2.p106–110 Table 1. Mean and standard deviation of PMN, macrophage, and lymphocyte expressions Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v50.i2.p106–110 Hendrijantini, et al./Dent. J. (Majalah Kedokteran Gigi) 2017 June; 50(2): 106–110 108 whereas the lymphocytes had a circular or spherical nucleus cell with dark blue chromatin and a surrounding thin, light blue cytoplasm. adherens, chemotaxis and phagocytosis. Furthermore, PMN cells are able to move actively, and in a short period of time can collect in large numbers in the wound area. PMN cells have a lifespan of 1-3 days in connective tissue.10 y p Subsequently, before a t-test analysis was performed, normality test, Saphiro Wilk test was conducted. Results of the Saphiro Wilk test showed that all data were normally distributed because p value was more than 0.05. Independent t-test then was carried out. Results of the independent t-test indicated that the p values of PMN cells between KI and PI groups, between KII and PIII groups, and between KIII and PIII groups were 0.020, 0.000, and 0.007, respectively. The results of the independent t-test also showed that the p values of macrophages between KI and PI groups, between KII and PII groups, and between KIII and PIII groups were 0.000, 0.000, and 0.001, sequentially. Meanwhile, the p values of lymphocytes between KI and PI groups, between KII and PII groups, and between KIII and PIII groups were 0.000, 0.000, and 0.055, respectively. materials and methodS Almost all of the independent t-test results in the control groups compared with the treatment groups indicated significant increase macrophages and lymphocytes differences (p<0.05), except between group PMN significant decrease (p>0.05). Thus, it can be said that no significant difference existed between the two groups. Another cell that plays a role in the inflammatory process is the macrophage cell which carries out several functions in the wound healing process, such as producing collagenase and elastase enzymes, generating cytokines, facilitating phagocytosis and angiogenesis processes, as well as stimulating granulation tissue formation in the proliferative phase.11 Macrophage cells in the inflammatory process can be distinguished by the origin of the tissue macrophage resident and monocytes undergoing differentiation.12 Monocytes in the blood vessels will be transported toward inflammatory tissues due to chemotaxis resulting from the response to chemoattractant. Chemoattractant consists partly of kemokin, a protein (8-14 kDa) that regulates cell travel through interaction with a 7-transmembrane subset G-protein pair receptor.13 Similarly, lymphocyte cells play a role in the inflammatory process. Lymphocytes result in both immune- mediated inflammation caused by infectious agents and non-immune inflammation. T and B lymphocytes migrate to the inflammatory area and direct neutrophils and other leukocytes.14 In the remodeling process, when the wound has been closed and the local infection has subsided, the leukocyte subtance most often found in the injured tissue is that of T cell which acts as an adaptive immune response cell.6 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v50.i2.p106–110 discussion The latter allow chitosan to be broken down into micromolecules so that they are easily absorbed by the body without causing toxicity, thereby enabling it to be used as an analgesic, anti-tumor, anti-microbial, anti-oxidant, and wound healing agent.22 Chitosan also contains a N-acetyl-D-glucosamine unit, polysaccharide similar to glucan, which accelerates cytokine production in order to stimulate repair of affected tissue.23 In addition, on the first day after dental extraction, the average number of PMN cells in Group KI was the highest compared with the other control groups (K II and K III) and the treatment groups on the other days (P II and P III) because PMN cells had already been active and assembled at a large number of lesion sites very rapidly, i.e. within hours.17 PMN cells are highly reactive to chemotactic products in the form of proteins produced by bacteria.16 Moreoever, the results of calculating macrophage cells on day 1 indicated that the treatment group (P I) had a higher number of such cells than the control group (K I). This may occur because the inflammatory cells that appear immediately post-incision are not only neutrophils, but also monocytes moving into the inflammatory area. However, the number of monocytes migrating to the wound area is not as high as that of neutrophils.18 The combination of 12% spirulina and 20% chitosan also contains more synthetic C-phycocyanin components which execute a greater immunomodulatory function than when applied alone.19 The results of the third day’s observation found that the number of PMN cells in Group PIII was lower than that in Group KIII. The number of PMN cells on day 3 was lower than that on Day 2. This happened because the number of PMN cells will usually decrease between day 3 and 7.18.24 This reduction is essential to preventing further damage to healthy body tissue. The body responds to a reduction in the production of PMN cells in order not to damage other tissues because PMN cells issue anti-microbial products that can damage healthy tissue of the body. Furthermore, the results of the independent t-test showed there to be a significant difference in the presence of lymphocytes between the K1 and P1 groups on the first post-extraction day. This may occur because phycocyanin pigments contained in spirulina may act as an anti- inflammatory by inhibiting proinflammatory cytokines, namely; TNF-α and IL-1β.15 Chitosan also plays a role in increasing lymphocyte cells. discussion The presence of PMN cells is very important as an indicator of the wound healing process since PMN cells are the first to appear in the acute inflammatory phase.9 PMN cells are cellular defenses that play an active role in the process of bacterial destruction through endothelial Results of the research on day 1 revealed that the presence of PMN cells in the control group was higher than in the treatment group. This occurred because phycocyanin and β-carotene contained in spirulina can decrease the A D Figure 1. Inflammatory cells in the tooth ext (A), group PI (B), group KII (C), gr arrows: PMN, yellow arrows: macro Figure 1. Inflammatory cells in the tooth extraction sockets of Cavia cobaya animals in g (A), group PI (B), group KII (C), group PII (D), group KIII (E), and group PIII ( arrows: PMN, yellow arrows: macrophage cells, green arrows: lymphocytes). B A C E D F Figure 1. Inflammatory cells in the tooth extraction sockets of Cavia cobaya animals in Group KI (A), Group PI (B), Group KII (C), Group PII (D), Group KIII (E), and Group PIII (F). (blue arrows: PMN, yellow arrows: macrophage cells, green arrows: lymphocytes). Figure 1. Inflammatory cells in the tooth ex (A), group PI (B), group KII (C), g arrows: PMN yellow arrows: macr Figure 1. Inflammatory cells in the tooth extraction sockets of Cavia cobaya animals in (A), group PI (B), group KII (C), group PII (D), group KIII (E), and group PIII arrows: PMN, yellow arrows: macrophage cells, green arrows: lymphocytes). Figure 1. Inflammatory cells in the tooth extraction sockets of Cavia cobaya animals in Group KI (A), Group PI (B), Group KII (C), Group PII (D), Group KIII (E), and Group PIII (F). (blue arrows: PMN, yellow arrows: macrophage cells, green arrows: lymphocytes). Hendrijantini, et al./Dent. J. (Majalah Kedokteran Gigi) 2017 June; 50(2): 106–110 109 109 in the bone marrow can reach a post-injury maximum within 24-48 hours. On the other hand, the process of PMN cell clearance can reach its peak 48 hours after the occurrence of the lesion. The number of PMN cells will then decrease as the chronic inflammatory phase is entered. Clearance can also occur when the PMN cells extravatase into the peripheral tissues. discussion A previous investigation into mice revealed that PMN cells can migrate back from peripheral tissue into the bloodstream through a process known as reverse transmigration.16 This suggests that the extravasation of PMN cells does not necessarily lead to the clearance of tissue. The clearance process reaches its peak on the second day. Consequently, the anti-inflammatory effect of the combination of 12% spirulina and 20% chitosan will reduce the number of PMN cells on the second day to a greater extent than on the first day.16 production of proinflammatory cytokines, TNF-α and IL- 1β. β-carotene in spirulina also has an anti-inflammatory effect through resistance to the production of nitric oxide and prostaglandin E2. Furthermore, β-carotene also inhibits the expression of INOS, COX2, TNFα and IL1β. Suppression of inflammatory mediators is due to NF-κB inhibition that restricts the nuclear translocation of subunits NF-κB p65.15 As a result, anti-inflammatory activity of spirulina can decrease the number of PMNs in the lesion site. TNF-α and IL-1β can also facilitate the movement of PMN cells to the site of the lesion and spur the production of endothelial adherens.16 It is intended that PMN cells easily pass through the gap between endothelial cells in capillary blood vessels to eliminate bacteria, so the reduction of proinflammatory cytokines can, in turn, lead to a decrease in the production of PMN cells.16 The number of lymphocytes in Group PII, based on the results of the second day of observation, was higher than that in Group KII. This difference is due to spirulina being able to increase the number of lymphocytes. Previous research has even shown spirulina to have an immune modulatory effect on lymphocytes by significantly increasing IFN-.2 production.21 The increased number of lymphocyte cells in the treatment group is also due to chitosan that exhibits biocompatible and biodegradable properties. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v50.i2.p106–110 discussion Rostiny, Kuntjoro M, Sitalaksmi RM, Salim S. Spirulina chitosan gel induction on healing process of cavia cobaya post extraction socket. Dent J (Maj Ked Gigi). 2014; 47(1): 19-24. 9. Azzahra H, Pujiastuti P, Purwanto P. Potensi ekstrak kulit buah manggis (Garcinia mangostana L.) buatan pabrik terhadap peningkatan aktivitas mikrobisidal sel neutrofil yang dipapar Streptococcus mutans. e-Jurnal Pustaka Kesehat. 2014; 2(1): 161-6. The phycocyanin and β-carotene substances contained in spirulina can be considered to be anti-inflammatory antioxidants that can accelerate the wound healing process. Phycyocyanin, according to previous in vitro and in vivo research using rat-fed animals, can inhibit TNF-α inflammatory cytokine secretion and act as an antioxidant.26 This strongly suggests that the anti-inflammatory activity of spirulina may cause TNF-α and IL-1β secretions to decrease.27 Similarly, the antioxidants in β-carotene contained in spirulina can improve the wound healing process.6 Chitin and chitosan are biopolymers that offer many benefits, including; a high level of biocompatibility, low toxicity, increased antibacterial activity and accelerated wound healing.28 Chitosan can also stimulate PMN cells to chemotaxyize the wound area due to the presence of IL-1, TNF-α, and bacterial products.29 10. Kumar V, Cotran RS, Robbins SL. Robbins buku ajar patologi volume 1. 7th ed. Jakarta: EGC; 2007. p. 35-56. 11. Sussman C, Bates-Jensen B. Wound care: a collaborative practice manual for health professionals. Philadelphia, USA: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2012. p. 33-9. 12. Murray PJ, Wynn TA. Protective and pathogenic functions macrophage subsets. Nat Rev Immunol. 2012; 11: 723-37. 13. Sánchez-Martín L, Estecha A, Samaniego R, Sánchez-Ramón S, Vega MÁ, Sánchez-Mateos P. The chemokine CXCL12 regulates monocyte-macrophage differentiation and RUNX3 expression. Blood. 2011; 117: 88-97. 14. Port CM. Essentials of pathophysiology. 4th ed. Philadelphia, USA: Wolters Kluwer; 2015. p. 49-61. 15. Quader SH, Islam SU, Saifullah A, Majumder MFU, Hanna J. In- vivo studies of the anti-inflammatory effects of Spirulina platensis. J Pharmacogn Phytochem. 2013; 2(4): 70-80. g y 16. Baratawidjaja KG. Imunologi dasar. 11th ed. Jakarta: Badan Penerbit Fakultas Kedokteran Universitas Indonesia; 2012. p. 34-46. 17. Tak T, Tesselaar K, Pillay J, Borghans JA, Koenderman L. What’s your age again? determination of human neutrophil half-lives revisited. J Leukoc Biol. 2013; 94: 595-601. In other words, spirulina and chitosan exert a synergistic effect when combined since chitosan plays a role in drug delivery, while spirulina has a therapeutic effect. Spirulina and chitosan can also interact intermolecularly to increase mechanical resistance. discussion A previous piece of research using mice orally induced with chitosan finds that the latter can stimulate the release of IL-10, IL-4, and TGF-β mRNA expressions in gastric mucosa, CD3 + T lymphocytes in the spleen, as well as natural killer cells (NK) in intestinal intraepithelial lymphocytes.20 The number of macrophages in Group PIII, based on the results of the third day’s observation, was still higher than that in Group KIII. Nevertheless, there was no significant difference in the post-extraction number of macrophages between the treatment groups on day 2 and day 3. This may be due to the inflammatory process beginning to enter the resolution phase during which a reduction of chemokine by the mechanism of proteolysis and chemokine sequestration occurs.25 Macrophages, as a result, begin to dominate the wound area from day 3 to day 7 after extraction. In addition, the number of PMN cells in Group PII, based on the results of the second day’s observation, was lower than that in Group KII. The decrease in PMN cells on the second day was higher than on the first day due to the homeostasis process where the number of PMN cells produced in the bone marrow must be balanced with that having clearance which had already worked on the system within the body. The number of PMN cells produced The number of lymphocyte cells in the treatment group, based on the results of the third day’s observation, appeared to increase compared with that of the control group. However, based on the results of the independent t-test, there was no significant difference in the number Hendrijantini, et al./Dent. J. (Majalah Kedokteran Gigi) 2017 June; 50(2): 106–110 110 of lymphocyte cells between the control group and the treatment group on day 3. During the chronic inflammatory process, the presence of lymphocyte can usually reach a peak between day 5 and day 10.15 Thus, 12% spirulina and 20% chitosan are expected to act as anti-inflammatories. This insignificant difference between the control group and the treatment group is due to the chronic inflammatory process having begun to subside and entering the maturation process which leads to regeneration. periodontitis cases: a placebo controlled clinical trial. J Clin Diagn Res. 2013; 7(10): 2330-3. periodontitis cases: a placebo controlled clinical trial. J Clin Diagn Res. 2013; 7(10): 2330-3. 7. Kim S. Chitin, chitosan, oligosaccharides and their derivatives. Florida: CRC Press; 2010. p. 3, 11. 8. discussion Both of these materials can even work together to provide more effective benefits than if applied alone. 18. Rodero MP, Licata F, Poupel L, Hamon P, Khosrotehrani K, Cambadiare C, Boissonnas A. In vivo imaging reveals a pioneer wave of monocyte recruitment into mouse skin wounds. PLoS One. 2014; 9(12): 1-9. 19. Madhyastha HK, Radha KS, Nakajima Y, Omura S, Maruyama M. uPA dependent and independent mechanisms of wound healing by C-phycocyanin. J Cell Mol Med. 2008; 12(6B): 2691-703. In addition, a high degree of chitosan acetylation will improve the hydrophobic properties of chitosan. The hydrophilic component is capable of diffusing through the chitosan polymer into the outer medium to be absorbed by the body.22 Finally, based on the above discussion, it can be concluded that 12% spirulina and 20% chitosan combination can not only decrease PMN expression, but also increase macrophages and lymphocytes in cavia cobaya animals on the first, second and third days after extraction. 20. Borges O, Borchard G, de Sousa A, Junginger HE, Cordeiro-da- Silva A. Induction of lymphocytes activated marker CD69 following exposure to chitosan and alginate biopolymers. Int J Pharm. 2007; 337: 254-64. 21. Karkos PD, Leong SC, Karkos CD, Sivaji N, Assimakopoulos DA. Spirulina in clinical practice: evidence-based human applications. Evid-based Complement Alternat Med. 2011; 2011: 1-5. Evid-based Complement Alternat Med. 2011; 2011: 1-5. 22. Aranaz I, Mengíbar M, Harris R, Panos I, Miralles B, Acosta N, Galed G, Heras A. Functional characterization of chitin and chitosan. 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https://openalex.org/W3013445004	https://europepmc.org/articles/pmc7230952?pdf=render	English	null	Spectrum of Genetic Variants Associated with Anterior Segment Dysgenesis in South Florida	Genes	2,020	cc-by	5,054	Received: 7 February 2020; Accepted: 25 March 2020; Published: 26 March 2020 Received: 7 February 2020; Accepted: 25 March 2020; Published: 26 March 2020 Abstract: Anterior segment dysgenesis (ASD) comprises a wide spectrum of developmental conditions aﬀecting the cornea, iris, and lens, which may be associated with abnormalities of other organs. To identify disease-causing variants, we performed exome sequencing in 24 South Florida families with ASD. We identiﬁed 12 likely causative variants in 10 families (42%), including single nucleotide or small insertion–deletion variants in B3GLCT, BMP4, CYP1B1, FOXC1, FOXE3, GJA1, PXDN, and TP63, and a large copy number variant involving PAX6. Four variants were novel. Each variant was detected only in one family. Likely causative variants were detected in 1 out of 7 black and 9 out of 17 white families. In conclusion, exome sequencing for ASD allows us to identify a wide spectrum of rare DNA variants in South Florida. Further studies will explore missing variants, especially in the black communities. Keywords: anterior segment dysgenesis; primary congenital glaucoma; exome sequencing Spectrum of Genetic Variants Associated with Anterior Segment Dysgenesis in South Florida Saradadevi Thanikachalam 1, Elizabeth Hodapp 2, Ta C. Chang 2 , Dayna Morel Swols 3, Filiz B. Cengiz 1, Shengru Guo 1, Mohammad F. Zafeer 1, Serhat Seyhan 1, Guney Bademci 1, William K. Scott 1,3, Alana Grajewski 2 and Mustafa Tekin 1,2,3,* 1 John P. Hussmann Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, USA; saradadevi.thanikachalam@uth.tmc.edu (S.T.); fbsakc@yahoo.com (F.B.C.); sguo@med.miami.edu (S.G.); mxz625@med.miami.edu (M.F.Z.); drserhatseyhan@gmail.com (S.S.); b d @ d d (G B ) @ d d (W K S ) 1 John P. Hussmann Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, USA; saradadevi.thanikachalam@uth.tmc.edu (S.T.); fbsakc@yahoo.com (F.B.C.); sguo@med.miami.edu (S.G.); mxz625@med.miami.edu (M.F.Z.); drserhatseyhan@gmail.com (S.S.); g.bademci@med.miami.edu (G.B.); w.scott@med.miami.edu (W.K.S.) g ( ) ( ) y g.bademci@med.miami.edu (G.B.); w.scott@med.miami.edu (W.K.S.) 2 Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA; hodapp@med.miami.edu (E.H.); t.chang@med.miami.edu (T.C.C.); agrajewski@med.miami.edu (A.G.) 3 Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, USA; dmorel@med.miami.edu hodapp@med.miami.edu (E.H.); t.chang@med.miami.edu (T.C.C.); agrajewski@med.miami.edu (A.G.) 3 Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, USA; dmorel@med.miami.edu * Correspondence: mtekin@med.miami.edu 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 Genes 2020, 11, 350; doi:10.3390/genes11040350 2.1. Subjects We studied 24 (22 simplex and 2 multiplex) unrelated ASD-aﬀected individuals (probands). Aﬀected or unaﬀected family members were available for the study in 13 families. Families were recruited through the Bascom Palmer Eye Institute at the University of Miami, Miami, Florida. Probands were consecutive patients seen by an ophthalmologist for clinical diagnosis and management of ASD. Clinical evaluation of all aﬀected individuals by an ophthalmologist included a slit lamp examination and dilated fundus exam. Further imaging and laboratory tests were performed when needed. DNA was extracted from peripheral leukocytes of each proband by standard protocols. 2.2. Genetic Screening We performed exome sequencing (ES) in the Hussman Institute for Human Genomics at the University of Miami. We used Agilent SureSelect Human All Exon 60 Mb V6 for in-solution enrichment of coding exons and ﬂanking intronic sequences following the manufacturer’s standard protocol (Agilent). A HiSeq 3000 instrument (Illumina) was used for sequencing and Genome Analysis Toolkit software package used for variant calling [5,6]. During the analysis, we focused on speciﬁc genes with putative pathogenic variants previously found in individuals with ASD (Supplementary Materials Table S1). We used Enlis genome software (https://www.enlis.com/) for annotation and variant ﬁltering. As recommended, we ﬁltered variants based on minor allele frequency of <0.005 in gnomAD (www.gnomad.broadinstitute.org) when considering a recessive mode of inheritance and <0.0005 when considering a dominant mode of inheritance [7,8]. Combined annotation dependent depletion (http://cadd.gs.washington.edu/) [9], MutationTaster (http://www.mutationtaster.org/) [10], and sorting intolerant from tolerant (http://sift.bii.a-star.edu.sg/) [11] in silico analysis tools were used for the pathogenicity prediction. Conservation of the variant was evaluated by using genomic evolutionary rate proﬁling (http://mendel.stanford.edu/SidowLab/downloads/gerp/) [12]. We used copy number inference from exome reads to detect copy number variants [13,14]. Sanger sequencing was performed to conﬁrm the variants, and when other family members were available only those that showed complete segregation with the phenotype in the entire family were considered pathogenic. We used the American College of Medical Genetics guidelines to interpret variant pathogenicity [15,16]. 1. Introduction Anterior segment dysgenesis (ASD) is a heterogeneous group of eye disorders aﬀecting the cornea, iris, lens, zonule, trabecular meshwork, Schlemm canal, and ciliary body. Primary defects in migration or diﬀerentiation of the mesenchymal cells may cause ASD, and in turn impede aqueous humor outﬂow and elevate intraocular pressure [1]. Increased intraocular pressure is a major risk factor for glaucoma [2]. About 50% of individuals with ASD develop glaucoma, which often manifests before the age of 40 years [3,4]. ASD can present with ophthalmic ﬁndings only, such as in Peters anomaly and isolated aniridia, or as part of a multisystemic condition, such as Axenfeld–Rieger (AR; MIM 601542, 601090, 601499), Peters plus (MIM 261540), or SHORT (MIM 269880; short stature-hyperextensibility of joints or hernia or both-ocular depression-Rieger anomaly-teething delay) syndromes. Primary congenital glaucoma (PCG) is included in the ASD spectrum due to the presumed abnormal trabecular meshwork and Schlemm canal development [1,2]. In large families with multiple aﬀected members, ASD is usually inherited as an autosomal dominant trait, though autosomal recessive inheritance has been reported [1]. Well-known ASD genes Genes 2020, 11, 350; doi:10.3390/genes11040350 www.mdpi.com/journal/genes www.mdpi.com/journal/genes 2 of 10 Genes 2020, 11, 350 are CYP1B1 (MIM 601771), FOXC1 (MIM 601090), FOXE3 (MIM 601094), PAX6 (MIM 607108), and PITX2 (MIM 601542) [1]. Mutations in PAX6, PITX2, and FOXC1 do not always correlate with speciﬁc ASD phenotypes. Patients with AR syndrome and PCG may have FOXC1 mutations. PAX6 mutations can occur in both Peters anomaly and aniridia, and CYP1B1 mutations may be the cause of Peters anomaly and PCG. Phenotype or genotype alone is insuﬃciently precise to classify or diagnose ASD [2]. In the present study, we have investigated the genetic origin of isolated or syndromic ASD in the diverse population of South Florida. 3. Results Based on the clinical evaluations, seven probands were considered to have syndromes associated with ASD (AR, Peters plus, and oculo–dento–digital syndromes (MIM 164200)), and 17 were diagnosed with isolated eye anomalies (Supplementary Materials Table S2). On average, each exome had 99.2%, 95.3%, and 89.4% of mappable bases of the Gencode deﬁned exome represented by coverage of 1×, 5×, and 10× reads for ES, respectively. The average read depth was 71.9× and the coverage and average read depth are considered adequate for exome sequencing [17,18]. We detected nine pathogenic or likely pathogenic variants and three variants of uncertain signiﬁcance (VUS) that potentially explain the observed phenotypes in 10 probands out of 24 (42%) (Figure 1, Table 1, Table 2, Supplementary Materials Figure S1 and Table S3 show phenotypic features of unsolved probands). 3 of 10 Genes 2020, 11, 350 enes 2020, 11, x FOR PEER REVIEW 3 of 11 Figure 1. Pedigrees of the studied families, electropherograms, and segregation of the variants. Sanger sequencing traces represents identified variant positions (red arrow). * These individuals were not phenotypically evaluated therefore expected dominant transmission or de novo occurrence could not be demonstrated. Figure 1. Pedigrees of the studied families, electropherograms, and segregation of the variants. Sanger sequencing traces represents identiﬁed variant positions (red arrow). * These individuals were not phenotypically evaluated therefore expected dominant transmission or de novo occurrence could not be demonstrated. Figure 1. Pedigrees of the studied families, electropherograms, and segregation of the variants. Sanger sequencing traces represents identified variant positions (red arrow). * These individuals were not phenotypically evaluated therefore expected dominant transmission or de novo occurrence could not be demonstrated. Figure 1. Pedigrees of the studied families, electropherograms, and segregation of the variants. Sanger sequencing traces represents identiﬁed variant positions (red arrow). * These individuals were not phenotypically evaluated therefore expected dominant transmission or de novo occurrence could not be demonstrated. 4 of 10 Genes 2020, 11, 350 Table 1. Summary of the identiﬁed variants in this study. 3. Results Family ID Gene Transcript Inh Zyg cDNA Amino Acid Change gnomAD CADD GERP RS MutationTaster SIFT ACMG ACMG Guidelines Reference 1 PAX6 NM_000280.4 AD HT Large deletion (~266,752 bp) Large deletion N/A N/A N/A N/A N/A LP PVS1 Aradhya, 2012 [19] 3 FOXC1 NM_001453.2 AD HT c.316C>T p.Q106* N/A 38 3.8599 DC N/A P PVS1, PM2, PP3, PP5 Dhaene, 2011 [20] 5 TP63 NM_003722.4 AD HT c.1028G>A p.R343Q N/A 33 5.8299 DC DM LP PS3, PM2, PM5, PP3 Ianakiev, 2000 [21] 6 BMP4 NM_130851.3 AD HT c.521dupG p.F175Lfs8 N/A 35 5.1999 DC N/A P PVS1, PM2, PP3 This study 8 PXDN NM_012293.2 AR HT c.3821T>C p.L1274P N/A 25.8 5.4099 DC DM VUS PM2, PP3 This study HT c.2276C>T p.S759L 0.00001204 32 5.63 DC DM VUS PM2, PP3, This study 10 B3GLCT NM_194318.3 AR HT c.660+1G>A Splice 0.0007602 34 6.0799 DC N/A P PVS1, PP3, PP5 Lesnik Oberstein, 2006 [22] HT c.1234C>T p.R412 N/A 36 3.23 DC N/A P PVS1, PM2, PP3, PP5 Weh, 2014 [23] 11 FOXC1 NM_001453.2 AD HT c.254dupC p.L86Afs220 N/A 33 0.5139 DC N/A P PVS1, PM1, PM2 This study 12 GJA1 NM_000165.4 AD HT c.119C>T p.A40V N/A 25.5 6.1599 DC DM P PS3, PM1, PM2, PM6, PP2, PP3, PP5 Paznekas, 2003 [24] 14 CYP1B1 NM_000104.3 AR HM c.535delG p.A179Rfs18 0.00004797 24.2 2.5599 DC N/A P PVS1, PM2, PP5 Belmouden, 2002 [25] 18 FOXE3 NM_012186.2 AR HM c.291C>G p.I97M 0.00002015 22.4 1.1799 DC DM VUS PM2, PP3 Quiroz-Casian, 2018 [26] ACMG: American College of Medical Genetics guidelines, AD: autosomal dominant, AR: autosomal recessive, CADD: combined annotation dependent depletion, DC: disease-causing, DM: damaging, GERP RS: genomic evolutionary rate proﬁling rejected substitution, gnomAD: genome aggregation database, HM: homozygous, HT: heterozygous, Inh: inheritance, LP: likely pathogenic, N/A: not available, P: pathogenic, SIFT: sorting intolerant from tolerant, VUS: variants of uncertain signiﬁcance, Zyg: zygosity. 18 FOXE3 NM_012186.2 AR HM c.291C>G p.I97M 0.00002015 22.4 1.1799 DC DM VUS PM2, PP3 Quiroz-Casian, 2018 [26] ACMG: American College of Medical Genetics guidelines, AD: autosomal dominant, AR: autosomal recessive, CADD: combined annotation dependent depletion, DC: disease-causing, DM: damaging, GERP RS: genomic evolutionary rate proﬁling rejected substitution, gnomAD: genome aggregation database, HM: homozygous, HT: heterozygous, Inh: inheritance, LP: likely pathogenic, N/A: not available, P: pathogenic, SIFT: sorting intolerant from tolerant, VUS: variants of uncertain signiﬁcance, Zyg: zygosity. Table 2. Phenotypic features of probands with causative variants. 3. Results Family-Individual ID Sex Simplex/Multiplex Age (Years) Ethnicity Eye Phenotype Additional Clinical Features Gene 1-II:1 M Sx 9 Black, non-Hispanic Aniridia with glaucoma - PAX6 3-II:1 M Sx 11 White, non-Hispanic AR with glaucoma - FOXC1 5-II:1 M Sx 9 White, Hispanic Peters anomaly OD Syndactyly of third and fourth toes in the left foot, vesicoureteral reﬂux, cleft lip and palate, and nasolacrimal abnormalities TP63 6-II:1 F Sx 9 White, Hispanic Peters anomaly OU - BMP4 8-II:1 M Sx 8 White, non-Hispanic Peters anomaly OU - PXDN 10-II:1 F Sx 8 White, Hispanic Peters anomaly OU - B3GLCT 11-II:1 F Sx 37 White, Hispanic AR with glaucoma - FOXC1 12-II:1 M Sx 13 White, Hispanic Microphthalmia with glaucoma Microdontia, underdeveloped alae nasi, syndactyly GJA1 14-II:1 M Sx 13 White, Hispanic Peters anomaly OU - CYP1B1 18-II:1 M Sx 6 White, Hispanic Peters anomaly OU - FOXE3 AR: Axenfeld–Rieger anomaly, ASD: anterior segment dysgenesis, F: female, M: male, OD: right eye, OS: left eye, OU: both eyes, Sx: simplex. Table 2. Phenotypic features of probands with causative variants. Genes 2020, 11, 350 5 of 10 4. Discussion In this study, we detected potentially causative variants in 42% of probands with ASD, which is higher than the reported proportion, which ranges from 10% to 25% [27]. Table 3 summarizes the characteristics of diﬀerent genetic studies on ASD. Potential explanations for a higher detection rate of causative variants in our cohort are ethnicities studied, diﬀerences in case selection, the number of genes analyzed, and sample size. Our cohort consisted of a unique demographic from South Florida, including large Hispanic and Caribbean populations. Earlier studies focused on European, Asian, African, and Middle Eastern populations [27,28]. We did not identify recurrent variants enriched in our cohort; the diﬀerence between ethnicities of our cohort and those of earlier studies does not appear to explain our higher detection rate. In our cohort, families with Hispanic ancestry appear to have a higher detection rate (Hispanic vs. non-Hispanic is 7/12 vs. 3/12). Additionally, the diﬀerence between whites and blacks is noticeable: only 1 out of 7 black families is solved while 9 of 17 white families studied are found to have potentially causative variants. The majority of our black families were from the Caribbean, suggesting that the underlying genetic factors of ASD in the Caribbean remain largely unknown. Another important diﬀerence between our study and previous studies is the spectrum of ASD being analyzed. We looked at a wide range of ASD conditions, such as Peters anomaly, aniridia, AR syndrome, and PCG. Some other studies focused on a speciﬁc phenotype, such as Peters anomaly [23] or primary open-angle glaucoma/primary angle-closure glaucoma [27]. Recognized gene variants for some focused phenotypes are present in smaller portions of aﬀected individuals, which likely contributes to higher detection rate in our study. We used ES to cover all genes previously associated with ASD and some previous studies used gene panels, which may not include all associated genes (Supplementary Materials Tables S1 and S4). While targeted next-generation sequencing gene panels potentially provide higher coverage for individual genes and lower cost, ES as a research tool reduces the need of development and validation of custom panels. Finally, our cohort is smaller in size compared to previous cohorts and may have a higher detection rate by chance. 6 of 10 Genes 2020, 11, 350 Table 3. Causative variant detection in published studies. Studies Sample Size Phenotypes Population Studied ES or Gene Panel Causative Variants Detected in ASD Weh et al. 4. Discussion [23] 27 Syndromic Peters anomaly: 20 Isolated Peters anomaly: 7 Children’s Hospital of Wisconsin (USA) Population subtypes were not mentioned ES 22.2% overall Huang et al. [27] 257 POAG: 125 PACG: 132 Chinese: 257 ES of 43 genes associated with ASD, microcornea or microphthalmia 10.9% overall POAG: 8.80% PACG: 12.9% Patel et al. [28] 277 MAC: 98 cases ASDA: 113 cases Other or syndromic: 8 cases RET: 49 cases Congenital cataracts and lens-associated conditions: 9 cases White European: 139 South Asian: 21 Black African: 7 Arabic or Middle Eastern: 5 Black Caribbean: 2 Unknown: 91 Mixed/unclassiﬁed: 12 Oculome panel of 429 known eye disease genes 24.5% overall Congenital cataracts and lens-associated conditions: 88.9% RET: 42.8% Other or syndromic: 37.5% ASD: 24.8% This study 24 Peters anomaly: 8 PCG: 6 AR: 5 Aniridia: 2 Congenital corneal dystrophy: 1 Microphthalmia with glaucoma: 1 White, Hispanic: 11 (7 solved) Black, Hispanic: 1 (0 solved) White, non-Hispanic: 6 (2 solved) Black, non-Hispanic: 6 (1 solved) ES of 92 genes associated with eye phenotypes 42% overall Peters anomaly: 75% Aniridia: 50% Others: 50% AR: 40% ASD: anterior segment dysgenesis, ASDA: anterior segment developmental anomalies including glaucoma, ES: exome sequencing, MAC: microphthalmia–anophthalmia–coloboma, PACG: primary angle-closure glaucoma, POAG: primary open-angle glaucoma, RET: retinal dystrophies. Table 3. Causative variant detection in published studies. 22.2% overall 10.9% overall POAG: 8.80% PACG: 12.9% 24.5% overall Congenital cataracts and lens-associated conditions: 88.9% RET: 42.8% Other or syndromic: 37.5% ASD: 24.8% 42% overall Peters anomaly: 75% Aniridia: 50% Others: 50% AR: 40% ASD: anterior segment dysgenesis, ASDA: anterior segment developmental anomalies including glaucoma, ES: exome sequencing, MAC: microphthalmia–anophthalmia–coloboma, PACG: primary angle-closure glaucoma, POAG: primary open-angle glaucoma, RET: retinal dystrophies. Genes 2020, 11, 350 7 of 10 Identiﬁed variants in PAX6, FOXC1, TP63, BMP4, B3GLCT, and GJA1 are considered likely pathogenic or pathogenic based on American College of Medical Genetics (ACMG) guidelines. It should be noted that while GJA1 variant was de novo we did not conﬁrm the parental origin. One proband with Peters anomaly was heterozygous for two VUSs in PXDN. One variant is a change from leucine to proline in position 1274. This variant aﬀects a conserved residue and is predicted to aﬀect protein function with a rare exome ensemble learner (REVEL) score of 0.776, which is a combination of 13 individual tools for pathogenicity prediction of missense variants [29]. 4. Discussion The second variant shows a change from serine to leucine in position 759. This missense variant is also predicted to make an impact on protein function with a REVEL score of 0.8399. Each parent is heterozygous for one variant suggesting that these two variants are in trans (Figure 1). Biallelic PXDN variants have been reported with various eye anomalies including microphthalmia, congenital cataracts, microcornea, sclerocornea, and glaucoma [30,31]. Therefore, it is possible that the identiﬁed variants are the cause of Peters anomaly in our patient. Similarly, one proband was homozygous for the FOXE3 p.I97M variant, which is a VUS. The allele frequency of this variant on gnomAD is 0.00002015. Multiple in silico prediction tools show a damaging eﬀect. This variant has been previously reported in a case with ASD [26]. Therefore, we consider the FOXE3 variant a likely cause of the eye phenotype in our proband. The observed phenotypes in the 10 probands and the variants identiﬁed are generally consistent with prior studies. However, in family ﬁve, the proband is heterozygous for a TP63 gene variant. Typically, TP63 mutations have been reported in Rapp–Hodgkin (MIM 129400), ADULT (acro–dermato–ungual–lacrimal–tooth; MIM 103285), EEC (ectrodactyly–ectodermal dysplasia–cleft lip/palate; MIM 604292), Hay–Wells (MIM 106260), limb–mammary (MIM 603543), and split hand/foot malformation (MIM 605289) syndromes. In addition to various systemic anomalies, eye ﬁndings of these syndromes include blue irides, photophobia, blepharophimosis, blepharitis, dacryocystitis, and lacrimal duct abnormalities [32–37]. Our proband was diagnosed with Peters anomaly in the right eye as well as with syndactyly of third and fourth toes in the left foot, vesicoureteral reﬂux, cleft lip and palate, possible glaucoma, and nasolacrimal abnormalities. All of these ﬁndings except for Peters anomaly have been reported in patients with TP63 variants. Another TP63 variant (p.R343W) aﬀecting the same amino acid residue has been reported in a patient with glaucoma and decreased central corneal thickness as well as ﬁndings consistent with lacrimo–auriculo–dento–digital syndrome (MIM 149730) [38]. Peters anomaly in our patient and decreased corneal thickness associated with glaucoma in the previously reported patient may suggest that the Arg343 residue of TP63 plays a role in corneal development. Some limitations of our study include the variety of ASD diagnoses among our patient sample. Our study encompasses patients with Peters anomaly, aniridia, AR syndrome, and PCG. 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Genet. 2002, 11, 1185–1193. [CrossRef] [PubMed] 1. Gould, D.B.; John, S.W. Anterior segment dysgenesis and the developmental glaucomas are complex traits. Hum. Mol. Genet. 2002, 11, 1185–1193. [CrossRef] [PubMed] 2. Ito, Y.A.; Walter, M.A. Genomics and anterior segment dysgenesis: A review. Clin. Exp. Ophthalmol. 2014, 42, 13–24. [CrossRef] [PubMed] 3. Strungaru, M.H.; Dinu, I.; Walter, M.A. Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations. Investig. Ophthalmol. Vis. Sci. 2007, 48, 228–237. [CrossRef] [PubMed] 4. Rudnicka, A.R.; Mt-Isa, S.; Owen, C.G.; Cook, D.G.; Ashby, D. Variations in primary open-angle glaucoma prevalence by age, gender, and race: A Bayesian meta-analysis. Investig. Ophthalmol. Vis. Sci. 2006, 47, 4254–4261. [CrossRef] [PubMed] 5. Bademci, G.; Foster, J., 2nd; Mahdieh, N.; Bonyadi, M.; Duman, D.; Cengiz, F.B.; Menendez, I.; Diaz-Horta, O.; Shirkavand, A.; Zeinali, S.; et al. Comprehensive analysis via exome sequencing uncovers genetic etiology in autosomal recessive nonsyndromic deafness in a large multiethnic cohort. Genet. Med. 2016, 18, 364–371. [CrossRef] 6. McKenna, A.; Hanna, M.; Banks, E.; Sivachenko, A.; Cibulskis, K.; Kernytsky, A.; Garimella, K.; Altshuler, D.; Gabriel, S.; Daly, M.; et al. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010, 20, 1297–1303. [CrossRef] 7. Shearer, A.E.; Eppsteiner, R.W.; Booth, K.T.; Ephraim, S.S.; Gurrola, J., 2nd; Simpson, A.; Black-Ziegelbein, E.A.; Joshi, S.; Ravi, H.; Giuﬀre, A.C.; et al. Utilizing ethnic-speciﬁc diﬀerences in minor allele frequency to recategorize reported pathogenic deafness variants. Am. J. Hum. Genet. 2014, 95, 445–453. [CrossRef] 8. Lek, M.; Karczewski, K.J.; Minikel, E.V.; Samocha, K.E.; Banks, E.; Fennell, T.; O’Donnell-Luria, A.H.; Ware, J.S.; Hill, A.J.; Cummings, B.B.; et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 2016, 536, 285–291. [CrossRef] 9. Rentzsch, P.; Witten, D.; Cooper, G.M.; Shendure, J.; Kircher, M. CADD: Predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019, 47, D886–D894. [CrossRef] 10. Schwarz, J.M.; Cooper, D.N.; Schuelke, M.; Seelow, D. MutationTaster2: Mutation prediction for the deep-sequencing age. Nat. Methods 2014, 11, 361–362. [CrossRef] [PubMed] 11. Kumar, P.; Henikoﬀ, S.; Ng, P.C. Predicting the eﬀects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 2009, 4, 1073–1081. [CrossRef] [PubMed] 12. Cooper, G.M.; Stone, E.A.; Asimenos, G.; Program, N.C.S.; Green, E.D.; Batzoglou, S.; Sidow, A. 5. Conclusions We studied 24 families with ASD from South Florida and identiﬁed DNA variants potentially explaining 42% of our cohort. Further studies are required to compare diﬀerent ASD demographics and identify underlying genetic variants on a larger scale. 8 of 10 Genes 2020, 11, 350 Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/4/350/s1, Table S1: List of the genes used for the ﬁltering by using ES in our cohort. Table S2: Syndromic and isolated subjects in both solved and unsolved probands. Table S3: Phenotypic features of the unsolved probands. Table S4: Characteristics of common genes for anterior segment dysgenesis. Figure S1: Representation of the PAX6 gene deletion by using CoNiFER. Author Contributions: Conceptualization, S.T., W.K.S., G.B., T.C.C., E.H., A.G., and M.T; Data Curation, S.T., G.B., D.M.S., S.G., and M.T.; Methodology, F.B.C., S.G., M.F.Z., S.S., and G.B.; Software, G.B., and S.G.; Formal analysis, S.T., G.B., S.G., and M.T.; Writing—original draft preparation., S.T., G.B., and M.T.; Writing—review and editing, S.T., E.H., T.C.C., D.M.S., F.B.C., S.G., M.F.Z., S.S., G.B., W.K.S., A.G., and M.T.; Resources, T.C.C., E.H., A.G., W.K.S., and M.T.; Investigation, T.C.C., E.H., A.G., G.B., W.K.S., and M.T.; Funding acquisition, T.C.C., E.H., A.G., W.K.S. and M.T. All authors have read and agree to the published version of the manuscript. Funding: This research was funded by the Samuel & Ethel Balkan International Pediatric Glaucoma Center, the James Annenberg La Vea Charitable Trust, the University of Miami Institute for Advanced Studies of the Americas, and the John T. and Winifred Hayward Foundation. Acknowledgments: We are grateful to the participating families and Jill Jensen La Vea for her generous philanthropic support. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Distribution and intensity of constraint in mammalian genomic sequence. Genome Res. 2005, 15, 901–913. [CrossRef] [PubMed] 13. Bademci, G.; Diaz-Horta, O.; Guo, S.; Duman, D.; Van Booven, D.; Foster, J., 2nd; Cengiz, F.B.; Blanton, S.; Tekin, M. Identiﬁcation of copy number variants through whole-exome sequencing in autosomal recessive nonsyndromic hearing loss. Genet. Test. Mol. Biomark. 2014, 18, 658–661. [CrossRef] [PubMed] 9 of 10 Genes 2020, 11, 350 14. Krumm, N.; Sudmant, P.H.; Ko, A.; O’Roak, B.J.; Malig, M.; Coe, B.P.; Project, N.E.S.; Quinlan, A.R.; Nickerson, D.A.; Eichler, E.E. Copy number variation detection and genotyping from exome sequence data. Genome Res. 2012, 22, 1525–1532. [CrossRef] [PubMed] 15. Richards, S.; Aziz, N.; Bale, S.; Bick, D.; Das, S.; Gastier-Foster, J.; Grody, W.W.; Hegde, M.; Lyon, E.; Spector, E.; et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015, 17, 405–424. [CrossRef] [PubMed] 16. Brandt, T.; Sack, L.M.; Arjona, D.; Tan, D.; Mei, H.; Cui, H.; Gao, H.; Bean, L.J.H.; Ankala, A.; Del Gaudio, D.; et al. Adapting ACMG/AMP sequence variant classiﬁcation guidelines for single-gene copy number variants. Genet. Med. 2019, 22, 336–344. [CrossRef] 17. Jennings, L.J.; Arcila, M.E.; Corless, C.; Kamel-Reid, S.; Lubin, I.M.; Pfeifer, J.; Temple-Smolkin, R.L.; Voelkerding, K.V.; Nikiforova, M.N. Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists. J. Mol. Diagn. 2017, 19, 341–365. [CrossRef] 18. Xu, Y.; Jiang, H.; Tyler-Smith, C.; Xue, Y.; Jiang, T.; Wang, J.; Wu, M.; Liu, X.; Tian, G.; Wang, J.; et al. Comprehensive comparison of three commercial human whole-exome capture platforms. Genome Biol. 2011, 12, R95. [CrossRef] 19. Aradhya, S.; Lewis, R.; Bonaga, T.; Nwokekeh, N.; Staﬀord, A.; Boggs, B.; Hruska, K.; Smaoui, N.; Compton, J.G.; Richard, G.; et al. Exon-level array CGH in a large clinical cohort demonstrates increased sensitivity of diagnostic testing for Mendelian disorders. Genet. Med. 2012, 14, 594–603. [CrossRef] 20. D’Haene, B.; Meire, F.; Claerhout, I.; Kroes, H.Y.; Plomp, A.; Arens, Y.H.; de Ravel, T.; Casteels, I.; De Jaegere, S.; Hooghe, S.; et al. Expanding the spectrum of FOXC1 and PITX2 mutations and copy number changes in patients with anterior segment malformations. Investig. Ophthalmol. Vis. Sci. 2011, 52, 324–333. [CrossRef] 21. References Ianakiev, P.; Kilpatrick, M.W.; Toudjarska, I.; Basel, D.; Beighton, P.; Tsipouras, P. Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am. J. Hum. Genet. 2000, 67, 59–66. [CrossRef] [PubMed] 22. Lesnik Oberstein, S.A.; Kriek, M.; White, S.J.; Kalf, M.E.; Szuhai, K.; den Dunnen, J.T.; Breuning, M.H.; Hennekam, R.C. Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase. Am. J. Hum. Genet. 2006, 79, 562–566. [CrossRef] [PubMed] 23. Weh, E.; Reis, L.M.; Happ, H.C.; Levin, A.V.; Wheeler, P.G.; David, K.L.; Carney, E.; Angle, B.; Hauser, N.; Semina, E.V. Whole exome sequence analysis of Peters anomaly. Hum. Genet. 2014, 133, 1497–1511. [CrossRef] [PubMed] 24. Paznekas, W.A.; Boyadjiev, S.A.; Shapiro, R.E.; Daniels, O.; Wollnik, B.; Keegan, C.E.; Innis, J.W.; Dinulos, M.B.; Christian, C.; Hannibal, M.C.; et al. Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am. J. Hum. Genet. 2003, 72, 408–418. [CrossRef] 25. Belmouden, A.; Melki, R.; Hamdani, M.; Zaghloul, K.; Amraoui, A.; Nadiﬁ, S.; Akhayat, O.; Garchon, H.J. A novel frameshift founder mutation in the cytochrome P450 1B1 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco. Clin. Genet. 2002, 62, 334–339. [CrossRef] [PubMed] 26. Quiroz-Casian, N.; Chacon-Camacho, O.F.; Barragan-Arevalo, T.; Nava-Valdez, J.; Lieberman, E.; Salgado-Medina, A.; Navas, A.; Graue-Hernandez, E.O.; Zenteno, J.C. Sclerocornea-Microphthalmia-Aphakia Complex: Description of Two Additional Cases Associated With Novel FOXE3 Mutations and Review of the Literature. Cornea 2018, 37, 1178–1181. [CrossRef] 27. Huang, X.; Xiao, X.; Jia, X.; Li, S.; Li, M.; Guo, X.; Liu, X.; Zhang, Q. Mutation analysis of the genes associated with anterior segment dysgenesis, microcornea and microphthalmia in 257 patients with glaucoma. Int. J. Mol. Med. 2015, 36, 1111–1117. [CrossRef] 28. Patel, A.; Hayward, J.D.; Tailor, V.; Nyanhete, R.; Ahlfors, H.; Gabriel, C.; Jannini, T.B.; Abbou-Rayyah, Y.; Henderson, R.; Nischal, K.K.; et al. The Oculome Panel Test: Next-Generation Sequencing to Diagnose a Diverse Range of Genetic Developmental Eye Disorders. Ophthalmology 2019, 126, 888–907. [CrossRef] 29. Ioannidis, N.M.; Rothstein, J.H.; Pejaver, V.; Middha, S.; McDonnell, S.K.; Baheti, S.; Musolf, A.; Li, Q.; Holzinger, E.; Karyadi, D.; et al. REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants. Am. J. Hum. Genet. 2016, 99, 877–885. [CrossRef] Genes 2020, 11, 350 10 of 10 10 of 10 30. Choi, A.; Lao, R.; Ling-Fung Tang, P.; Wan, E.; Mayer, W.; Bardakjian, T.; Shaw, G.M.; Kwok, P.Y.; Schneider, A.; Slavotinek, A. © 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/). References Novel mutations in PXDN cause microphthalmia and anterior segment dysgenesis. Eur. J. Hum. Genet. 2015, 23, 337–341. [CrossRef] 31. Khan, K.; Rudkin, A.; Parry, D.A.; Burdon, K.P.; McKibbin, M.; Logan, C.V.; Abdelhamed, Z.I.; Muecke, J.S.; Fernandez-Fuentes, N.; Laurie, K.J.; et al. Homozygous mutations in PXDN cause congenital cataract, corneal opacity, and developmental glaucoma. Am. J. Hum. Genet. 2011, 89, 464–473. [CrossRef] 32. Bougeard, G.; Hadj-Rabia, S.; Faivre, L.; Sarafan-Vasseur, N.; Frebourg, T. The Rapp-Hodgkin syndrome results from mutations of the TP63 gene. Eur. J. Hum. Genet. 2003, 11, 700–704. [CrossRef] 33. Salinas, C.F.; Montes, G.M. Rapp-Hodgkin syndrome: Observations on ten cases and characteristic hair changes (pili canaliculi). Birth Defects Orig. Artic. Ser. 1988, 24, 149–168. [PubMed] 34. Chatterjee, M.; Neema, S.; Mukherjee, S. Rapp Hodgkin Syndrome. Indian Dermatol. Online J. 2017, 8, 215–216. [CrossRef] [PubMed] 35. Gonzalez, F.; Loidi, L.; Abalo-Lojo, J.M. Novel variant in the TP63 gene associated to ankyloblepharon-ectodermal dysplasia-cleft lip/palate (AEC) syndrome. Ophthalmic Genet. 2017, 38, 277–280. [CrossRef] [PubMed] 36. Sutton, V.R.; van Bokhoven, H. TP63-Related Disorders. In GeneReviews ((R)); Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J.H., Stephens, K., Amemiya, A., Eds.; University of Washington: Seattle, WA, USA, 1993. 37. Sutton, V.R.; Plunkett, K.; Dang, D.X.; Lewis, R.A.; Bree, A.F.; Bacino, C.A. Craniofacial and anthropometric phenotype in ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (Hay-Wells syndrome) in a cohort of 17 patients. Am. J. Med. Genet. A 2009, 149, 1916–1921. [CrossRef] [PubMed] 38. Simpson, A.; Avdic, A.; Roos, B.R.; DeLuca, A.; Miller, K.; Schnieders, M.J.; Scheetz, T.E.; Alward, W.L.; Fingert, J.H. LADD syndrome with glaucoma is caused by a novel gene. Mol. Vis. 2017, 23, 179–184. [PubMed] © 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/W4212851080	https://eprints.gla.ac.uk/265765/1/265765.pdf	English	null	Investigating the uptake, effectiveness and safety of COVID-19 vaccines: protocol for an observational study using linked UK national data	BMJ open	2,022	cc-by	14,009	Strengths and limitations of this study Introduction The novel coronavirus SARS-CoV-2, which emerged in December 2019, has caused millions of deaths and severe illness worldwide. Numerous vaccines are currently under development of which a few have now been authorised for population-level administration by several countries. As of 20 September 2021, over 48 million people have received their first vaccine dose and over 44 million people have received their second vaccine dose across the UK. We aim to assess the uptake rates, effectiveness, and safety of all currently approved COVID-19 vaccines in the UK. copyright. on February 22, 2022 at University of Glasgow. http://bmjopen.bmj.com/ Downloaded from copyright. on February 22, 2022 at University of Glasgow. http://bmjopen.bmj.com/ Downloaded from copyright. on February 22, 2022 at University of Glasgow. http://bmjopen.bmj.com/ Downloaded from copyright. on February 22, 2022 at University of Glasgow. http://bmjopen.bmj.com/ Downloaded from copyright. on February 22, 2022 at University of Glasgow. http://bmjopen.bmj.com/ Downloaded from ►We will use national data for each UK nation and across the UK general population. ►Rapid and robust real-time estimates on uptake, ef- fectiveness and safety of COVID-19 vaccines will be provided using data from existing national pandemic platforms in the UK. ►This is an observational study and analyses are, therefore, potentially susceptible to residual or un- measured confounders. ►This is an observational study and analyses are, therefore, potentially susceptible to residual or un- measured confounders. ►Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2021-050062). on February 22, 2022 at University of Glasgow. Protected by open.bmj.com/ on February 22, 2022 at University of Glasgow. Protected by open.bmj.com/ on February 22, 2022 at University of Glasgow. Protected by open.bmj.com/ on February 22, 2022 at University of Glasgow. Protected by open.bmj.com/ on February 22, 2022 at University of Glasgow. Protected by open.bmj.com/ Methods and analysis We will use prospective cohort study designs to assess vaccine uptake, effectiveness and safety against clinical outcomes and deaths. Test- negative case–control study design will be used to assess vaccine effectiveness (VE) against laboratory confirmed SARS-CoV-2 infection. Self-controlled case series and retrospective cohort study designs will be carried out to assess vaccine safety against mild-to-moderate and severe adverse events, respectively. Individual-level pseudonymised data from primary care, secondary care, laboratory test and death records will be linked and analysed in secure research environments in each UK nation. BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 Protocol Open access Investigating the uptake, effectiveness and safety of COVID-19 vaccines: protocol for an observational study using linked UK national data Eleftheria Vasileiou ‍ ‍ ,1 Ting Shi ‍ ‍ ,1 Steven Kerr,1 Chris Robertson,2,3 Mark Joy,4 Ruby Tsang ‍ ‍ ,4 Dylan McGagh,4 John Williams,4 Richard Hobbs,4 Simon de Lusignan ‍ ‍ ,4 Declan Bradley,5 Dermot OReilly,5 Siobhan Murphy,5 Antony Chuter,6 Jillian Beggs,6 David Ford,7 Chris Orton ‍ ‍ ,7 Ashley Akbari ‍ ‍ ,7 Stuart Bedston,7 Gareth Davies,7 Lucy J Griffiths ‍ ‍ ,7 Rowena Griffiths,7 Emily Lowthian ‍ ‍ ,7 Jane Lyons ‍ ‍ ,7 Ronan A Lyons ‍ ‍ ,7 Laura North,7 Malorie Perry,8 Fatemeh Torabi,7 James Pickett,9 Jim McMenamin,3 Colin McCowan,10 Utkarsh Agrawal,10 Rachael Wood ‍ ‍ ,1,11 Sarah Jane Stock ‍ ‍ ,1,11 Emily Moore,3 Paul Henery ‍ ‍ ,12 Colin R Simpson,1,13 Aziz Sheikh 1 To cite: Vasileiou E, Shi T, Kerr S, et al. Investigating the uptake, effectiveness and safety of COVID-19 vaccines: protocol for an observational study using linked UK national data. BMJ Open 2022;12:e050062. doi:10.1136/ bmjopen-2021-050062 Strengths and limitations of this study Univariate and multivariate logistic regression models will be carried out to estimate vaccine uptake levels in relation to various population characteristics. VE estimates against laboratory confirmed SARS-CoV-2 infection will be generated using a generalised additive logistic model. Time-dependent Cox models will be used to estimate the VE against clinical outcomes and deaths. The safety of the vaccines will be assessed using logistic regression models with an offset for the length of the risk period. Where possible, data will be meta-analysed across the UK nations. Ethics and dissemination We obtained approvals from the National Research Ethics Service Committee, Southeast Scotland 02 (12/SS/0201), the Secure Anonymised Information Linkage independent Information Governance Review Panel project number 0911. Concerning English data, University of Oxford is compliant with the General Data Protection Regulation and the National Health Service (NHS) Digital Data Security and Protection Policy. This is an approved study (Integrated Research Application ID 301740, Health Research Authority (HRA) Research Ethics Committee 21/HRA/2786). The Oxford-Royal College of General Practitioners Clinical Informatics Digital Hub meets NHS Digital’s Data Security and Protection Toolkit requirements. In Northern Ireland, the project was approved by the Honest Broker Governance Board, project number 0064. Findings will be made available to national policy-makers, presented at conferences and published in peer-reviewed journals. Received 09 February 2021 Accepted 19 January 2022 ebruary 22, 2022 at University of Glasgow. Protected by ebruary 22, 2022 at University of Glasgow. Protected by ebruary 22, 2022 at University of Glasgow. Protected by ebruary 22, 2022 at University of Glasgow. Protected by ebruary 22, 2022 at University of Glasgow. Protected by For numbered affiliations see end of article. Correspondence to Dr Eleftheria Vasileiou; eleftheria.vasileiou@ed.ac.uk © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ. Investigating the uptake, effectiveness and safety of COVID-19 vaccines: protocol for an observational study using linked UK national data copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. http://bmjo ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. http://bmjo ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. http://bmjo ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. http://bmjo ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. http://bmjo ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Eleftheria Vasileiou ‍ ‍ ,1 Ting Shi ‍ ‍ ,1 Steven Kerr,1 Chris Robertson,2,3 Mark Joy,4 Ruby Tsang ‍ ‍ ,4 Dylan McGagh,4 John Williams,4 Richard Hobbs,4 Simon de Lusignan ‍ ‍ ,4 Declan Bradley,5 Dermot OReilly,5 Siobhan Murphy,5 Antony Chuter,6 Jillian Beggs,6 David Ford,7 Chris Orton ‍ ‍ ,7 Ashley Akbari ‍ ‍ ,7 Stuart Bedston,7 Gareth Davies,7 Lucy J Griffiths ‍ ‍ ,7 Rowena Griffiths,7 Emily Lowthian ‍ ‍ ,7 Jane Lyons ‍ ‍ ,7 Ronan A Lyons ‍ ‍ ,7 Laura North,7 Malorie Perry,8 Fatemeh Torabi,7 James Pickett,9 Jim McMenamin,3 Colin McCowan,10 Utkarsh Agrawal,10 Rachael Wood ‍ ‍ ,1,11 Sarah Jane Stock ‍ ‍ ,1,11 Emily Moore,3 Paul Henery ‍ ‍ ,12 Colin R Simpson,1,13 Aziz Sheikh ‍ ‍ 1 copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from p p The Pfizer-BioNTech and Oxford-AstraZeneca were among the first vaccines approved by national regulatory authorities such as the UK’s Medicines and Healthcare products Regulatory Agency.5 Based on prelicensure clin- ical trials, the Pfizer-BioNTech vaccine was 95% effective at preventing laboratory-confirmed COVID-19 illness in individuals without evidence of previous infection.6 The Oxford-AstraZeneca vaccine has also reported significant efficacy of 64% and 70% after one and two doses, respec- tively, against symptomatic disease.7 As of the 8 January 2021, the UK has also approved a third COVID-19 vaccine manufactured by Moderna which has shown 94% efficacy against confirmed SARS-CoV-2 infection after receipt of second dose.8 All three vaccines were well tolerated with mild-to-moderate side effects mostly reported.6–8 In March 2021, the European Medicines Agency reported extremely rare but serious side effects including blood clots and bleeding following administration of the Oxford-AstraZeneca vaccine.9 10 As a result, the Joint Committee on Vaccination and Immunisation (JCVI) of the UK Government has recommended that healthy adults aged 18–39 years old should not be offered the Oxford-AstraZeneca vaccine but the Pfizer-BioNTech or Moderna vaccines instead.11 Rare cases of cardiac inflam- mation including myocarditis and pericarditis following immunisation with the Pfizer-BioNTech and Moderna vaccines have also been reported12 and are thus closely monitored by national and international regulatory agencies. on February 22, 2022 at University of Glasgow. Protected by mj.com/ As of 20 September 2021, over 48 million people have received their first vaccine dose and over 44 million people have received their second vaccine dose across the UK13 based on JCVI’s vaccination priority list which targets those most at risk of severe COVID-19 illness (eg, older adults and people with comorbidities).14 INTRODUCTION Correspondence to Dr Eleftheria Vasileiou; eleftheria.vasileiou@ed.ac.uk The first human cases infected by the novel coronavirus SARS-CoV-2 pathogen were Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 1 Open access events. These postmarketing observational studies will add additional value to the prelicensure clinical trials as they can assess real-life effects of the COVID-19 vaccines and the impact of the vaccination programme at popula- tion levels.15 All UK-licensed vaccines have demonstrated high efficacy in clinical trials; however, more evidence is needed about the type, level and duration of protection for different segments of the population. The recom- mended time period between administration of the first and second doses of the Pfizer-BioNTech6 and Oxford- AstraZeneca7 vaccines are 3 and 6 weeks, respectively. The UK government decided to lengthen this gap to up to 12 weeks for both vaccines.14 This is because of the desire to provide some degree of protection to as many people as possible and the decision was supported by findings from the Oxford-AstraZeneca vaccine trial, which have shown that vaccine efficacy was higher (65%) in the group that was administered the second dose more than 6 weeks after the first dose, compared with the group given the second dose less than 6 weeks after the first dose (53%).7 16 A simu- lation study has also shown that high vaccine coverage even with less efficacious vaccines (due to partial immu- nisation) can lead to a greater reduction of SARS-CoV-2 infection levels compared with a lower vaccine coverage with more efficacious vaccines.17 On the other hand, some experts have expressed their concerns that delay between doses could increase the risk of vaccine-resistant strains emerging due to a partially immunised population.16 The provision of timely estimates on the protection conferred between doses is thus urgently needed. The assessment of vaccine-induced adverse events also needs to be carried out, particularly for rare adverse events that are usually only detectable in large population studies.18 The rate of vaccine receipt by demographic, socioeconomic and other epidemiological characteristics also needs to be measured. INTRODUCTION In the UK, the ‘Understanding Society’ COVID-19 survey asked 12 035 participants (in November 2020) their likelihood of vaccine uptake and reason for hesitancy.19 High levels of hesitancy were found in women (21%), younger adults aged 16–24 years old (27%), those with lower education levels (19%) and in black (72%) and Pakistani/Bangladeshi (42%) ethnic groups.19 This is particularly concerning given that ethnic minority groups are some of the subgroups of the population that have been most at risk in this pandemic.19 The identification of key factors related to vaccine uptake may be useful in efforts to increase uptake and subsequently maximise the impact of the COVID-19 vaccination programme in the UK. detected in Wuhan, China in December 2019.1 On 11 March 2020, WHO declared the COVID-19 as a global pandemic, which as of 20 September 2021 has caused more than 228 million infections and four million deaths worldwide.1 The need for vaccines against this novel virus triggered an emergency response by governments, pharmaceutical companies and research institutions to develop, licence and manufacture COVID-19 vaccines at scale. Dozens of COVID-19 vaccines are currently under development with some vaccines now having successfully completed their prelicensure clinical trials and been approved for population vaccine administration.2 3 The speed with which the world’s first vaccines3 were available for mass administration at the end of 2020 is unprece- dented given that typically it takes years for a vaccine to be available for use at a population level.4 events. These postmarketing observational studies will add additional value to the prelicensure clinical trials as they can assess real-life effects of the COVID-19 vaccines and the impact of the vaccination programme at popula- tion levels.15 All UK-licensed vaccines have demonstrated high efficacy in clinical trials; however, more evidence is needed about the type, level and duration of protection for different segments of the population. The recom- mended time period between administration of the first and second doses of the Pfizer-BioNTech6 and Oxford- AstraZeneca7 vaccines are 3 and 6 weeks, respectively. INTRODUCTION The UK government decided to lengthen this gap to up to 12 weeks for both vaccines.14 This is because of the desire to provide some degree of protection to as many people as possible and the decision was supported by findings from the Oxford-AstraZeneca vaccine trial, which have shown that vaccine efficacy was higher (65%) in the group that was administered the second dose more than 6 weeks after the first dose, compared with the group given the second dose less than 6 weeks after the first dose (53%).7 16 A simu- lation study has also shown that high vaccine coverage even with less efficacious vaccines (due to partial immu- nisation) can lead to a greater reduction of SARS-CoV-2 infection levels compared with a lower vaccine coverage with more efficacious vaccines.17 On the other hand, some experts have expressed their concerns that delay between doses could increase the risk of vaccine-resistant strains emerging due to a partially immunised population.16 The provision of timely estimates on the protection conferred between doses is thus urgently needed. The assessment of vaccine-induced adverse events also needs to be carried out, particularly for rare adverse events that are usually only detectable in large population studies.18 The rate of vaccine receipt by demographic, socioeconomic and other epidemiological characteristics also needs to be measured. In the UK, the ‘Understanding Society’ COVID-19 survey asked 12 035 participants (in November 2020) their likelihood of vaccine uptake and reason for hesitancy.19 High levels of hesitancy were found in women (21%), younger adults aged 16–24 years old (27%), those with lower education levels (19%) and in black (72%) and Pakistani/Bangladeshi (42%) ethnic groups.19 This is particularly concerning given that ethnic minority groups are some of the subgroups of the population that have been most at risk in this pandemic.19 The identification of key factors related to vaccine uptake may be useful in efforts to increase uptake and subsequently maximise the impact of the COVID-19 vaccination programme in the UK. Overview of linked databases and study population We will use pseudonymised individual level data routinely collected at primary and secondary care visits, linked with mortality, laboratory and vaccination data across the UK. Unique national datasets will be developed and hosted within secure research environments in each UK nation with standardised individual-level analyses run across data- sets. Pooled estimates across the UK nations will also be calculated. For England, data from the Royal College of General Practitioners Research and Surveillance Centre (RCGP RSC) (approximately 5.4 million people)23 will be used. We will also access and analyse national coverage data from Northern Ireland (approximately 1.9 million people). For Scotland, data will be derived from the Early Pandemic Evaluation and Enhanced Surveillance of COVID-19 (approximately 5.4 million people).24 For Wales, data from the Controlling COVID-19 through enhanced population surveillance and intervention (ConCOV) (approximately 3.2 million people)25 26 and the Secure Anonymised Information Linkage (SAIL) Databank27 will be used. See table 1 for details on data sources from each UK nation. Aim and objectives Timely postlicensure monitoring of coverage, protec- tion and safety of these newly introduced vaccines is imperative.15 Specifically, robust observational epidemi- ological studies are required to measure coverage rates in the population in relation to demographic and other population characteristics, assess effectiveness against infection, severe illness and deaths, and to detect adverse The aims of this study are to assess the uptake, effec- tiveness, and safety of the currently licensed COVID-19 vaccines (Pfizer-BioNTech, Oxford-AstraZeneca and Moderna) in the UK using linked healthcare and admin- istrative data. We will also seek to assess any additional licensed vaccines during the course of this study. Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 2 copyright. on February 22, 2022 at University of Gla http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access (NHS) Digital Data Security and Protection approved method.29 30 NHS Digital will link additional data to these pseudonyms. (NHS) Digital Data Security and Protection approved method.29 30 NHS Digital will link additional data to these pseudonyms. Our primary objectives are to: (1) measure variation in vaccine uptake in relation to population characteris- tics; (2) assess vaccine effectiveness (VE) against infec- tion, transmission,20 severe outcomes and deaths; and (3) identify the risk of adverse events following immunisation (AEFIs) in each UK nation. Our secondary objectives are to provide UK-wide pooled estimates of each primary objective. Wales Withing SAIL and in collaboration with the ConCOV project,25 26 data will be linked and anonymised from NHS sources via a mature split-file system. All identifiable data will stay within the NHS, and will be linked to the Welsh Demographic Service Dataset (WDSD), pseudony- mised and encrypted within NHS Wales by Digital Health and Care Wales, with pseudonymised demographics supplied to SAIL at Swansea University,27 where the non- identifiable clinical data are held. SAIL will link the demo- graphic data to relevant clinical data, and then further encrypt the linked data before presenting to research teams within a secure virtual desktop. Scotland For the Scottish data, Public Health Scotland (PHS) will carry out the data linkage and offer a secure environment for researchers to access and analyse the pseudonymised individual level data.24 The Community Health Index number (a unique identifier for each resident receiving healthcare) will be used to link individuals’ data which will be replaced by a study ID. Exposure (vaccination) data England In England, data on vaccination will derive from general practitioners (GPs) and the National Immunisation Management Service (NIMS).31 NIMS is the System of Record for the NHS COVID-19 vaccination programme in England developed by NHS Digital.31 NIMS will collect any demographic, GP and employee (for NHS) data to identify groups of the population eligible for vaccination. Data collected from NIMS will also feed back into GP systems so that an individual’s electronic health record is updated regarding to their vaccination history.31 Vaccina- tion data in GP records will be recorded using the System- atised Nomenclature of Medicine (SNOMED) Clinical Terms (CT).32 RCGP RSC now uses SNOMED CT for all its key variables including vaccine data.33 A key part of these curated variables are those for COVID-19; there have been three iterations of these and we have carefully curated case definitions.34–36 on February 22, 2022 at University of Glasgow. Protected by mj.com/ METHODS Study design A prospective cohort study design will be used to measure variations in vaccine uptake and assess VE against severe illness, deaths, secondary SARS-CoV-2 infection due to household transmission. A test-negative design (TND) case-control study will be carried out to assess VE against laboratory confirmed COVID-19 infection. In the TND, cases and controls will be those with a positive and nega- tive test for COVID-19, respectively.21 A self-controlled case series (SCCS) study design will be used to assess the risk of AEFI. The SCCS study will be used to deter- mine the relative incidence of adverse events for exposed time periods (periods following vaccine administration) compared with unexposed time periods (prevaccination or postvaccination periods unrelated to vaccination) in individuals who present with the outcome of interest.22 For more severe and event dependent safety outcomes, a retrospective cohort study will be considered. England English primary care data will be held in the Oxford- Royal College of General Practitioners Clinical Infor- matics Digital Hub.28 Data will be pseudonymised as close to source as possible using an National Health Service Northern Ireland copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from For the Northern Irish data, the Honest Broker Service (HBS) will carry out the data linkage and offer a secure environment for researchers to access and analyse the pseudonymised individual-level data. The Healthcare Number will be used to link individuals’ data though replaced with an anonymous study ID in the analysis dataset. copyright. on February 22, 2022 at Un http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access Table 1 Available UK datasets for each data item of interest Data item England Northern Ireland Scotland Wales Exposures Pfizer-BioNTech vaccine GP, NIMS VMS GP, TVMT/PHS, SIRS CVVD Oxford-AstraZeneca vaccine GP, NIMS VMS GP, TVMT/PHS, SIRS CVVD Moderna vaccine GP, NIMS VMS GP, TVMT/PHS, SIRS CVVD Outcomes Laboratory confirmed SARS-CoV-2 infection GP, Pillar 1 and 2 SGSS, PHE Pillar 1 and Pillar 2 ECOSS PATD (Pillar 1, 2 and 3 data from all NHS and private labs), CVLF testing and results data COVID-19-related GP consultation GP NA GP WLGP COVID-19-related emergency department consultation GP, ECDS Symphony, NIRAES SMR01 EDDD and EDDS COVID-19-related hospital admission GP, SUS Admissions and discharge dataset SMR01 PEDW COVID-19-related ICU admission GP, CHESS Admissions and discharge dataset SICSAG CDDS, ICCD and ICNC COVID-19-related death GP, ONS, SSRS NHAIS NRS ADDE and ADDE (ONS mortality), CDDS and WDSD Secondary SARS-CoV-2 infection due to household transmission RCGP RSC household key Pillar 1 and Pillar 2 dataset ECOSS PATD (Pillar 1, 2 and 3 data from all NHS and private labs), CVLF testing and results data Maternity outcomes GP, MSDS NIMATS COPS study ADBE (ONS births), MIDS and NCCH Patient characteristics and confounders Age GP NHAIS GP C19_COHORT20 Sex GP NHAIS GP C19_COHORT20 Socioeconomic status Postal code to IMD NHAIS GP C19_COHORT20 Ethnicity GP, SUS VMS Census 2011 National ethnicity spine (made up of 20 EHR data sources and the ONS Census 2011) Underlying medical condition GP EPD GP GP, WLGP, PEDW, CVSP Type of settlement (urban/rural) GP NHAIS GP C19_COHORT20 Type of settlement (eg, private home, care home or social housing) GP NHAIS GP C19_COHORT20, CARE Smoking status GP NA GP WLGP Body Mass Index GP NA GP WLGP Prescribed medications GP EPD GP, PIS, HEPMA WLGP, WDDS Other non-COVID-19 vaccines (eg, influenza, pneumococcal) GP VMS GP WLGP, NCCH Occupation (eg, healthcare workers, front- line workers, essential workers) GP where recorded Pillar 1 and 2 To be confirmed HWRA, SWAC History of healthcare utilisation (eg, GP consultations, hospital admissions) GP, SUS Admissions and discharge dataset GP, SMR01 PEDW, WLGP ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS, Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database; GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS, National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19 Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS, Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database; GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS, National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19 Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS, Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database; GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS, National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19 Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. copyright. on February 22, 2022 at Un http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of G http://bmjopen.bmj.com/ MJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ ed as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS, Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database; GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS, National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19 Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. Northern Ireland In Northern Ireland, GP practices and Trust vaccination sites are currently delivering the COVID-19 vaccination 3 Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 copyright. on February 22, 2022 at University of Glasgow http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Laboratory confirmed outcomes England In Northern Ireland, the Admissions and Discharge dataset will be used to collate data relating to admitted patient care delivered by HSC hospitals in Northern Ireland, generated by the patient administration systems within each hospital. These data are held centrally by the HSC Regional Data Warehouse.43 For England, most community testing (called Pillar 2) is in the GP record, though for this study we will addi- tionally link to resources held by NHS Digital this is the Second Generation Surveillance System, this also included hospital tests.39 copyright. on February 22, 2022 at University of Glasgow http://bmjopen.bmj.com/ wnloaded from Scotland For Northern Ireland, the Pillar 1 dataset is extracted from the Laboratory Information Systems in Northern Ireland hospitals on a daily basis into a central repository in the Health and Social Care (HSC) Regional Data Warehouse, which is maintained by the HSC Business Services Organ- isation.39 It contains details of COVID-19 antigen tests carried out in each of the hospital laboratories, including those processed on behalf of primary care, social care and community settings. Pillar 2 data are processed by NHS Digital and extracts for NI residents are sent to the NI HSC Regional Data Warehouse.39 In Scotland, data on patients receiving care in general or acute hospitals are recorded in the Scottish Morbidity Record 01 (SMR01).24 The International Statistical Clas- sification of Diseases and Related Health Problems, 10th Revision (ICD-10) codes are used to index any diagnoses recorded in the patient’s medical notes by a clinician.24 Consistent and high level (>90%) of data accuracy have been shown in recent data quality reports for the SMR01 database.20 Data on adult patients admitted to general intensive care units (ICUs) will derive from the Scottish Intensive Care Society Audit Group database.24 on February 22, 2022 at University of Glasgow. Protected by j.com/ copyright. on February 22, 2022 at University of Glas http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access Clinical outcomes arising from a patient consultation.24 Additional data on vaccination will also be available via the Turas Vaccination Management Tool (TVMT), which is a web-based tool that enables front-line vaccinators to capture and create real-time patient vaccination records. PHS is currently collating vaccination data from Turas.38 Scheduled vacci- nations for children may also be recorded in the Scottish Immunisation Recall System (SIRS) database. Data on vaccinations administered at schools and not in GPs may thus derive from SIRS.24 arising from a patient consultation.24 Additional data on vaccination will also be available via the Turas Vaccination Management Tool (TVMT), which is a web-based tool that enables front-line vaccinators to capture and create real-time patient vaccination records. PHS is currently collating vaccination data from Turas.38 Scheduled vacci- nations for children may also be recorded in the Scottish Immunisation Recall System (SIRS) database. Data on vaccinations administered at schools and not in GPs may thus derive from SIRS.24 Data on primary care consultations for COVID-19 illness will be accessed via GP electronic health records in each UK nation. copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from England In England, we will link to secondary care data through collections held by NHS Digital. Hospital data are held in two forms: Hospital Episode Statistics (HES), which are the long-term validated record; and Secondary Uses Services which is an extract of contemporary operational data which after validation will become HES. We plan to use HES and also have an extract of intensive care data. We will access these data through NHS Digital’s Data Access Request Service.40 We also have SARS-CoV-2 virology data that were collected through the sentinel surveillance system.41 The English primary care sentinel system within RCGP RSC has a strong working relation- ship with Public Health England (PHE), with whom we have worked closely for over half a century.42 Scotland In Wales, data on all interactions with secondary care including Accident and Emergency (A&E) events (Emer- gency Department Dataset), inpatient hospital admis- sions (Patient Episode Database for Wales), intensive care (Critical Care DataSet—CDDS and Intensive Care National Audit and Research Centre) and outpatient appointments and activity (Outpatient Database for Wales) are all available within SAIL Databank.27 There are also a collection of specialised services and condition specific secondary care such as cancer which are available within SAIL.27 For Scotland, laboratory results from Scottish diagnostic and reference laboratories are captured by the Electronic Communication of Surveillance in Scotland (ECOSS) database, which can be used for surveillance and research purposes.24 Laboratory results from NHS and community (Lighthouse laboratory) testing centres will also be avail- able through ECOSS. Wales In Wales, the vaccine programme is administered and recorded nationally in the all Wales Immunisation System (WIS) and is available in SAIL through the COVID-19 Vaccine Data. This is a separate independent system and data source to the GP data, which is also available in SAIL with vaccination recorded using Read codes.27 Scotland programme. Vaccination data from GP practices and Trust vaccination sites are stored in a central Vaccination Management System and made available via the HBS secure research environment.37 In Scotland, GPs usually facilitate vaccination programmes and record any data related to vaccine administration. GPs use Read codes to code and record relevant information Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 4 Wales For Wales, national coverage of Pillar 1, 2 and 3 data from all NHS and private laboratories will be available, as well as national lateral flow testing data.39 We will also pursue to access genome sequencing data in a proportion of laboratory tests positive for SARS- CoV-2 available from national sequencing centres where possible. Deaths England In England, NHS Digital’s Personal Demographic Service flags the date of death in the GP record. We have previously Deaths England In England, NHS Digital’s Personal Demographic Service flags the date of death in the GP record. We have previously 5 Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 copyright. on February http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at University of Glas http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access Community Child Health and Maternity Indicators DataSet. Data sources for Wales will be sought via the SAIL Databank.27 Community Child Health and Maternity Indicators DataSet. Data sources for Wales will be sought via the SAIL Databank.27 used these data of death to report excess mortality, both overall44 45 and in people with known COVID-19 status.46 We will augment these data with the certificated cause of death provided by the Office for National Statistics (ONS), which will be linked for us at individual pseud- onymised patient level by NHS Digital. used these data of death to report excess mortality, both overall44 45 and in people with known COVID-19 status.46 We will augment these data with the certificated cause of death provided by the Office for National Statistics (ONS), which will be linked for us at individual pseud- onymised patient level by NHS Digital. Northern Ireland In Northern Ireland, the National Health Applications and Infrastructure Services (NHAIS) will be used for mortality data.47 NHAIS receives regular updates from General Register Office on fact and cause of death. ICD-10 codes on deaths records derive from diagnoses recorded by the certifying doctor on the death certificate.47 Exposure definitions copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ ublished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from i Data on the currently licensed COVID-19 vaccines, including Pfizer-BioNTech, Oxford-AstraZeneca and Moderna, will be derived from GPs, NIMS, TVMT/PHS, WIS and HSC Trusts databases. For the first vaccine dose (partial vaccination), an individual will be defined as exposed or vaccinated from day 14 after receiving the first dose between the period of 8 December 2020 and until the end of follow-up. For the second vaccine dose (full vaccination), an individual will be defined as exposed or vaccinated from day 14 after receiving the second dose during the study period. Exposed or vaccinated groups will be stratified by the following time intervals: (1) 0–13 days after dose 1; (2) 14–20 days after dose 1; (3) 21–27 days after dose 1; (4) 28–34 days after dose 1; (5) 35–41 days after dose 1; (6) >42 days after dose 1; (7) 0–13 days after dose 2 and (8) >14 days after dose 2. England In England, we will use a customised ‘sliding window’ to capture pregnancy data.49 Specifically, an algorithm that accurately inferred pregnancies will be used by adopting an ontological approach for case finding.49 The ontolog- ical approach will thus be used to identify pregnancies and associated complications using a systematic approach to derive this information from routinely collected admin- istrative health data which will be available via the RCGP RSC.49 on February 22, 2022 at University of Glasgow. Protected by mj.com/ Northern Ireland In Northern Ireland, data will be accessed via the Northern Ireland Maternity System.50 Pregnancy and neonatal outcomes Capturing vaccine exposure in pregnancy is important.48 As no trial to date has included pregnant women this type of study is the only opportunity to explore safety and effectiveness in pregnant women and their babies. Scotland In Scotland, the death registry within National Records of Scotland records information included in the death certificates.24 ICD-10 codes on deaths records derive from diagnoses recorded by the certifying doctor on the death certificate.24 y y Controls or unvaccinated will be defined as those who have not yet received a COVID-19 vaccine or have only received one vaccine dose. Controls who become vaccinated with any vaccine (ie, including one dose of Moderna) or receive a second vaccine will then be assigned within the exposure group. As a result, follow-up of the exposure period will be censored for both the vaccinated and control recipient if the control meets the criteria to be classified as exposed (receiving a first dose when compared with the unvaccinated group and receiving a second dose when compared with the partially/one dose vaccinated group). Maximum follow-up period will correspond to the latest event date depending on the outcome of interest. Similar vaccinated and unvaccinated groups and periods will be determined for the Moderna vaccine.52 Scotland In Scotland, pregnancy and neonatal outcomes in Scot- tish participants will be identified through the COVID-19 in Pregnancy in Scotland study.51 VE outcomes Wales In Wales, multiple sources of mortality data including information from the National Population Spine (WDSD), ONS death data (ADDE and ADDD) and a national NHS master patient index record (Consolidated Death Data Source—CDDS) will be accessed to retrieve complete, harmonised details on cause and associated mortality details.27 Pregnancy and neonatal outcomes Adverse events following immunisation Adverse events to be monitored are derived from the safety results of the prelicensure vaccine clinical trials, common side effects related to influenza vaccines and an unpublished study protocol of an ongoing observational study.6 7 53 54 These include use of health services such as GP or out-of-hours GP consultation, A&E department attendance, inpatient hospital admission and admission to ICU for suspected adverse events. Safety of vaccines in pregnant women will also be considered once vaccines are widely administered in this group of the population. AEFI by specific vaccine type will also be considered. A full list of candidate AEFI is available in in online supple- mental material, appendices 1 and 2. Northern Ireland In Northern Ireland, data will be accessed via the Northern Ireland Maternity System.50 Northern Ireland At-risk underlying medical conditions Based on the QCOVID algorithm,63 we will consider the following conditions: (1) cardiovascular condi- tions (atrial fibrillation, heart failure, stroke, peripheral vascular disease, coronary heart disease, congenital heart disease); (2) diabetes (type 1 and type 2); (3) respiratory conditions (asthma, rare respiratory conditions (cystic fibrosis, bronchiectasis or alveolitis), chronic obstructive pulmonary disease, pulmonary hypertension or pulmo- nary fibrosis); (4) cancer (blood cancer, chemotherapy, lung or oral cancer, marrow transplant, radiotherapy); (5) neurological conditions (cerebral palsy, Parkinson’s disease, rare neurological conditions (motor neuron disease, multiple sclerosis, myasthenia, Huntington’s chorea), epilepsy, dementia, learning disability, severe mental illness) and (6) other conditions (liver cirrhosis, osteoporotic fracture, rheumatoid arthritis or systemic lupus erythematosus, sickle cell disease, venous thrombo- embolism, solid organ transplant, renal failure (chronic kidney disease stages 3–5 with or without dialysis or trans- plant).63 Body mass index will also be considered.63 p g A COVID-19 hospital or ICU admission will be defined based on either a RT-PCR confirmed positive test for SARS-CoV-2 in the 28 days prior to admission or based on an ICD-10 code for COVID-19 (U07.1 or U07.2) in any diagnostic position. A COVID-19 death will be defined as COVID-19 as the underling ICD-10 cause of death recorded on the death certificate, or death from any cause within 28 days of a positive RT-PCR test for SARS-CoV-2. History of COVID-19 infection Any suspected or confirmed COVID-19 infections in the previous last 6 months (prior to 1 December 2020) will be included. copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from social/council housing for each UK nation if data are available. social/council housing for each UK nation if data are available. suspected or confirmed COVID-19 illness; (3) A&E attendance related to suspected or confirmed COVID-19 illness at presentation; (4) hospital admissions related to confirmed COVID-19 illness; (5) ICU admissions related to confirmed COVID-19 illness and (6) deaths related to suspected or confirmed COVID-19 illness. We will also explore the effects of vaccines on secondary SARS-CoV-2 infection due to household transmission. VE against maternal and neonatal COVID-19 related outcomes will be explored once the vaccines are more widely available to pregnant women. suspected or confirmed COVID-19 illness; (3) A&E attendance related to suspected or confirmed COVID-19 illness at presentation; (4) hospital admissions related to confirmed COVID-19 illness; (5) ICU admissions related to confirmed COVID-19 illness and (6) deaths related to suspected or confirmed COVID-19 illness. We will also explore the effects of vaccines on secondary SARS-CoV-2 infection due to household transmission. VE against maternal and neonatal COVID-19 related outcomes will be explored once the vaccines are more widely available to pregnant women. Population characteristics, confounding factors and effect modifiers A number of key characteristics that could explain vari- ation in vaccine uptake will be considered. In addition, these characteristics could confound our planned anal- yses. We will determine and include these characteristics or potential confounders at the baseline of our study’s cohort which include (see table 1 for details on data sources for each UK nation): History of prescribed medications Based on the QCOVID algorithm,63 we will measure number (>4) of prescriptions from general practices for oral steroids, long acting β agonists or leukotrienes, immu- nosuppressants in previous 6 months prior to the start of the study cohort (1 December 2020). Prior or concomi- tant usage of the novel oral anticoagulants, warfarin and heparin will also be measured. History of non-COVID-19 vaccination Receipt of influenza or pneumococcal vaccination during the 2020–2021 season (1 September to 30 November 2020) available in a binary format (yes or no) will be included which could be predictive of COVID-19 vaccination. on February 22, 2022 at University of Glasgow. Protected by mj.com/ History of prescribed medications History of healthcare utilisation Sex at birth will be included in a binary format (females and males). Age will be included in bands that will be determined based on available vaccination data. Socio- economic status will be assessed by the following national versions of area level deprivation indices: (1) Index of Multiple Deprivation (IMD), 2019 version for England55 56 ; (2) the Scottish IMD (SIMD)24; (3) the Welsh IMD57 and (4) the Northern Ireland Multiple Deprivation Measure.58 For England, we will maximise the capture of ethnicity data through the use of a customised ontology.59 Ethnicity data for the other UK nations will also be included if available. Number of GP consultations and hospital admissions in the 6 months before the start of the study cohort (December 1, 2020) will also be measured as a proxy of severity of pre-existing medical conditions. Geographic In England, the RSC will use the ONS data on population density and classify households as rural, urban (town and city) or conurbation.60 The RSC also has a unique house- hold key, so we can report household incidence of respi- ratory and other infectious conditions.61 In Scotland, settlement type will be included using the urban/rural sixfold classification (UR6).24 In Wales, urban and rural household classification that is based on the Lower-layer Super Output Area of the person’s residence information will be used.26 In Northern Ireland, NISRA’s Statistical Classification and Delineation of Settlements will be used to determine settlement type.62 Type of residence will also be considered such as private residence, care home and Smoking status Smoking status will be included through four catego- ries: smoker, ex-smoker, non-smoker and ‘not recorded’. Smoking status will not be determined for Northern Ireland due to no access to primary care data. copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ s 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Wales Protective effects of the vaccines will be assessed against the following outcomes: (1) RT-PCR laboratory confirmed COVID-19 infection; (2) GP consultations related to In Wales, pregnancy and neonatal outcomes will be iden- tified from linked Annual District Birth Extract, National Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 6 copyright. on February 22, 2022 at University of Glasgow. P http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 22, 2022 at Universit http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access Vaccine effectiveness TND case–control study for laboratory-confirmed outcomes TND case control study for laboratory confirmed outcomes Vaccine status will be compared between cases (patients with a positive test for COVID-19) and controls (patients with a negative test for COVID-19) using a TND case– control study.24 The main advantage of the TND studies compared with traditional case–control studies is that it minimises confounding factors from health care-seeking behaviour, which means both cases and controls have similar likelihood of seeking healthcare when having symptoms indicative of COVID-19 illness.64 Selection bias can still arise if study participants are not recruited based on predefined criteria (eg, signs/symptoms indicative to COVID-19 illness) but based on clinician- ordered test.64 In this scenario, clinicians may be more likely to carried out a test on patients that are more likely to have COVID-19 illness (outcome) or not being vaccinated.64 This will result to biased sampling (non- representativeness) of the study participants from the source population which could lead to overestimation of the VE estimates.64 VE is estimated based on the OR using the formula VE=1 OR. OR is defined as the odds of a SARS-CoV-2 infection among the vaccinated group divided by the odds of a SARS-CoV-2 infection among the unvaccinated group. A generalised additive logistic on February 22, 2022 at University of Glasgow. Protected by mj.com/ Additional sensitivity or post hoc analyses such as using different time intervals following administration of the vaccine to define exposure will also be explored for all study outcomes related to VE. Prospective cohort study for vaccine uptake Overall proportion of individuals that receive the vaccine, stratified by sociodemographic, medical and other char- acteristics will be reported using a prospective cohort study. We will also consider reporting uptake levels within certain subgroups of the population where possible (eg, ethnic minorities and healthcare and other front-line workers, care home residents and pregnant women). We will also seek to examine patterns related to the number of eligible individuals that were offered vaccine but refused to be vaccinated if data are available. Univari- able and multivariable logistic regression models will be carried out to estimate the coefficient of each predictor variable in the model and their 95% CIs, as well as the OR for vaccine uptake. Prospective cohort study for clinical outcomes and deaths A prospective cohort study will be used to estimate VE against clinical outcomes and deaths in vaccinated and unvaccinated individuals respectively. Time-dependent Cox models will provide the adjusted rate ratios for these outcomes. VE will be calculated according to VE=1 RR. VE estimates will be adjusted for potential confounders and effect modifiers including age, sex, underlying medical condition, SES, history of healthcare utilisation, medi- cation and non-COVID-19 vaccination. Other potential confounders and effect modifiers may also be explored. Recall and misclassification bias will be minimised in our planned prospective cohort studies as we will use data from national linked datasets which allow rapid analysis of vaccination and clinical outcomes data derived from electronic health records. Nevertheless, unmeasured confounding can still influence the VE estimates (given the observational nature of these study designs) despite attempts to provide VE adjusted for potential confounders as mentioned above. Vaccine effectiveness Vaccine effectiveness TND case–control study for laboratory-confirmed outcomes Individual patient-level analyses Vaccine uptake Prospective cohort study for vaccine uptake Statistical analysis We will report summary statistics of baseline character- istics for the individuals in the study as of 1 December Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 7 copyright. on February 22, 2022 at University of Glas http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access 2020. These will be also stratified by vaccine status and respective study outcome of interest. Relevant measures of tendency (eg, mean, median, proportion) and vari- ability (eg, SD, IQR) will be calculated. Relative estimates (eg, OR and rate ratio (RR)) and their respective 95% CI for risk of outcomes of interest will also be included for different population strata. Missing data will be reported as percentages of total, imputation will be considered if possible, and sensitivity analysis will be carried out to examine if the nature of the missing data mechanism affects the study findings. All statistical hypotheses tests will be two tailed with significance level set at 5% for all outcomes of interest. Statistical computing language R will be used to conduct all planned statistical analyses. model will be used to estimate the OR and VE. Strati- fied VE estimates by vaccine type, dose, dosing schedules, COVID-19 strain, age group and underlying medical condition will also be considered. The analyses in English databases will be carried out in collaboration with PHE, following their guidance, as the RSC is a major data and sampling source for PHE.65 It will be important to avoid the confusion that might arise from different approaches to analysing RSC data, and to benefit from shared expertise in monitoring VE.66 67 We have a shared protocol for this season’s analysis.68 Like- wise, in Northern Ireland, we will work closely with the Public Health Agency. In Scotland, all planned analyses will also be carried out in collaboration with PHS. In Wales, analyses will be carried out in collaboration with PHW, who are involved with other colleagues in Wales in the national rollout and evaluation of the vaccine deployment, and are unique placed to provide expertise, and are key contributors as part of the existing ConCOV project in Wales.26 copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ 1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Pooled analyses In our study, we will initially provide estimates on vaccine uptake, effectiveness, and safety for each UK nation. We will also provide pooled estimates across the UK nations. A generic inverse variance method for meta-analysis will be used. Heterogeneity of our pooled estimates will be assessed using the standard χ2 and the I2 statistic.71 Forest plots will be used to visualise any statistical heterogeneity in our pooled estimates for the four nations. Individual nation’s ORs or RRs and their 95% CIs will be used to estimate the pooled VE estimates. Measured and unmea- sured heterogeneity is highly probable across the UK nations. Effect estimates from random-effect models will thus only be considered. ETHICS AND DISSEMINATION For Scotland, approvals have been obtained by the National Research Ethics Service Committee (REC), South East Scotland 02 (REC number: 12/SS/0201) and the Public Benefit and Privacy Panel for Health and Social Care (reference number: 1920–0279). For Wales, the data used in this study are available in the SAIL Databank at Swansea University, Swansea, UK. All proposals to use SAIL data are subject to review by an independent Infor- mation Governance Review Panel (IGRP). Before any data can be accessed, approval must be given by the IGRP. The IGRP gives careful consideration to each project to ensure proper and appropriate use of SAIL data. When access has been approved, it is gained through a privacy- protecting safe haven and remote access system referred to as the SAIL Gateway. SAIL has established an applica- tion process to be followed by anyone who would like to access data via SAIL.27 Similarly, in Northern Ireland, the project was approved by the HSC HBS Governance Board (project number 0064), and accessed through the online secure research platform. Findings will be presented at conferences, published in peer-reviewed journals and to the funders and government COVID-19 advisory bodies as appropriate. Strengthening the Reporting of Observa- tional Studies in Epidemiology and Reporting of studies Conducted using Observational Routinely-collected Data (via the COVID-19 extension) checklists will guide our study findings reporting.75 76 We will also consider using the European Network of Centres for Pharmacoepidemi- ology and Pharmacovigilance checklist.77 A sensitivity analysis by previous history of SARS-CoV-2 infection will also be considered. We will explore if previous SARS-CoV-2 infection is associated with any AEs observed following a COVID-19 vaccination. on February 22, 2022 at University of Glasgow. Protected by mj.com/ Patient and public involvement and engagement The exact duration (in days) of the risk and control intervals will be determined for each AEFI outcome based on severity level (mild-to-moderate, severe and typical onset) and vaccine type separately (see online supple- mental material, appendix 3). We will use the Benjamini- Hochberg procedure to control the False Discovery rate of testing a large number of hypotheses related to each prespecified adverse events of interest.70 Subgroup anal- yses by vaccine type, dose and dosing schedules will also be considered. Patient and public involvement and engagement (PPIE) members (Antony Chuter, Alex Brownrigg and Jillian Beggs) have been involved since the beginning of this project. The research proposal for the Wales analysis has also been reviewed by members of the public. Their contribution includes defining research questions, inter- pretation and dissemination of study findings. As part of the ConCOV project26 in Wales, PPIE members have been involved in the design and evaluation of findings, as well as presentations made to the independent SAIL consumer panel group made up of lay members. It is possible that sample selection bias could be induced in the SCCS if inclusion in the study is related nontrivially to the adverse outcome of interest. This may be partic- ularly true for severe adverse events. For example, if an individual has a cardiac arrest then they are less likely to be vaccinated and thus less likely to be included in the study. We will therefore carry out a retrospective cohort or an event-dependent exposure version of SCCS study for severe outcomes. Vaccine safety SCCS and retrospective cohort studies for adverse events Protected by http://bmjopen.bmj.com/ blished as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Vaccine safety SCCS and retrospective cohort studies for adverse events The risk of any vaccine-related adverse events will be assessed using a SCCS study design.24 This study design tests whether the risk of an adverse event is higher at post- vaccination period compared with other periods that are temporarily unrelated to vaccine administration.69 The main advantage of this case series method over other methods of analysis is that it only includes individuals who have been vaccinated. As a result, adequate statistical power can often be obtained with relatively small sample sizes. Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 8 copyright. on February 22, 2022 at Univers http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from copyright. on February 2 http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access copyright. on February 22, 2022 at University of Glasgow. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from In addition, all confounders (eg, sex, genetics, SES, location, underlying condition) that do not vary with time over the observation period are implicitly controlled for.69 The number of adverse events in a pre-defined risk interval will be compared with predefined control intervals. Risk interval refers to postvaccine administra- tion period over the observation period of the study and control intervals refer to prevaccine and postvaccine administration over the study’s observation period. Risk and control intervals will also be determined in relation to vaccine dose administration (eg, between first and second doses of the vaccines). A clearance or wash-out interval between the risk and control intervals will also be applied. 0.89 against COVID-19 hospitalisation at 28–34 days post- vaccination, with a SD of 0.06. Assuming our VE estimates are asymptotically normally distributed, this gives almost 100% power to detect a VE of over 0.5. The number of COVID-10 vaccines doses required to detect a relative risk of 5.0 is at least 10 000 doses for a relatively common adverse outcome (eg, myocardial infraction with a background incidence rate of 1400 per 100 000 person years in men older than 85 years old) and more than a million doses are needed to detect a rela- tive risk of 1.5 for a rare adverse outcome (eg, myocardial infraction with a background incidence rate of 28 per 100 000 person years among those 18–34 years).73 74 copyright. on February 22, 2022 at University of Glasgow. Author affiliations Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation (BHF) and the Wellcome Trust; and Administrative Data Research UK which is funded by the Economic and Social Research Council (grant ES/ S007393/1). Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation (BHF) and the Wellcome Trust; and Administrative Data Research UK which is funded by the Economic and Social Research Council (grant ES/ S007393/1). 1The University of Edinburgh, Usher Institute, Edinburgh, UK 2Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK 3Public Health Scotland, Glasgow, UK 4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK 4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK Competing interests AS is a member of the Scottish Government Chief Medical Officer’s COVID-19 Advisory Group. RAL reports grants from MRC during the conduct of the study. SJS reports grants from Wellcome Trust, during the conduct of the study; grants from National Institute of Healthcare Research HTA, grants from Tommy's Charity and grants from Chief Scientist for Scotland, outside the submitted work. SdeL is Director of the Royal College of General Practitioners Research and Surveillance Centre. He has received grant funding through his University from AstraZeneca, Eli Lilly, GSK MSD, Seqirus and Takeda. He has been members of advisory boards for AstraZeneca, Sanofi, and Seqirus. DB is jointly employed by Queen’s University Belfast, the Public Health Agency and the Department of Health (Northern Ireland), and he is currently or has been a member of COVID-19 government advisory groups, including the Scientific Advisory Group for Emergencies (SAGE), its subgroups, and the UK Vaccine Effectiveness Expert Panel. All other authors report no conflicts of interest. 5School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, UK 6BREATHE – The Health Data Research Hub for Respiratory Health, London, UK 7Population Data Science, Swansea University Medical School, Swansea, UK 8Vaccine Preventable Disease Programme, Public Health Wales, Cardiff, UK 10School of Medicine, University of St Andrews, St Andrews, UK 11Public Health Scotland, Edinburgh, UK 12MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow, UK 13 13Wellington School of Health, Faculty of Health, Victoria University of Wellington, Wellington, New Zealand Patient consent for publication Not applicable. Twitter Eleftheria Vasileiou @elvasileiou, Simon de Lusignan @Lusignan_S, Chris Orton @chrisortonSUHIG, Ashley Akbari @AshleyAkbari, Sarah Jane Stock @ sarahjanestock and Aziz Sheikh @DrAzizSheikh Provenance and peer review Not commissioned; externally peer reviewed. Author affiliations Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Acknowledgements England: Patients and general practices who provide samples and agree to share data with the Oxford-RCGP RSC. EMIS, TPP, In-Practice Systems Wellbeing software for supporting the RSC data extraction. Public Health England for support and collaboration. Wales: This work uses data provided by patients and collected by the NHS as part of their care and support. We would also like to acknowledge all data providers who make anonymised data available for research. We wish to acknowledge the collaborative partnership that enabled acquisition and access to the de-identified data, which led to this output. The collaboration was led by the Swansea University Health Data Research UK team under the direction of the Welsh Government Technical Advisory Cell (TAC) and includes the following groups and organisations: the Secure Anonymised Information Linkage (SAIL) Databank, Administrative Data Research (ADR) Wales, NHS Wales Informatics Service (NWIS), Public Health Wales, NHS Shared Services Partnership and the Welsh Ambulance Service Trust (WAST). All research conducted has been completed under the permission and approval of the SAIL independent Information Governance Review Panel (IGRP) project number 0911. Northern Ireland: The authors would like to acknowledge the help provided by the staff of the Honest Broker Service (HBS) within the Business Services Organisation Northern Ireland (BSO). The HBS is funded by the BSO and the Department of Health (DoH). The authors alone are responsible for the interpretation of the data and any views or opinions presented are solely those of the author and do not necessarily represent those of the BSO. copyright. on February 22, 2022 at University of http://bmjopen.bmj.com/ ary 2022. ORCID iDs Eleftheria Vasileiou http://orcid.org/0000-0001-6850-7578 Ting Shi http://orcid.org/0000-0002-4101-4535 Ruby Tsang http://orcid.org/0000-0002-2520-526X Simon de Lusignan http://orcid.org/0000-0001-5613-6810 Chris Orton http://orcid.org/0000-0002-9561-2493 Ashley Akbari http://orcid.org/0000-0003-0814-0801 Lucy J Griffiths http://orcid.org/0000-0001-9230-624X Emily Lowthian http://orcid.org/0000-0001-9362-0046 Jane Lyons http://orcid.org/0000-0002-4407-770X Ronan A Lyons http://orcid.org/0000-0001-5225-000X Rachael Wood http://orcid.org/0000-0003-4453-623X Sarah Jane Stock http://orcid.org/0000-0003-4308-856X Paul Henery http://orcid.org/0000-0003-0380-738X Aziz Sheikh http://orcid.org/0000-0001-7022-3056 Contributors AS conceived this study and commented on several drafts of the protocol. EV wrote the first draft of the protocol with assistance from TS and SK. SdeL, RT, MJ and DMcC, JW and RH contributed about the English sentinel system and commented on the research methods. DB, DO and SM contributed about the Northern Ireland data and commented on the research methods. AC and JB were involved in the study design and commented on introduction and research methods from a patient and public perspective. DF, CO, AA, SB, GD, LJG, RG, EL, JL, RAL, LN, MP and FT contributed about the Wales data and commented on the research methods. JP contributed on the study design and commented on several drafts. JM, CM, UA, RW, SJS, EM, PH, CRS and CR contributed about the Scotland data and comments on research methods. All authors contributed to the study design. All authors contributed to drafting the protocol. All authors revised the manuscript for important intellectual content. All authors gave final approval of the version to be published. Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 Sample size We are basing sample size calculations on Scottish testing and vaccination data because it is currently the only UK nation with full national data coverage at the time of writing. Based on previous work,72 we estimated a VE of 9 Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 copyright. on February 22, 2022 at University of Glasgow. P http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 Author affiliations Downloaded from Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. Open access Open access Author affiliations 1The University of Edinburgh, Usher Institute, Edinburgh, UK 2Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK 3Public Health Scotland, Glasgow, UK 4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK 5School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, UK 6BREATHE – The Health Data Research Hub for Respiratory Health, London, UK 7Population Data Science, Swansea University Medical School, Swansea, UK 8Vaccine Preventable Disease Programme, Public Health Wales, Cardiff, UK 9Health Data Research, London, UK 10School of Medicine, University of St Andrews, St Andrews, UK 11Public Health Scotland, Edinburgh, UK 12MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow, UK 13Wellington School of Health, Faculty of Health, Victoria University of Wellington, Wellington, New Zealand Author affiliations 1The University of Edinburgh, Usher Institute, Edinburgh, UK 2Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK 3Public Health Scotland, Glasgow, UK 4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK 5School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, UK 6BREATHE – The Health Data Research Hub for Respiratory Health, London, UK 7Population Data Science, Swansea University Medical School, Swansea, UK 8Vaccine Preventable Disease Programme, Public Health Wales, Cardiff, UK 9Health Data Research, London, UK 10School of Medicine, University of St Andrews, St Andrews, UK 11Public Health Scotland, Edinburgh, UK 12MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow, UK 13Wellington School of Health, Faculty of Health, Victoria University of Wellington, Wellington, New Zealand copyright. on February 22, 202 http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Available: https://www.gov.uk/government/publications/ priority-groups-for-coronavirus-covid-19-vaccination-advice- from-the-jcvi-30-december-2020/joint-committee-on- vaccination-and-immunisation-advice-on-priority-groups-for-covid- 19-vaccination-30-december-2020 37 COVID-19 vaccination programme in Northern Ireland. Available: https://www.nidirect.gov.uk/articles/covid-19-vaccination- programme-northern-ireland 38 Turas vaccination management tool. Available: https://learn.nes.nhs. scot/42708/turas-vaccination-management-tool 39 UK Government. COVID-19 testing data: methodology note. Available: https://www.gov.uk/government/publications/coronavirus- covid-19-testing-data-methodology/covid-19-testing-data- methodology-note 15 Dhanda S, Osborne V, Lynn E, et al. Postmarketing studies: can they provide a safety net for COVID-19 vaccines in the UK? BMJ Evid Based Med 2022;27:1-6. 16 Iacobucci G, Mahase E. Covid-19 vaccination: what's the evidence for extending the dosing interval? BMJ 2021;372:n18. 40 NHS Digital. Data access Request service (DARS). Available: https:// digital.nhs.uk/services/data-access-request-service-dars/dars- products-and-services 17 Patel MD, Rosenstrom E, Ivy JS, et al. Association of simulated COVID-19 vaccination and Nonpharmaceutical interventions with infections, hospitalizations, and mortality. JAMA Netw Open 2021;4:e2110782. 41 de Lusignan S, Dorward J, Correa A, et al. Risk factors for SARS- CoV-2 among patients in the Oxford Royal College of general practitioners research and surveillance centre primary care network: a cross-sectional study. Lancet Infect Dis 2020;20:1034–42. 18 Berlin JA, Glasser SC, Ellenberg SS. Adverse event detection in drug development: recommendations and obligations beyond phase 3. Am J Public Health 2008;98:1366–71. 42 de Lusignan S, Correa A, Smith GE, et al. RCGP research and surveillance centre: 50 years' surveillance of influenza, infections, and respiratory conditions. Br J Gen Pract 2017;67:440–1. 19 Robertson E, Reeve KS, Niedzwiedz CL, et al. Predictors of COVID-19 vaccine hesitancy in the UK household longitudinal study. Brain Behav Immun 2021;94:41–50. p y 43 Department of Health, Northern Ireland. Hospital statistics. Available: www.health-ni.gov.uk/articles/inpatient-and-day-case-activity ; 20 Madewell ZJ, Yang Y, Longini IM, et al. Household transmission of SARS-CoV-2: a systematic review and meta-analysis. JAMA Netw Open 2020;3:e2031756. g y y 44 Joy M, Hobbs FDR, McGagh D, et al. Excess mortality from COVID-19 in an English sentinel network population. Lancet Infect Dis 2021;21:S1473-3099(20)30632-0. ( ) 45 de Lusignan S, Joy M, Oke J, et al. Disparities in the excess risk of mortality in the first wave of COVID-19: cross sectional study of the English sentinel network. 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Available: https://www.strobe-statement.org/ index.php?id=strobe-home 76 RECORD. Available: http://www.record-statement.org/ 64 Fukushima W, Hirota Y. Basic principles of test-negative design in evaluating influenza vaccine effectiveness. Vaccine 2017;35:4796–800. 64 Fukushima W, Hirota Y. Basic principles of test-negative design in evaluating influenza vaccine effectiveness. Vaccine 2017;35:4796–800. 77 European network of centres for pharmacoepidemiology and pharmacovigilance. Available: http://www.encepp.eu/index.shtml 12 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance plemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material BMJ Open Supplementary Material Supplementary Material Very common (≥1/10), common (≥1/100 to <1/10), uncommon (≥1/1,000 to <1/100), rare (≥1/10,000 to <1/1,000), very rare (<1/10,000), not known (cannot be estimated). Very rare events of neuroinflammatory disorders have been reported following vaccination with the Oxford AstraZeneca vaccine, but a causal relationship has not been established. doi: 10.1136/bmjopen-2021-050062 :e050062. 12 2022; BMJ Open , et al. Vasileiou E Supplementary Material Appendix 1: Adverse Events Following Immunisation (AEFI) reported in pre-licensure COVID- 19 vaccine trials Adverse event Pfizer-BioNTech (BNT162b2) Oxford-AstraZeneca (AZD1222) Moderna Abdominal pain Uncommon Acute peripheral facial paralysis (/palsy) Rare (37 days after dose 1, 3-48 days after dose 2) Rare (22-32 days after dose 2) Anaphylaxis* Not known Not known Arthralgia Very common Very common Very common Chills Very common Very common Very common Decreased appetite Uncommon Dizziness Uncommon Facial swelling Rare (1-2 days post- vaccination in vaccinees with history of injection of dermatological fillers) Fatigue Very common Very common Very common Headache Very common Very common Very common Hyperhidrosis Uncommon Hypersensitivity Not known Not known Influenza-like illness Common Injection site bruising Very common Injection site erythema Very common Common Injection site induration Common Injection site pain Very common Very common Very common Injection site pruritus Uncommon Very common Uncommon Injection site rash Common Injection site redness Common Injection site swelling Very common Very common Very common Injection site tenderness Very common Injection site urticaria Common Injection site warmth Very common Insomnia Uncommon Lymphadenopathy* Uncommon Uncommon Very common Malaise Uncommon Very common Myalgia Very common Very common Very common Nausea** Common Very common Very common Pain in extremity Uncommon Pruritus Uncommon Pyrexia*** Very common Very common Very common Rash Uncommon Common Vomiting Common * Anaphylaxis reported in post-marketing setting for Moderna vaccine. Appendix 1: Adverse Events Following Immunisation (AEFI) reported in pre-licensure COVID- 19 vaccine trials 1 1 doi: 10.1136/bmjopen-2021-050062 :e050062. 12 2022; BMJ Open , et al. Vasileiou E BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material BMJ Open Injection site bruising includes injections site haematoma * Lymphadenopathy captured as axillary lymphadenopathy on the same side as the injection site for Moderna vaccine. ** A higher frequency of pyrexia observed after dose 2 for the Pfizer BioNTech vaccine. *** Pyrexia includes feverishness Note. Appendix 2: List of AEFI for influenza vaccines 2 doi: 10.1136/bmjopen-2021-050062 :e050062. 12 2022; BMJ Open , et al. Vasileiou E BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance rial placed on this supplemental material which has been supplied by the author(s) BMJ Open BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance placed on this supplemental material which has been supplied by the author(s) Supplemental material BMJ Open Appendix 3: Schematic presentation of the self-controlled case series study design 3 doi: 10.1136/bmjopen-2021-050062 :e050062. 12 2022; BMJ Open , et al. Vasileiou E
https://openalex.org/W2262205322	https://www.nature.com/articles/srep14610.pdf	English	null	The role of shear in crystallization kinetics: From suppression to enhancement	Scientific reports	2,015	cc-by	5,997	The role of shear in crystallization kinetics: From suppression to enhancement received: 28 June 2015 accepted: 27 August 2015 Published: 29 September 2015 David Richard & Thomas Speck In many technical applications crystallization proceeds in the presence of stresses and flows, hence the importance to understand the crystallization mechanism in simple situations. We employ molecular dynamics simulations to study the crystallization kinetics of a nearly hard sphere liquid that is weakly sheared. We demonstrate that shear flow both enhances and suppresses the crystallization kinetics of hard spheres. The effect of shear depends on the quiescent mechanism: suppression in the activated regime and enhancement in the diffusion-limited regime for small strain rates. At higher strain rates crystallization again becomes an activated process even at densities close to the glass transition. The making of a material is a process that often involves phase transformations. How a material trans- forms from one stable phase to another has wide-spread consequences: for technological applications such as metal alloys1, colloids2–4, and the graphite-diamond transition of carbon5; but also for the nucleation of ice in clouds6 affecting our climate. Phase transformations have been studied primarily after a sudden change of thermodynamic conditions but with dynamics that obey detailed balance. More realistically however, such transformations proceed away from thermal equilibrium and in the presence of external mechanical stresses and flows, for example when preparing colloidal crystals through spin-coating7. To study the basic physical mechanisms, here we consider as a model system a supersaturated liquid of hard spheres8–10 and generate a non-vanishing stress through constantly shearing the liquid.t p g g g y g q Hard spheres (or more generally, hard particles11) are a paradigm for a wide range of soft materials in which entropy dominates over energetic forces and is one of the best-studied model systems in statistical mechanics. Moreover, many aspects of liquids are dominated by the packing of atoms. In the absence of shear, even though there are no direct forces between particles, hard spheres crystallize at sufficiently high packing fractions φ > φf with φf ≃ 0.492 when the entropy of the crystal has become higher than that of the disordered liquid. Experimentally, the limit of true hard spheres can be approached using colloidal suspensions12, which allow the direct observation of nucleation events3,13. The exact microscopic pathway from liquid to solid remains the focus of intensive research. Institut für Physik, Johannes Gutenberg-Universität Mainz, Staudingerweg 7-9, 55128 Mainz, Germany. Correspondence and requests for materials should be addressed to T.S. (email: thomas.speck@uni-mainz.de) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 28 June 2015 accepted: 27 August 2015 Published: 29 September 2015 Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 Results h b As has been done in previous studies9,10, we do not simulate true hard spheres but particles interacting pairwise via the Weeks-Chandler-Andersen potential, which is mapped onto hard spheres through an effective diameter d (see Methods). We calculate the crystallization rate density k (in Brownian units τBd3) for different values of the packing fraction φ and strain rate γ. To this end we bracket the crystallization time τ τ τ = ( + )/ < > 2 x x x (the spread is included in the error estimation) by the two times τ< x τ ( ) > x such that the size n of the largest cluster obeys n < ns (n > ns) for all τ < < t x τ ( > ) > t x . The exact value nc < ns ≪ N of the threshold ns is not important as long as it is larger than the critical nucleus size nc and much smaller than the system size. We found that ns = 200 is a convenient value, which in the following is used for all packing fractions studied. For the lowest density we have performed a committor analy- sis24,25 and checked that the probability to commit to the solid state is unity for n ≥ 200. For the highest density n ≃ 200 roughly corresponds to the onset of the linear growth regime. From 100 independent runs, the crystallization rate is then determined as k = (V〈 τx〉 )−1, where V is the volume of the simulation box. In the quiescent case (γ =  0) the rate is a non-monotonic function of the packing fraction, see Fig. 1a: It has a maximum around φ ≃ 0.56 and is decreasing fast when going to lower densities (lower supersat- uration). The calculated rates agree well with previous simulation results9,10 and within errors also with experimental data (for a detailed discussion see ref. 12. Note that densities even closer to freezing can be studied using rare-event sampling methods8,10 but predict absolute rates that are several orders of mag- nitude too small compared to experimental results12. A comprehensive explanation of this discrepancy is still an open issue.h The crystallization rate also decreases at higher densities due to two competing factors: going to higher densities the free energy barrier against nucleation decreases while at the same time the diffusivity of particles also decreases. The role of shear in crystallization kinetics: From suppression to enhancement The classical picture has been that of a one-step processes in which a rare fluctuation leads to densification and the emergence of a nucleus with the same symmetry as the solid14. More recent work has observed different pre-cursors during the initial stage that have a different symmetry than the final solid. This leads to a picture of crystallization as a two-step9,15, or even three-step3, process. Increasing the density, the entropic barrier to nucleation shrinks with dynamics that effectively become arrested on experimental time scales for packing fractions φ > φg with φg ≃ 0.58. How the crystallization pathway then changes is currently under investigation16.i g g y p y g y g Weakly sheared liquids still crystallize but with kinetics that are significantly altered. Understanding and controlling these kinetics at least qualitatively is of technological importance in the search of new protocols to process materials. For example, growing crystals without defects requires a small growth rate once the critical nucleus has formed. Typically this implies conditions close to coexistence2, where, however, the nucleation time (the time for the critical nucleus to appear) becomes prohibitively large. No consensus has emerged so far regarding the role of shear in numerical17–20 and experimental studies21–23 Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 1 www.nature.com/scientificreports/ Figure 1. Crystallization kinetics. (a) Crystallization rate k vs. the (effective) packing fraction φ for γ =  0. The arrow indicates the melting point φm ≃ 0.545 of hard spheres. Closed symbols show our work employing molecular dynamics (MD), open symbols refer to results obtained in refs 9,10 employing Brownian dynamics (BD). (b) Crystallization rate k vs. the square of the strain rate γ2 for the different packing fractions (colors agree with a). The solid lines are fits to Equation (1). Inset: The fitted values for A as a function of packing fraction φ, where the solid line is A ∝ (φ − φ0) with φ0 ≃ 0.56. If not shown then error bars in this figure are smaller than the symbol size. Figure 1. Crystallization kinetics. (a) Crystallization rate k vs. the (effective) packing fraction φ for γ =  0. The arrow indicates the melting point φm ≃ 0.545 of hard spheres. Closed symbols show our work employing molecular dynamics (MD), open symbols refer to results obtained in refs 9,10 employing Brownian dynamics (BD). (b) Crystallization rate k vs. The role of shear in crystallization kinetics: From suppression to enhancement the square of the strain rate γ2 for the different packing fractions (colors agree with a). The solid lines are fits to Equation (1). Inset: The fitted values for A as a function of packing fraction φ, where the solid line is A ∝ (φ − φ0) with φ0 ≃ 0.56. If not shown then error bars in this figure are smaller than the symbol size. with and without attractive interactions. On one hand the suppression of crystallization has been reported for weak, moderate, and high strain rates17,19,22, which is characterized by a decrease of the structural order in the sheared crystal22, larger critical nuclei and nucleation barriers compared to the quiescent case17, and finally the break-up and melting of solid clusters19. On the other hand, weak shear can induce crystallization in the disordered liquid and glassy state18,23. To complicate matters, an optimal strain rate has been reported19–21, where at first crystallization is promoted but hampered at larger strain rates. We will show that all of these features are reproduced in a sheared hard sphere liquid. Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 Results h b The non-monotonicity thus reflects the turnover from an activated process at lower densities, where a sufficiently large solid cluster has to be nucleated to overcome interfacial tension, to a crystallization process that is limited by diffusion26. In colloidal suspensions and complex fluids it is the mobility of particles that is rate-limiting, while in atomistic fluids it is the conduction of the released latent heat. Activated regime. We now apply a steady shear flow leading to a non-vanishing stress. The shear flow is weak so that for the range of strain rates studied the system remains in the linear response Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 2 www.nature.com/scientificreports/ Figure 2. (a) Committor analysis for packing fraction φ ≃ 0.539. Scatter plot showing the correlations between the committor PB and the size n of the largest cluster for the quiescent (gray) and shear driven case (red) with strain rate γτ . 0 07 B . The shaded area indicates n ≥ ns = 200, for which the probability that solid clusters continue to grow has reached PB ≃ 1. The dashed lines limit the transition state ensemble, for which the distribution of nucleus sizes is shown in the inset. (b) Effective chemical potential for packing fractions φ < φ0. The crystallization rates k for several strain rates collapse when plotted against an effective chemical potential. The dashed line is a guide to the eye. Figure 2. (a) Committor analysis for packing fraction φ ≃ 0.539. Scatter plot showing the correlations Figure 2. (a) Committor analysis for packing fraction φ ≃ 0.539. Scatter plot showing the correlations between the committor PB and the size n of the largest cluster for the quiescent (gray) and shear driven case (red) with strain rate γτ . 0 07 B . The shaded area indicates n ≥ ns = 200, for which the probability that solid clusters continue to grow has reached PB ≃ 1. The dashed lines limit the transition state ensemble, for which the distribution of nucleus sizes is shown in the inset. (b) Effective chemical potential for packing fractions φ < φ0. The crystallization rates k for several strain rates collapse when plotted against an effective chemical potential. The dashed line is a guide to the eye. Figure 2. (a) Committor analysis for packing fraction φ ≃ 0.539. Results h b Scatter plot showing the correlations between the committor PB and the size n of the largest cluster for the quiescent (gray) and shear driven case (red) with strain rate γτ . 0 07 B . The shaded area indicates n ≥ ns = 200, for which the probability that solid clusters continue to grow has reached PB ≃ 1. The dashed lines limit the transition state ensemble, for which the distribution of nucleus sizes is shown in the inset. (b) Effective chemical potential for packing fractions φ < φ0. The crystallization rates k for several strain rates collapse when plotted against an effective chemical potential. The dashed line is a guide to the eye. regime and the temperature is approximately constant, see Supplementary Figure 1. Since we consider a non-equilibrium situation we require a thermostat in order to remove the dissipated heat. We have been particularly careful in selecting this thermostat in order to minimize the deviation of the kinetic energy from the bath temperature while not perturbing collective dynamics. Supplementary Figure 2 demonstrates that the kinetic energy remains unperturbed also during crystallization, indicating that the latent heat is removed sufficiently fast by the thermostat. Note that the strain rates studied here are much smaller than what is required for shear-induced ordering27.h q g In Fig. 1b, we observe for packing fractions φ ≤ 0.56 a strong suppression of crystallization. The rate ecreases as a function of the square of the strain rate, φ γ φ φ γ ( , ) ≈ ( ) + ( ) , ( )   k k A ln ln 1 0 2 φ γ φ φ γ ( , ) ≈ ( ) + ( ) ,   k k A ln ln 0 2 ( ) 1 where k0 is the quiescent rate. Such a quadratic dependence to lowest order is expected from symmetry considerations alone since the rate should be invariant under the inversion γ γ ( −) of the flow profile. Moreover, the fitted expansion coefficients A < 0 for the lowest densities are well described by a linear function A ∝ (φ − φ0) with φ0 ≃ 0.56 well below the glassy regime. Given the strong dependence of the quiescent rate k0 on density, this simple result is somewhat surprising. Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 Results h b This would imply that under shear there are two effective chemical potentials: one determines the nucleation of the new phase and one determines the final coex- istence in the steady state. Both cannot be the same, which is in striking contrast to the quiescent liquid. Diffusion-limited regime. We finally study the regime φ ≥ 0.56. In Fig. 3a the mean crystallization time 〈 τx〉 is plotted for the largest packing fraction φ ≃ 0.587, which indeed shows an enhancement of crystallization for weak shear flow. For consistency, we maintain the same criteria for determining the crystallization time although now multiple solid clusters appear and grow. While at low supersaturation the distinction between the actual nucleation time (random waiting time to reach the critical cluster size) and crystallization time (random waiting time τx to reach ns) is negligible, this is no longer the case. At this density, the solidification process is limited by the rate with which additional particles “attach” (there are no direct attractive forces but arranging into ordered structures locally increases the entropy). Shear flow increases the diffusion both parallel and perpendicular to the direction of the shear flow30, and thus speeds up the attachment while the barrier to nucleate small solid clusters is still small. However, we observe that increasing γ the mean crystallization time reaches a minimum before it again increases for higher strain rates.f g In Fig. 3b the evolution of the largest cluster is shown for φ ≃ 0.587. To separate the effects of nucle- ation and growth, the single trajectories have been shifted by τx before averaging. In the quiescent liquid, the growth of the largest nucleus is gradually and slow before 〈 n〉 becomes an approximately linear function of time for 〈 n〉 > ns. Hence, the value ns = 200 is also a good estimate for the beginning of the linear growth regime. In the presence of shear flow, the initial increase of 〈 n〉 is less gradual and the following growth is more rapid. Interestingly, for both non-zero strain rates the curves lie on top of each other while the crystallization rates differ by a factor of two. The reason is revealed in Fig. 3c, which shows that the distribution of the single crystallization times τx is strongly affected by the shear flow. Results h b These Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 3 www.nature.com/scientificreports/ configurations constitute the transition state ensemble, for which the distribution of cluster sizes are shown in the inset of Fig. 2(a). The mean values nc ≃ 36 for γ =  0 and nc ≃ 68 for γτ . 0 07 B are esti- mates of the critical cluster sizes for the quiescent and sheared liquid, respectively. Moreover, PB ≃ 1 for n ≥ ns, which shows that the threshold is sufficiently large. For higher densities critical cluster sizes are smaller and we use the same threshold for all densities. Effective chemical potential. Classical nucleation theory predicts a crystallization rate k0 = κexp(− Δ G/kBT) with two contributions: a kinetic pre-factor κ and an exponential free energy bar- rier Δ G ∝ \|Δ μ\|−2. Here, T is the temperature, kB is Boltzmann’s constant, and Δ μ < 0 is the difference in chemical potential of the solid and the liquid. The proportionality depends on the interfacial tension and geometric factors due to the average shape of the critical nuclei.flh g g p Both contributions to the rate are affected when turning on the shear flow. The pre-factor is deter- mined by the fluctuations and correlations between solid-like particles in the meta-stable liquid, the reduction of which is not strong enough to explain the observed exponential reduction of the crystal- lization rate. Alternatively, it has been proposed that the suppression can be accounted for through an effective free energy with an increased barrier17. To test this idea, we convert packing fractions to chem- ical potential differences using a linear fit to the data of ref. 10. Indeed, plotting the rates as a function of an effectively reduced chemical potential difference μ μ αγ μ Δ = Δ ( + ) < Δ ( )  1 2 eff 2 ( ) 2 (while keeping the interfacial tension constant) with constant α ≃ − 15, all rates for different strain rates collapse onto a single curve as shown in Fig. 2(b). This demonstrates that nucleation under shear remains a rare, collective fluctuation to overcome a single barrier. Interestingly, Butler and Harrowell have argued for the non-equilibrium coexistence of a sheared liquid and the solid that the magnitude of the chemical potential difference is likely to increase28,29. Results h b The density φ0 approximately indicates the crossover from a regime where crystallization is suppressed by shear flow to a regime where it is enhanced with A > 0 (at least for weak shear). The location of this crossover agrees with the location for the qualitative change from activated to diffusion-limited. Committor analysis. We now analyze in more detail the lowest density φ ≃ 0.539 studied to gain some insights into the transition state ensemble, which comprises the configurations at the top of the free energy barrier separating the liquid from the solid. To this end we perform a committor analysis24,25 computing the probability PB to commit to the solid state, which is a good reaction coordinate to unam- biguously describe the process of crystallization. Specifically, PB is computed from several (20 in our case) fleeting trajectories – short runs of length τf – for every configuration along a stored trajectory with randomized velocities. The fleeting time τ τ τ ≪ 18 f B x is much shorter than the nucleation time τx with mean τ τ 750 x B, but large enough to allow the system to unambiguously reach the threshold (ns = 200). The committor for one configuration is calculated as the ratio between the number of fleeting trajectories that reach the solid divided by the total number of trials fired from that configuration. Such an analysis can also be performed in a driven system. Some care has to be taken regarding the initial velocities, which here are taken from the equilibrium Maxwell-Boltzmann distribution. Due to the small strain rates, the perturbation of the steady state is negligible compared to the fleeting time τf (cf. Supplementary Figure 3). pp y g Figure 2(a) shows for φ ≃ 0.539 that the correlations between PB and the cluster size n are similar for both the quiescent and the sheared liquid. For the latter, equal PB typically have larger cluster sizes which indicates that smaller clusters are more likely to break up under shear. This intuitively agrees with the observation of a suppression of nucleation. Of particular interest are configurations for which PB ~ 0.5, i.e., for which the chances to fall back to the liquid or to surmount the barrier are equal. Results h b In the quiescent liquid the barrier to nucleation has basically vanished and we observe multiple nuclei (see snapshot Fig. 3d). The mean nucleation time 〈 τx〉 ≃ 16.5τB is small (compare 〈 τx〉 ≃ 750τB for φ ≃ 0.539) while the distribution is approximately Gaussian, which qualitatively agrees with a diffusive evolution under a constant force given by the chemical potential difference between liquid and solid. Going to a small strain rate γτ . 0 15 B , the distribution of nucleation times remains a Gaussian but becomes more narrow with a smaller mean. At this strain rate solid clusters thus appear more frequently and grow faster compared to the quiescent liquid (see Fig. 3e). At higher strain rate γτ . 0 3 B the distribution of τx becomes very broad with a pronounced tail, which implies a delayed induction of solid clusters and the qualitative change to activated behavior. Once these have formed, however, clusters grow faster than in the absence of shear flow (Fig. 3f). Conclusionshf The effect of weakly shearing a hard sphere liquid on crystallization strongly depends on the density: At low supersaturation the driving force of crystallization, the difference of the chemical potential between liquid and solid, is also small. Here the forces exerted by the shear flow might overcome the gain of entropy and lead to a larger probability for small solid clusters to lose particles (or even break up). Larger Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 4 www.nature.com/scientificreports/ Figure 3. Shear-enhanced crystallization for φ ≃ 0.587. (a) Mean crystallization time 〈 τx〉 vs. the strain rate γ (black, other packing fractions are shown using the same colors as in Fig. 1). The solid lines show the quadratic fits, the dashed line is a guide to the eye. (b) Evolution of average cluster size 〈 n〉 as a function of shifted time. (c) Histograms of crystallization times for three strain rates and their respective means. Solid lines are Gaussian fits. (d–f) Snapshots after t = 5τB starting from the same initial particle configuration for (d) the quiescent liquid, (e) at strain rate γτ . 0 15 B , and (f) at strain rate γτ . 0 3 B . Red particles have been identified as solid-like, green particles as pre-structured (i.e., particles with high local order but less orientational “bonds” with their neighbors). For clarity, liquid-like particles are shown with a reduced size. Figure 3. Shear-enhanced crystallization for φ ≃ 0.587. (a) Mean crystallization time 〈 τx〉 vs. the strain rate γ (black, other packing fractions are shown using the same colors as in Fig. 1). The solid lines show the quadratic fits, the dashed line is a guide to the eye. (b) Evolution of average cluster size 〈 n〉 as a function of shifted time. (c) Histograms of crystallization times for three strain rates and their respective means. Solid lines are Gaussian fits. (d–f) Snapshots after t = 5τB starting from the same initial particle configuration for (d) the quiescent liquid, (e) at strain rate γτ . 0 15 B , and (f) at strain rate γτ . 0 3 B . Red particles have been identified as solid-like, green particles as pre-structured (i.e., particles with high local order but less orientational “bonds” with their neighbors). For clarity, liquid-like particles are shown with a reduced size. Conclusionshf clusters are not affected, which, in combination, leads to a shift of the critical cluster size that can be interpreted as an effectively larger barrier to nucleation. No enhancement of crystallization occurs in this regime. This result might also shed some light onto the controversy regarding the discrepancy between experimental and simulation results at low supersaturations. As pointed out by Russo et al.31, sedimenta- tion of not fully density-matched colloidal particles has an influence on the crystallization rate. However, the actual mechanism is not known and combined with the suppression of crystallization found here seems to rule out shear-induced effects.fhf f At high supersaturation there is a combination of two effects: (i) The diffusion of particles in the surrounding liquid is increased. This enhanced exploration of configuration space accelerates the growth of solid clusters. (ii) The shear flow disrupts the subtle order in the liquid and thus suppresses the forma- tion of small solid clusters. At larger strain rates this suppression is so strong that crystallization again becomes an activated process comparable to crystallization at lower supersaturation in the quiescent liquid. We expect that these physical mechanisms are valid beyond hard spheres also in the presence of short-ranged interactions. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Random configurations of dense hard spheres without preformed structures were created with the algorithm by Clarke and Wiley34, where the non-overlapping distance between particles was chosen to be equal to the effective diameter d. These configurations are thermalized in a short run without shear at high collision frequency Γ ≃ 100 before the production runs at given strain rate with Γ ≃ 10. g q y p g To distinguish between liquid-like and solid-like particles, we follow ref. 10. We employ the local bond order parameter35 ( ) ∑ θ ϕ ( ) = ( ) , ( ) , = ( ) , , , q i N i Y 1 3 l m n j N i l m i j i j 1 n ( ) 3 for particle i, where Yl,m(θ, φ) are spherical harmonics and Nn is the number of neighbors within distance rij < 1.5σ. We construct a bond network through the scalar product for particle i, where Yl,m(θ, φ) are spherical harmonics and Nn is the number of neighbors within distance rij < 1.5σ. We construct a bond network through the scalar product ( , ) = ∑ ( ) ( ) (∑ \| ( )\|) (∑ \| ( )\|) ( ) =− , , =− , / =− , / ⁎ d i j q i q j q i q j 4 m l l l m l m m l l l m m l l l m 1 2 1 2 ( ) 4 sing l = 6 with d(i, j) ≥ 0.7 defining a bond. A particle is defined as “solid-like” (“pre-structured”) if the umber of bonds is ≥ 9 (≥ 6), and clusters are constructed from mutually bonded solid-like particles. using l = 6 with d(i, j) ≥ 0.7 defining a bond. A particle is defined as “solid-like” (“pre-structured”) if the number of bonds is ≥ 9 (≥ 6), and clusters are constructed from mutually bonded solid-like particles. References Blaak, R., Auer, S., Frenkel, D. & Löwen, H. Crystal nucleation of colloidal suspensions under shear. Phys. Rev. Lett. 93 (2004). 18. Mokshin, A. V. & Barrat, J.-L. Shear-induced crystallization of an amorphous system. Phys. Rev. E 77, 021505 (2008).f 19. Cerdà, J. J., Sintes, T., Holm, C., Sorensen, C. M. & Chakrabarti, A. Shear effects on crystal nucleation in colloidal suspe Phys. Rev. E 78, 031403 (2008). y 20. Lander, B., Seifert, U. & Speck, T. Crystallization in a sheared colloidal suspension. J. Chem. Phys. 138, 224907 (2013). 21. Holmqvist, P., Lettinga, M. P., Buitenhuis, J. & Dhont, J. K. G. Crystallization kinetics of colloidal spheres under stationary shear flow. Langmuir 21, 10976–10982 (2005). l 22. Wu, Y. L., Derks, D., van Blaaderen, A. & Imhof, A. 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Prediction of absolute crystal-nucleatio y 9. Kawasaki, T. & Tanaka, H. Formation of a crystal nucleus from liquid. Proc. Natl. Acad. Sci. USA. 107, 14036–14041 (2010 0. Filion, L., Ni, R., Frenkel, D. & Dijkstra, M. Simulation of nucleation in almost hard-sphere colloids: The discrepancy betwee experiment and simulation persists. J. Chem. Phys. 134, 134901 (2011). 1. Damasceno, P. F., Engel, M. & Glotzer, S. C. Predictive self-assembly of polyhedra into complex structures. Science 337, 453–457 (2012). 2. Palberg, T. Crystallization kinetics of colloidal model suspensions: recent achievements and new perspectives. J. Phys.: Condens Matter 26, 333101 (2014).i 3. Gasser, U., Weeks, E. R., Schofield, A., Pusey, P. N. & Weitz, D. A. Real-space imaging of nucleation and growth in colloida crystallization. Science 292, 258–262 (2001). y 4. Becker, R. & Döring, W. Kinetische behandlung der keimbildung in übersättigten dämpfen. Ann. Phys. 24, 719–752 (1935). 15. Schilling, T., Schöpe, H. J., Oettel, M., Opletal, G. & Snook, I. Precursor-mediated crystallization process in suspensions of hard spheres. Phys. Rev. Lett. 105, 025701 (2010). 16. Sanz, E. et al. Avalanches mediate crystallization in a hard-sphere glass. Proc. Natl. Acad. Sci. USA. 111, 75–80 (2014). 17. Methods W d We study a model liquid composed of N = 5000 monodisperse, nearly hard spheres in a periodic box with volume V employing standard NVT molecular dynamics32. Particles interact via the Weeks-Chandler-Andersen potential (WCA) ε σ σ ( ) = ( / ) −( / ) + / u r r r 4 [ 1 4] 12 6 for r < 21/6σ. The temperature is held constant via the stochastic Lowe-Andersen thermostat33, which is characterized by the bath collision frequency Γ . This thermostat has desirable properties, in particular it is Galilean invar- iant and conserves angular momentum. The WCA potential can be mapped onto hard spheres by means of an effective diameter d10, which we compute from φ ρ = π d f HS 6 f WCA 3 with freezing packing fraction φ . 0 492 f HS of hard spheres and freezing density ρ . 0 712 f WCA of the WCA liquid10. The packing fraction is φ = πd N V 6 3 . As time scale we employ the Brownian time τB = d2/D0, where D0 is the bare dif- fusion coefficient in the infinitely dilute system. The system is driven through Lees-Edwards periodic boundary conditions leading to a linear flow profile γ / =  d v dy x . Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 5 References 1. Porter, D. A., Easterling, K. E. & Sherif, M. Phase Transformations in Metals and Alloys (CRC Press, 2008). www.nature.com/scientificreports/ 34. Clarke, A. S. & Wiley, J. D. Numerical simulation of the dense random packing of a binary mixture of hard spheres: Amorphous metals. Phys. Rev. B 35, 7350 (1987). 35. Steinhardt, P. J., Nelson, D. R. & Ronchetti, M. Bond-orientational order in liquids and glasses. Phys. Rev. B 28, 784–805 (1983). 34. Clarke, A. S. & Wiley, J. D. Numerical simulation of the dense random packing of a binary mixture of hard spheres: Amorphous metals. Phys. Rev. B 35, 7350 (1987). 35. Steinhardt, P. J., Nelson, D. R. & Ronchetti, M. Bond-orientational order in liquids and glasses. Phys. Rev. B 28, 784–805 (1983). Acknowledgements Acknowledgementsi Acknowledgements We acknowledge financial support by DFG through collaborative research center TRR 146, and ZDV Mainz for computing time on the MOGON supercomputer. Author Contributions Author Contributions D.R. and T.S. designed the research and wrote the manuscript, D.R. wrote the code, performed simulations, and analyzed the data. All authors reviewed the manuscript. References Mobility and diffusion of a tagged particle in a driven colloidal suspension. EPL 92, 5 (2010).h 31. Russo, J., Maggs, A. C., Bonn, D. & Tanaka, H. The interplay of sedimentation and crystallization in hard-sphere suspensions. Soft Matter 9, 7369–7383 (2013). ft ( ) 32. Allen, M. P. & Tildesley, D. J. Computer Simulation of Liquids (Clarendon Press, New York, NY, USA, 1989). y p f q 33. Koopman, E. A. & Lowe, C. P. Advantages of a lowe-andersen thermostat in molecular dynamics simulations. J. Chem. Phys. 124, 204103 (2006). Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 6 www.nature.com/scientificreports/ Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 Additional Information upplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Richard, D. and Speck, T. The role of shear in crystallization kinetics: From suppression to enhancement. Sci. Rep. 5, 14610; doi: 10.1038/srep14610 (2015). How to cite this article: Richard, D. and Speck, T. The role of shear in crystallization kinetics: From suppression to enhancement. Sci. Rep. 5, 14610; doi: 10.1038/srep14610 (2015). 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 Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 5:14610 \| DOI: 10.1038/srep14610 7
https://openalex.org/W4200080762	https://www.frontiersin.org/articles/10.3389/fendo.2021.750135/pdf	English	null	Table_1.docx	null	2,021	cc-by	6,925	ORIGINAL RESEARCH published: 25 November 2021 doi: 10.3389/fendo.2021.750135 Keywords: type 1 diabetes mellitus, ketosis, ketoacidosis, NK cells subset, differentially expressed genes Reviewed by: Reviewed by: Alpna Tyagi, University of Colorado Anschutz Medical Campus, United States Joubert Kharlyngdoh, Rush University Medical Center, United States Rush University Medical Center, United States *Correspondence: Datong Deng 13855134251@163.com Qiu Zhang aynfmk@163.com Results: A total of 70 DEGs were identiﬁed between T1DM patients recovered from ketosis or ketoacidosis and healthy control blood samples in GSE44314. Among the DEGs, 10 hub genes were screened out. The mature NK cell-speciﬁc gene co-expression network for DEGs in T1DM was built using NetworkAnalyst tools. DEGs between activated CD56+CD16+ NK cells and CD56brightCD16- NK cells were identiﬁed from GSE1511. After intersection, 13 overlapping genes between GSE44314 and GSE1511 microarray datasets were screened out, in which 7 hub genes were identiﬁed. Additionally, 59 target miRNAs were predicted according to the 7 hub genes. After validating with the exosome miRNA expression proﬁle dataset of GSE97123, seven differentially expressed miRNAs (DEmiRNAs) in plasma-derived exosome were selected. Finally, a mRNA–miRNA network was constructed, which was involved in the T1DM ketosis or ketoacidosis process. Specialty section: This article was submitted to Diabetes: Molecular Mechanisms, a section of the journal Frontiers in Endocrinology Received: 30 July 2021 Accepted: 05 November 2021 Published: 25 November 2021 ORIGINAL RESEARCH published: 25 November 2021 doi: 10.3389/fendo.2021.750135 Objectives: Alterations in natural killer (NK) cells activity cause damage to pancreatic islets in type 1 diabetes mellitus (T1DM). The aim of this study is to identify T1DM ketosis- or ketoacidosis-related genes in activated CD56+CD16+ NK cells. Edited by: Ahmet Uc¸ar, S¸is¸li Hamidiye Etfal Education and Research Hospital, Turkey Edited by: Ahmet Uc¸ar, S¸is¸li Hamidiye Etfal Education and Research Hospital, Turkey Methods: Microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were analyzed using the GEO2R tool. Enrichment analyses were performed using Metascape online database and GSEA software. Cell-speciﬁc gene co-expression network was built using NetworkAnalyst tools. Cytoscape software was used to identify hub genes and construct co-expressed networks. Target miRNAs were predicted based on the DIANA-micro T, miRDB, and miRWalk online databases. Identiﬁcation and Enrichment Analysis of Differentially Expressed Genes in T1DM Ketosis or Ketoacidosis The dataset GSE44314 contained ﬁve classical T1DM recovered from ketosis or ketoacidosis and six healthy controls. There was no difference in gender (male: 20.0% vs. 33.3%, p > 0.05) or age (53.8 ± 7.6 vs. 47.0 ± 6.4, p > 0.05) between the two groups in GSE44314 (Table S1). As shown in Figure 1A, a total of 70 DEGs were identiﬁed, including 32 upregulated genes and 38 downregulated genes. The GO and KEGG enrichment analyses were performed using Metascape online tools. In enriched functional category terms, ﬁve enriched GO terms and four KEGG pathways were identiﬁed (Figure 1B). A network of GO and KEGG enriched terms colored by clusters is shown in Figure 1C. MCODE algorithm was applied to this network to identify neighborhoods where proteins were densely connected (Figure 1D). Citation: Shi R, Dai F, He Y, Sun L, Xu M, Deng D and Zhang Q (2021) Comprehensive Analyses of Type 1 Diabetes Ketosis- or Ketoacidosis- Related Genes in Activated CD56+CD16+ NK Cells. Front. Endocrinol. 12:750135. doi: 10.3389/fendo.2021.750135 Conclusion: This work identiﬁed seven hub genes in activated CD56+CD16+ NK cells and seven miRNAs in plasma-derived exosome as potential predictors of T1DM ketoacidosis, which provided a novel insight for the pathogenesis at the transcriptome level. November 2021 \| Volume 12 \| Article 750135 1 Frontiers in Endocrinology \| www.frontiersin.org Ketoacidosis Genes in NK Cells Shi et al. INTRODUCTION NK cell subset samples. The miRNA (in plasma-derived exosomes) expression proﬁle GSE97123 included 12 T1DM and 12 control subjects. The differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRNAs) were analyzed using online tool GEO2R (https://www.ncbi.nlm.nih.gov/geo/ geo2r/). The p-value < 0.05 and \|log2 (Fold-Change)\| ≥1 were considered criteria for DEGs. Overlapping genes were identiﬁed by the Venn diagram webtool (http://bioinformatics.psb.ugent.be/ webtools/Venn/). Diabetic ketoacidosis (DKA) is one kind of serious acute hyperglycemic complication of type 1 diabetes mellitus (T1DM) (1). DKA can be a preliminary sign of T1DM and is associated with high morbidity and mortality without optimal treatment. The cost of hospital treatment for patients experiencing DKA exceeds the cost ofroutinetreatment for diabetes (2).The most common causes of DKA include inadequate dose of insulin, infection, intercurrent illness, and drugs (3). For some patients, DKA is an initial manifestation of T1DM (4). Therefore, early identiﬁcation of DKA-related geneexpression isofgreat signiﬁcance for prevention. The development of T1DM is associated with various immune cell populations as it is a multifactorial autoimmune disease (5–8). Natural killer (NK) cells as a crucial component of immune system are also involved in this process. The inhibitory receptors and activating receptors expressed on human NK cells have been demonstrated to play a vital role in pathological situations (9, 10). Additionally, NK cells can secrete cytokine participating in kinds of immune regulation. Recent studies focus on the association between NK cells and autoimmune diabetes. The frequencies and counts of NK cells in peripheral blood were reported to be altered in newly diagnosed T1DM patients (11, 12). The peripheral blood of LADA patients also exhibited a signiﬁcant decrease in NK cells frequency (13). Therefore, NK cells might serve as a primary immune regulator in T1DM due to their immunoregulatory properties. However, only the activated NK cell subset is abundant with cytokine producers (14). Diabetic ketoacidosis (DKA) is one kind of serious acute hyperglycemic complication of type 1 diabetes mellitus (T1DM) (1). DKA can be a preliminary sign of T1DM and is associated with high morbidity and mortality without optimal treatment. The cost of hospital treatment for patients experiencing DKA exceeds the cost ofroutinetreatment for diabetes (2).The most common causes of DKA include inadequate dose of insulin, infection, intercurrent illness, and drugs (3). For some patients, DKA is an initial manifestation of T1DM (4). Therefore, early identiﬁcation of DKA-related geneexpression isofgreat signiﬁcance for prevention. Construction of a PPI Network A PPI (protein–protein interaction) network was built with Search Tool for the Retrieval of Interacting Genes (STRING) database (http://string-db.org/) (22). The cytoHubba plugin in Cytoscape software (version 3.7.2) was used to select hub genes and construct mRNA–miRNA network. Human NK cells are divided into different populations based on their cell surface density of CD56 and CD16 (15). The CD56brightCD16- NK cells are inactivated and low cytotoxic NK cells, while the CD56+CD16+ NK cells are activated NK cells in inﬂamed tissue (16). Inﬂammation has a broad role in T1DM, which contributes to the induction and ampliﬁcation of immune assault against islet b cells (17). As activated CD56+CD16+ NK cells in T1DM have been investigated scarcely, this study was to explore potential genes in activated CD56+CD16+ NK cells associated with T1DM ketosis or ketoacidosis. Exosomes are important mediators in cell communication. Evidence shows a distinct exosome miRNA signature in T1DM (18). Since a better understanding of exosome miRNA may provide novel insight into DKA, exosome miRNAs related to NK cells in T1DM will also be explored in our study. Prediction and Validation of Target miRNA Online miRNA databases DIANA-micro T, miRDB, and miRWalk were used to predict target miRNAs based on hub genes (23–25). In addition, the DEmiRNA in GSE97123 was analyzed. Only the overlapping miRNA that was found in all the above three databases and veriﬁed with the GSE97123 dataset was considered as target miRNA. Enrichment Analysis The Metascape online database (http://metascape.org) was used for Gene Ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs (19). The enriched KEGG pathway and GO terms were selected with a criterion of p < 0.05. The gene expression information of all T1DM was uploaded to GSEA software to assess the distribution trend of genes, which determine their contribution to the phenotype (20). Cell-speciﬁc gene co-expression network was built using NetworkAnalyst tools (21). g p g g p The development of T1DM is associated with various immune cell populations as it is a multifactorial autoimmune disease (5–8). Natural killer (NK) cells as a crucial component of immune system are also involved in this process. The inhibitory receptors and activating receptors expressed on human NK cells have been demonstrated to play a vital role in pathological situations (9, 10). Additionally, NK cells can secrete cytokine participating in kinds of immune regulation. Recent studies focus on the association between NK cells and autoimmune diabetes. The frequencies and counts of NK cells in peripheral blood were reported to be altered in newly diagnosed T1DM patients (11, 12). The peripheral blood of LADA patients also exhibited a signiﬁcant decrease in NK cells frequency (13). Therefore, NK cells might serve as a primary immune regulator in T1DM due to their immunoregulatory properties. However, only the activated NK cell subset is abundant with cytokine producers (14). GSEA Analysis of T1DM Ketosis- or Ketoacidosis-Related Genes were PID_TXA2PATHWAY, REACTOME_LONG_TERM_ POTENTIATION, PID_EPO_PATHWAY, TERAO_AOX4_ TARGETS_HG_UP, WP_GABA_RECEPTOR_SIGNALING, WP_PDGF_PATHWAY, and ISSAEVA_MLL2_TARGETS. were PID_TXA2PATHWAY, REACTOME_LONG_TERM_ POTENTIATION, PID_EPO_PATHWAY, TERAO_AOX4_ TARGETS_HG_UP, WP_GABA_RECEPTOR_SIGNALING, WP_PDGF_PATHWAY, and ISSAEVA_MLL2_TARGETS. The gene in expression proﬁle was analyzed using Molecular Signatures Database at a holistic level via GSEA software. A total of 2,565 gene sets were upregulated in the classical T1DM samples compared to healthy controls. A total of 1,046 gene sets were signiﬁcantly enriched at FDR < 0.25 and 1,194 gene sets were signiﬁcantly enriched at nominal p-value < 0.01. The enrichment results of GSEA analysis showed pathways enriched in the T1DM ketosis or ketoacidosis samples (Table S2). As shown in Figure 2, the top seven gene sets with NES>2.5 Microarray Data Acquisition and Processing The gene expression proﬁles including GSE44314, GSE1511, and GSE97123 were downloaded from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/).The microarray proﬁle dataset GSE44314, deposited by Shinsuke et al., was conducted on blood samples from ﬁve classical type 1A diabetes recovered from ketosisorketoacidosisandsixhealthycontrols.Thegeneexpression dataset GSE1511, provided by Mandelboim et al., was selected for containing CD56brightCD16- and activated CD56+CD16+ human November 2021 \| Volume 12 \| Article 750135 Frontiers in Endocrinology \| www.frontiersin.org 2 Shi et al. Ketoacidosis Genes in NK Cells A B D C FIGURE 1 \| Differentially expressed genes in T1DM ketosis or ketoacidosis. (A) Volcano plot for differentially expressing genes between T1DM recovered from ketosis or ketoacidosis and healthy control samples. Red dots represented upregulated genes, while green dots represented downregulated genes. (B) Heatmap of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. (C) Network of GO and KEGG enriched terms colored by clusters. (D) MCODE algorithm was applied to this network to identify neighborhoods. Each MCODE network was assigned a unique color. GO enrichment analysis was applied to each MCODE network to assign “meanings” to the network component. B D D FIGURE 1 \| Differentially expressed genes in T1DM ketosis or ketoacidosis. (A) Volcano plot for differentially expressing genes between T1DM recovered from ketosis or ketoacidosis and healthy control samples. Red dots represented upregulated genes, while green dots represented downregulated genes. (B) Heatmap of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. (C) Network of GO and KEGG enriched terms colored by clusters. (D) MCODE algorithm was applied to this network to identify neighborhoods. Each MCODE network was assigned a unique color. GO enrichment analysis was applied to each MCODE network to assign “meanings” to the network component. Identiﬁcation and Cluster Analysis of Overlapping Genes Overlapping Genes As shown in Figure 5A, there were 13 overlapping genes (COX7B, HLA-DQA1, RPS3A, PFDN1, PSMA6, KIAA0101, HLA-DQB1, GZMK, NDUFA4, CKLF, LTF, TIMM8B, and IER3IP1) between DEGs in T1DM and DEGs in the activated CD56+CD16+ NK cell subset. Based on the STRING online database, a PPI network with an interaction score >0.4 was constructed (Figure 5B). The hub genes (COX7B, HLA-DQA1, RPS3A, PFDN1, PSMA6, HLA-DQB1, and NDUFA4) were identiﬁed using Cytoscape software (Figure 5C). Enriched KEGG pathways included “Downstream TCR signaling” and “Cytoprotection by HMOX1”, which were identiﬁed using Metascape online tools (Figure 5D). Hub Gene Identiﬁcation of T1DM Ketosis or Ketoacidosis A PPI network with 63 nodes and 28 edges was obtained using the STRING tools (Figure S1). The CytoHubba plugin in Cytoscape software was used to cluster network genes to FIGURE 2 \| The whole gene expression value of T1DM ketosis or ketoacidosis and control samples analyzed by GSEA software. The top seven gene sets were enriched with criterion of NES>2.5, FDR < 0.25, and p-value < 0.01. FIGURE 2 \| The whole gene expression value of T1DM ketosis or ketoacidosis and control samples analyzed by GSEA software. The top seven gene sets were enriched with criterion of NES>2.5, FDR < 0.25, and p-value < 0.01. FIGURE 2 \| The whole gene expression value of T1DM ketosis or ketoacidosis and control samples analyzed by GSEA software enriched with criterion of NES>2.5, FDR < 0.25, and p-value < 0.01. November 2021 \| Volume 12 \| Article 750135 3 Frontiers in Endocrinology \| www.frontiersin.org Ketoacidosis Genes in NK Cells Shi et al. identify hub genes in network. The network with an interaction score >0.4 was built according to the STRING online database (Figure 3A). The edge represented the link between different genes. As shown in Figures 3B, a key module with eight upregulated genes (RPS15A, RPS7, RPL9, RPS3A, NDUFA4, MRPL43, PSMA6, and PFDN1) and two downregulated genes (GART and TUBB2A) was identiﬁed. It was consistent with Figure 1D in which one module was made up of RPS15A, RPS7, RPL9, and RPS3A. In addition, cell-speciﬁc gene co-expression network was built using NetworkAnalyst tools. The mature NK cell-speciﬁc gene co-expression network for DEGs in T1DM ketosis or ketoacidosis is shown in Figure 3C. establishment of protein localization to organelle, small molecule biosyntheticprocess,cellcyclecheckpoint,virallifecycle,andpeptide biosynthetic process (Table 1). The summary of enrichment analysis in PaGenBase conﬁrmed tissue and cell speciﬁc for these genes (Figure 4C and Table 2). NK Cell Subset The microarray expression dataset GSE1511 was analyzed to acquire DEGsbetweenCD56brightCD16-andactivatedCD56+CD16+human NK cell subsets (Figure 4A). A total of 2,671 DEGs were identiﬁed including 282 upregulated genes and 2,389 downregulated genes. Enriched GO terms and KEGG pathways were identiﬁed using Metascape online tools (Figure 4B). Cell cycle, Cellular responses to stress, Retinoblastoma Gene in Cancer, Mitotic Prometaphase, Transcriptional Regulation by TP53, Processing of Capped Intron- Containing Pre-mRNA, and Ciliary landscape were signiﬁcantly enriched in KEGG pathway. The enriched GO terms included DNA repair, cell division, mitochondrion organization, DNA replication, leukocyte activation involved in immune response, regulation of chromosome organization, mitochondrial translation, Prediction and Validation of Target miRNA The target miRNAs were predicted based on the DIANA-micro T, miRDB, and miRWalk online databases. A total of 1,718 target miRNAs to seven speciﬁcally expressed hub genes were obtained. There were 59 miRNAs after intersection (Figure 6A). The miRNA–mRNA regulatory network was constructed using CytoHubba (Figure 6B). DEmiRNAs in T1DM patients were acquired by analyzing the GSE97123 A C B FIGURE 3 \| PPI network analysis of DEGs in T1DM ketosis or ketoacidosis. The node represented gene and the edge represented link between genes. (A) PPI network of differentially expression genes in T1DM ketosis or ketoacidosis. (B) Cytoscape network clustering visualization of hub DEGs in T1DM ketosis or ketoacidosis samples. The node represented gene and the edge represented link between genes. The red/orange/yellow color meant the MMC score was highest/moderate high/mild high. (C) The mature NK cell-speciﬁc gene co-expression network for DEGs in T1DM ketosis or ketoacidosis. C A A C C B B FIGURE 3 \| PPI network analysis of DEGs in T1DM ketosis or ketoacidosis. The node represented gene and the edge represented link between genes. (A) PPI network of differentially expression genes in T1DM ketosis or ketoacidosis. (B) Cytoscape network clustering visualization of hub DEGs in T1DM ketosis or ketoacidosis samples. The node represented gene and the edge represented link between genes. The red/orange/yellow color meant the MMC score was highest/moderate high/mild high. (C) The mature NK cell-speciﬁc gene co-expression network for DEGs in T1DM ketosis or ketoacidosis. November 2021 \| Volume 12 \| Article 750135 Frontiers in Endocrinology \| www.frontiersin.org Shi et al. Shi et al. Ketoacidosis Genes in NK Cells A B C FIGURE 4 \| Differentially expressed genes in activated CD56+CD16+ human NK cell subset samples. NK Cell Subset (A) Volcano plot for differentially expressing genes between CD56brightCD16- and activated CD56+CD16+ human NK cell subset samples. Red dots represented signiﬁcantly upregulated genes, while green dots represented signiﬁcantly downregulated genes. (B) Heatmap of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. (C) The summary of enrichment analysis in PaGenBase conﬁrmed tissue and cell speciﬁc for these genes. A A B B C C FIGURE 4 \| Differentially expressed genes in activated CD56+CD16+ human NK cell subset samples. (A) Volcano plot for differentially expressing genes between CD56brightCD16- and activated CD56+CD16+ human NK cell subset samples. Red dots represented signiﬁcantly upregulated genes, while green dots represented signiﬁcantly downregulated genes. (B) Heatmap of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. (C) The summary of enrichment analysis in PaGenBase conﬁrmed tissue and cell speciﬁc for these genes. TABLE 1 \| Top clusters with their representative enriched GO terms (one per cluster). GO Category Description Count % Log10(p) Log10(q) GO:0006281 GO Biological Processes DNA repair 164 7.13 −47.59 −44.28 GO:0051301 GO Biological Processes Cell division 156 6.79 −39.36 −36.37 GO:0007005 GO Biological Processes Mitochondrion organization 146 6.35 −38.15 −35.18 GO:0006260 GO Biological Processes DNA replication 95 4.13 −33.93 −31.14 GO:0002366 GO Biological Processes Leukocyte activation involved in immune response 162 7.05 −32.62 −29.90 GO:0033044 GO Biological Processes Regulation of chromosome organization 85 3.70 −27.51 −25.12 GO:0032543 GO Biological Processes Mitochondrial translation 56 2.44 −25.58 −23.31 GO:0072594 GO Biological Processes Establishment of protein localization to organelle 124 5.39 −22.99 −20.89 GO:0044283 GO Biological Processes Small molecule biosynthetic process 138 6.00 −22.38 −20.32 GO:0000075 GO Biological Processes Cell cycle checkpoint 66 2.87 −21.88 −19.84 GO:0019058 GO Biological Processes Viral life cycle 87 3.78 −20.77 −18.79 GO:0043043 GO Biological Processes Peptide biosynthetic process 139 6.05 −19.94 −17.99 “Count” is the number of genes in the given ontology term. “%” is the percentage of all genes that are found in the given ontology term. “Log10(p)” is the p-value in log base 10. “Log10(q)” is the multi-test adjusted p-value in log base 10. TABLE 1 \| Top clusters with their representative enriched GO terms (one per cluster). November 2021 \| Volume 12 \| Article 750135 Frontiers in Endocrinology \| www.frontiersin.org 5 dataset, in which all miRNAs were obtained from plasma- derived exosome. There was no difference in gender (male: 50.0% vs. NK Cell Subset 50.0%, p > 0.05) or age (41.3 ± 2.9 vs. 46.4 ± 4.0, p > 0.05) between the two groups in GSE97123 (Table S3). After intersection with the above 59 predicted miRNAs, 7 miRNAs were screened out (Figure 6C). A co-expressed network on T1DM ketosis or ketoacidosis was built based on the predicted miRNA–RNA pairs (Figure 6D). TABLE 2 \| Summary of enrichment analysis in PaGenBase. GO Description Count % Log10(p) Log10(q) PGB:00041 Tissue-speciﬁc: blood 58 2.50 −14.00 −11.00 PGB:00016 Tissue-speciﬁc: thymus 43 1.90 −7.70 −5.70 PGB:00043 Cell-speciﬁc: CD56+ NK Cells 18 0.78 −5.80 −4.00 PGB:00048 Tissue-speciﬁc: bone marrow 40 1.70 −5.50 −3.80 PGB:00011 Tissue-speciﬁc: spleen 70 3.00 −5.20 −3.50 PGB:00046 Cell-speciﬁc: 721B lymphoblasts 21 0.91 −5.20 −3.50 PGB:00101 Tissue-speciﬁc: Colorectal adenocarcinoma 12 0.52 −4.10 −2.60 PGB:00060 Tissue-speciﬁc: retinoblastoma 19 0.83 −3.50 −2.10 PGB:00111 Cell-speciﬁc: ZR75_1 5 0.22 −2.40 −1.10 PGB:00026 Cell-speciﬁc: CD71+ Early Erythroid 22 0.96 −2.40 −1.10 PGB:00036 Cell-speciﬁc: MOLT4 20 0.87 −2.10 −0.91 A B D C FIGURE 5 \| Identiﬁcation and cluster analysis of overlapping genes. (A) Venn diagram of differentially expressed genes between the DEGs in T1DM ketosis or ketoacidosis and DEGs in the activated CD56+CD16+ human NK cell subset. (B) PPI network for the 13 overlapping genes. (C) Analysis of hub genes using cytoHubba for the overlapped genes. (D) Heatmap of Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. Shi et al. Ketoacidosis Genes in NK Cells Ketoacidosis Genes in NK Cells Shi et al. TABLE 2 \| Summary of enrichment analysis in PaGenBase. GO Description Count % Log10(p) Log10(q) PGB:00041 Tissue-speciﬁc: blood 58 2.50 −14.00 −11.00 PGB:00016 Tissue-speciﬁc: thymus 43 1.90 −7.70 −5.70 PGB:00043 Cell-speciﬁc: CD56+ NK Cells 18 0.78 −5.80 −4.00 PGB:00048 Tissue-speciﬁc: bone marrow 40 1.70 −5.50 −3.80 PGB:00011 Tissue-speciﬁc: spleen 70 3.00 −5.20 −3.50 PGB:00046 Cell-speciﬁc: 721B lymphoblasts 21 0.91 −5.20 −3.50 PGB:00101 Tissue-speciﬁc: Colorectal adenocarcinoma 12 0.52 −4.10 −2.60 PGB:00060 Tissue-speciﬁc: retinoblastoma 19 0.83 −3.50 −2.10 PGB:00111 Cell-speciﬁc: ZR75_1 5 0.22 −2.40 −1.10 PGB:00026 Cell-speciﬁc: CD71+ Early Erythroid 22 0.96 −2.40 −1.10 PGB:00036 Cell-speciﬁc: MOLT4 20 0.87 −2.10 −0.91 A TABLE 2 \| Summary of enrichment analysis in PaGenBase. NK Cell Subset GO Description Count % Log10(p) Log10(q) PGB:00041 Tissue-speciﬁc: blood 58 2.50 −14.00 −11.00 PGB:00016 Tissue-speciﬁc: thymus 43 1.90 −7.70 −5.70 PGB:00043 Cell-speciﬁc: CD56+ NK Cells 18 0.78 −5.80 −4.00 PGB:00048 Tissue-speciﬁc: bone marrow 40 1.70 −5.50 −3.80 PGB:00011 Tissue-speciﬁc: spleen 70 3.00 −5.20 −3.50 PGB:00046 Cell-speciﬁc: 721B lymphoblasts 21 0.91 −5.20 −3.50 PGB:00101 Tissue-speciﬁc: Colorectal adenocarcinoma 12 0.52 −4.10 −2.60 PGB:00060 Tissue-speciﬁc: retinoblastoma 19 0.83 −3.50 −2.10 PGB:00111 Cell-speciﬁc: ZR75_1 5 0.22 −2.40 −1.10 PGB:00026 Cell-speciﬁc: CD71+ Early Erythroid 22 0.96 −2.40 −1.10 PGB:00036 Cell-speciﬁc: MOLT4 20 0.87 −2.10 −0.91 A B D C FIGURE 5 \| Identiﬁcation and cluster analysis of overlapping genes. (A) Venn diagram of differentially expressed genes between the DEGs in T1DM ketosis or ketoacidosis and DEGs in the activated CD56+CD16+ human NK cell subset. (B) PPI network for the 13 overlapping genes. (C) Analysis of hub genes using cytoHubba for the overlapped genes. (D) Heatmap of Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. C B D C C B D FIGURE 5 \| Identiﬁcation and cluster analysis of overlapping genes. (A) Venn diagram of differentially expressed genes between the DEGs in T1DM ketosis or ketoacidosis and DEGs in the activated CD56+CD16+ human NK cell subset. (B) PPI network for the 13 overlapping genes. (C) Analysis of hub genes using cytoHubba for the overlapped genes. (D) Heatmap of Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched clusters colored by p-value. dataset, in which all miRNAs were obtained from plasma- derived exosome. There was no difference in gender (male: 50.0% vs. 50.0%, p > 0.05) or age (41.3 ± 2.9 vs. 46.4 ± 4.0, p > 0.05) between the two groups in GSE97123 (Table S3). After intersection with the above 59 predicted miRNAs, 7 miRNAs were screened out (Figure 6C). A co-expressed network on T1DM ketosis or ketoacidosis was built based on the predicted miRNA–RNA pairs (Figure 6D). November 2021 \| Volume 12 \| Article 750135 Frontiers in Endocrinology \| www.frontiersin.org 6 Shi et al. Ketoacidosis Genes in NK Cells A B D C FIGURE 6 \| Prediction and validation of target miRNA. (A) Venn diagram showing the numbers of predicted miRNAs based on the DIANA-micro T, miRDB, and miRWalk online databases. (B) The relationship between 7 hub genes and 59 miRNAs (miRNA–mRNA regulatory network). DISCUSSION still unclariﬁed (17). T cells are proved to inﬁltrate pancreatic islets, resulting in b cell destruction. However, NK cells are the ﬁrst type of cells to inﬁltrate islets without the need for previous activation (28). NK cells have been demonstrated to be involved in several steps of the immune-mediated attack (29). A more recent study shows that NK cell subsets are associated with the partial remission of T1DM (30). Based on CD56 and CD16 expression, human NK cells can be puriﬁed into different subpopulations, which have unique repertoires of the chemokine receptor (14, 15, 31). CD56+CD16+ NK cells can be activated by cytokine IL-21 in combination with IL-15 (32). Moreover, activated CD56+CD16+ NK cells have been demonstrated to be highly cytotoxic and strikingly distinct from other subsets (16). However, the study on activated CD56+CD16+ NK cells in T1DM ketone or ketoacidosis is rare. Our study analyzed DEGs between healthy controls and T1DM who recovered from ketosis or ketoacidosis, as well as the DEGs between activated CD56+CD16+ NK cells and CD56brightCD16- NK cells. In order to explore the correlation between T1DM ketone prone and activated CD56+CD16+ NK cells, the intersection of DEGs in the two data series was performed and 13 overlapped genes were screened out. Moreover, seven hub genes were picked out by PPI analysis. The seven hub genes in activated CD56+CD16+ NK cells might play a critical role in type 1A diabetes ketosis or ketoacidosis. Based on predicted miRNAs for the seven hub genes and miRNAs in the GSE97123 dataset, seven miRNAs in plasma-derived exosomes were picked out, which might become detectable biomarkers in plasma. In our study, DEGs analysis for T1DM with a history of ketone or ketoacidosis and activated CD56+CD16+ NK cells were combined to identify potential predicted genes for T1DM ketoacidosis. After PPI analysis, seven key genes were identiﬁed from 13 overlapping genes, which formed two modules. One module was formed with HLA-DAQ1 and HLA-DBQ1. It is well known that human leukocyte antigen (HLA) class II gene alleles at the DQA1 and DQB1 loci are the major genetic determinants for T1DM (27). According to the GeneCards database (https://www.genecards.org/), ﬁve other genes in the other module are involved in cell metabolism. Cytochrome C Oxidase Subunit 7B (COX7B) is the last enzyme in the mitochondrial electron transport chain that drives oxidative phosphorylation. Mitochondrial Complex Associated (NDUFA4) codes protein that has NADH dehydrogenase activity and oxidoreductase activity. NK Cell Subset (C) Venn diagram showing the intersection between predicted miRNAs and DEmiRNAs in T1DM patients from the GSE97123 dataset. (D) The relationship between seven hub genes and seven validated miRNAs (miRNA–mRNA regulatory network). A B B C D D C FIGURE 6 \| Prediction and validation of target miRNA. (A) Venn diagram showing the numbers of predicted miRNAs based on the DIANA-micro T, miRDB, and miRWalk online databases. (B) The relationship between 7 hub genes and 59 miRNAs (miRNA–mRNA regulatory network). (C) Venn diagram showing the intersection between predicted miRNAs and DEmiRNAs in T1DM patients from the GSE97123 dataset. (D) The relationship between seven hub genes and seven validated miRNAs (miRNA–mRNA regulatory network). Frontiers in Endocrinology \| www.frontiersin.org DISCUSSION Ribosomal Diabetes increases ketone production and decreases ketone clearance (26). Genetic factors play a dominant role in the progress. The association between genes and the risk for DKA is still unclariﬁed (27). The type 1A diabetes in GSE4431 have been recovered from ketosis or ketoacidosis and treated with insulin intensively for at least 1 month. Therefore, samples from T1DM were free of metabolic derangements and represented the genetic difference for ketosis or ketoacidosis prone. A total of 70 DEGs were identiﬁed in our study. Additionally, most of them were clustered according to mature NK cell-speciﬁc gene co- expression network. This was in line with a previous study, which reported that NK cells participated in the process of T1DM (28). Type 1A diabetes is mediated by islet-associated autoimmunity, in which immune cells play a critical role, while the mechanism is November 2021 \| Volume 12 \| Article 750135 Frontiers in Endocrinology \| www.frontiersin.org 7 Shi et al. Ketoacidosis Genes in NK Cells Protein S3A (RPS3A) is a protein coding gene. Among its related pathways are metabolism of proteins and HIV Life Cycle. Prefoldin Subunit 1 (PFDN1)-related pathways are metabolism of proteins and cooperation of Prefoldin and TriC/CCT in actin and tubulin folding. Proteasome 20S Subunit Alpha 6 (PSMA6) is a protein coding gene. Among its related pathways are Cellular Senescence (REACTOME) and RET signaling. In summary, seven hub genes were identiﬁed from two datasets. In addition, seven target miRNAs were predicted and validated using GEO expression proﬁling data. Finally, a miRNA–mRNA network was constructed. Our study provided a reliable comprehensive analysis on the DEGs proﬁle in activated CD56+CD16+ NK cells for T1DM ketoacidosis, which provided a novel insight for the pathogenesis at transcriptome. The result of enrichment analysis for the seven hub genes included downstream TCR signaling and cytoprotection by HMOX1. T cell receptors (TCRs) play a vital role in T-cell function and immunological synapse formation. TCR activation promotes a number of signaling cascades that determine cell fate through regulating cytokine production, cell survival, proliferation, and differentiation. TCR signaling events drive the progression of T1DM by affecting T-cell development (33). Sequencing of TCR gene in pancreatic islets of T1DM donors reveals repeat clonal expansion and supports the existence of public TCRs shared among T1DM (34–37). FUNDING This study was supported by the National Natural Science Foundation of China (No. 82100845) and the Cultivation Plan of First Afﬁliated Hospital of Anhui Medical University for National Natural Science Foundation of China Youth Science Foundation in 2021 (2021kj03). Exosome miRNA proﬁling in different body ﬂuids have been reported to have potential in the diagnosis of disease (40). Exosomes, as small single-stranded non-coding RNAs, have been demonstrated to take part in islet autoimmunity (41). Emerging studies focus on the relationship between exosomes and T1DM development (42). Our study predicted target miRNAs according to seven hub genes. Moreover, the predicted miRNAs were veriﬁed with the data from GSE97123, which were derived from plasma exosome. Therefore, the predicted miRNAs in our study could be accurate and detectable predictors for early ketoacidosis. The role of miRNA as a key regulator of mRNA has been well established (43). Accordingly, a co-expressed network of mRNAs–miRNAs was constructed in our study. Nevertheless, our results were based on bioinformatics analysis, which was the limitation of this study. Future research is needed to explore the clinical application value of these potential biomarkers. DISCUSSION Heme Oxygenase 1 (HOMX1) is an essential enzyme in heme catabolism and cleaves heme to form biliverdin, which is subsequently converted to bilirubin by biliverdin reductase, and carbon monoxide, a putative neurotransmitter. Heme oxygenase 1 (HO-1), also named heme oxygenase-1 (HO-1), expression upregulation in high glucose plus oxidized LDL-treated primary peritoneal macrophages from wild-type mice and Nrf2/HO-1 can be a therapeutic target for diabetic nephropathy (38, 39). It is consistent with our enrichment result that downstream TCR signaling and cytoprotection by HMOX1 may be involved in T1DM with ketosis or ketoacidosis. Therefore, the seven key genes in activated CD56+CD16+ NK cells might play a vital role in T1DM ketosis or ketoacidosis. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2021.750135/ full#supplementary-material Supplementary Figure 1 \| A PPI network for T1DM ketosis or ketoacidosis obtained using the STRING tools. Supplementary Table 1 \| The Characteristic of 5 classical T1DM recovered from ketosis or ketoacidosis and 6 healthy controls samples in GSE44314. Supplementary Table 2 \| The enrichment results of GSEA analysis for pathways enriched in the T1DM ketosis or ketoacidosis samples. Supplementary Table 3 \| The Characteristic of 12 T1DM and 12 healthy controls samples in GSE97123. Supplementary Table 3 \| The Characteristic of 12 T1DM and 12 healthy controls samples in GSE97123. AUTHOR CONTRIBUTIONS RS was involved in the overall study, designed the analysis plan, analyzed the data, and wrote the manuscript. FD, YH, LS, and MX collected the data and analyzed the data. DD and QZ contributed to the discussion and reviewed the manuscript. All authors contributed to the article and approved the submitted version. 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 at: https://www.ncbi.nlm. nih.gov/geo/, GSE44314; https://www.ncbi.nlm.nih.gov/geo/, GSE1511; https://www.ncbi.nlm.nih.gov/geo/, GSE97123. 4. Wang ZH, Kihl-Selstam E, Eriksson JW. Ketoacidosis Occurs in Both Type 1 and Type 2 Diabetes–A Population-Based Study From Northern Sweden. 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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 Shi, Dai, He, Sun, Xu, Deng and Zhang. 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. 43. Carthew RW. Gene Regulation by microRNAs. Curr Opin Genet Dev (2006) 16(2):203–8. doi: 10.1016/j.gde.2006.02.012 Frontiers in Endocrinology \| www.frontiersin.org November 2021 \| Volume 12 \| Article 750135 REFERENCES Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in November 2021 \| Volume 12 \| Article 750135 Frontiers in Endocrinology \| www.frontiersin.org 10
https://openalex.org/W2635658300	https://curis.ku.dk/ws/files/199170326/1_s2.0_S000632231732245X_main.pdf	English	null	A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder	Biological psychiatry	2,018	cc-by	10,009	university of copenhagen university of copenhagen A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder Martin, Joanna; Walters, Raymond K.; Demontis, Ditte; Mattheisen, Manuel; Lee, S. Hong; Robinson, Elise; Brikell, Isabell; Ghirardi, Laura; Larsson, Henrik; Lichtenstein, Paul; Eriksson, Nicholas; 23andMe Research Team; Psychiatric Genomics Consortium: ADHD Subgroup; iPSYCH–Broad ADHD Workgroup; Werge, Thomas; Nordentoft, Merete Published in: Biological Psychiatry university of copenhagen A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder Martin, Joanna; Walters, Raymond K.; Demontis, Ditte; Mattheisen, Manuel; Lee, S. Hong; Robinson, Elise; Brikell, Isabell; Ghirardi, Laura; Larsson, Henrik; Lichtenstein, Paul; Eriksson, Nicholas; 23andMe Research Team; Psychiatric Genomics Consortium: ADHD Subgroup; iPSYCH–Broad ADHD Workgroup; Werge, Thomas; Nordentoft, Merete Published in: Biological Psychiatry Download date: 24. Oct. 2024 Citation for published version (APA): Martin, J., Walters, R. K., Demontis, D., Mattheisen, M., Lee, S. H., Robinson, E., Brikell, I., Ghirardi, L., Larsson, H., Lichtenstein, P., Eriksson, N., 23andMe Research Team, Psychiatric Genomics Consortium: ADHD Subgroup, iPSYCH–Broad ADHD Workgroup, Werge, T., & Nordentoft, M. (2018). A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder. Biological Psychiatry, 83(12), 1044-1053. https://doi.org/10.1016/j.biopsych.2017.11.026 A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder Martin, Joanna; Walters, Raymond K.; Demontis, Ditte; Mattheisen, Manuel; Lee, S. Hong; Robinson, Elise; Brikell, Isabell; Ghirardi, Laura; Larsson, Henrik; Lichtenstein, Paul; Eriksson, Nicholas; 23andMe Research Team; Psychiatric Genomics Consortium: ADHD Subgroup; iPSYCH–Broad ADHD Workgroup; Werge, Thomas; Nordentoft, Merete Published in: Biological Psychiatry Published in: Biological Psychiatry Published in: Biological Psychiatry DOI: 10.1016/j.biopsych.2017.11.026 DOI: 10.1016/j.biopsych.2017.11.026 Publication date: 2018 Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Document license: CC BY Document license: CC BY Document license: CC BY Citation for published version (APA): Martin, J., Walters, R. K., Demontis, D., Mattheisen, M., Lee, S. H., Robinson, E., Brikell, I., Ghirardi, L., Larsson, H., Lichtenstein, P., Eriksson, N., 23andMe Research Team, Psychiatric Genomics Consortium: ADHD Subgroup, iPSYCH–Broad ADHD Workgroup, Werge, T., & Nordentoft, M. (2018). A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder. Biological Psychiatry, 83(12), 1044-1053. https://doi.org/10.1016/j.biopsych.2017.11.026 Citation for published version (APA): Martin, J., Walters, R. K., Demontis, D., Mattheisen, M., Lee, S. H., Robinson, E., Brikell, I., Ghirardi, L., Larsson, H., Lichtenstein, P., Eriksson, N., 23andMe Research Team, Psychiatric Genomics Consortium: ADHD Subgroup, iPSYCH–Broad ADHD Workgroup, Werge, T., & Nordentoft, M. (2018). A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deficit/Hyperactivity Disorder. Biological Psychiatry, 83(12), 1044-1053. https://doi.org/10.1016/j.biopsych.2017.11.026 Download date: 24. Oct. 2024 Archival Report Biological Psychiatry A Genetic Investigation of Sex Bias in the Prevalence of Attention-Deﬁcit/Hyperactivity Disorder Joanna Martin, Raymond K. Walters, Ditte Demontis, Manuel Mattheisen, S. Hong Lee, Elise Robinson, Isabell Brikell, Laura Ghirardi, Henrik Larsson, Paul Lichtenstein, Nicholas Eriksson, 23andMe Research Team, Psychiatric Genomics Consortium: ADHD Subgroup, iPSYCH–Broad ADHD Workgroup, Thomas Werge, Preben Bo Mortensen, Marianne Giørtz Pedersen, Ole Mors, Merete Nordentoft, David M. Hougaard, Jonas Bybjerg-Grauholm, Naomi R. Wray, Barbara Franke, Stephen V. Faraone, Michael C. O’Donovan, Anita Thapar, Anders D. Børglum, and Benjamin M. Neale ABSTRACT The ﬁrst robust genome-wide signiﬁcant single nucleotide polymorphisms (SNPs) were recently identiﬁed in a GWAS meta-analysis of 20,183 ADHD cases and 35,191 controls/ pseudocontrols (7). While such efforts are beginning to shed light on ADHD biology, secondary genome-wide analyses can address important issues regarding the etiological and clinical heterogeneity of ADHD. In children, boys show a two to seven times higher ADHD diagnosis rate than girls (1,8). The male excess is more pronounced in the community hood (2). The r lence are unclea at elucidating th Sex-Speciﬁc One possibility from male ADH not detected an ADHD heritabili the same gene both sexes. If A SEE COMMENTARY ON PAGE e5 1044 ª 2017 Society of Biological Psychiatry. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal Attention-deﬁcit/hyperactivity disorder (ADHD) is a common (w5% childhood prevalence), highly heritable (70%–80%) neurodevelopmental disorder (1,2). Recent genome-wide association studies (GWASs) implicate thousands of genetic risk variants across the allele frequency spectrum (3–7). The ﬁrst robust genome-wide signiﬁcant single nucleotide polymorphisms (SNPs) were recently identiﬁed in a GWAS meta-analysis of 20,183 ADHD cases and 35,191 controls/ pseudocontrols (7). While such efforts are beginning to shed light on ADHD biology, secondary genome-wide analyses can address important issues regarding the etiological and clinical heterogeneity of ADHD. Attention-deﬁcit/hyperactivity disorder (ADHD) is a common (w5% childhood prevalence), highly heritable (70%–80%) neurodevelopmental disorder (1,2). Recent genome-wide association studies (GWASs) implicate thousands of genetic risk variants across the allele frequency spectrum (3–7). The ﬁrst robust genome-wide signiﬁcant single nucleotide polymorphisms (SNPs) were recently identiﬁed in a GWAS meta-analysis of 20,183 ADHD cases and 35,191 controls/ pseudocontrols (7). While such efforts are beginning to shed light on ADHD biology, secondary genome-wide analyses can address important issues regarding the etiological and clinical heterogeneity of ADHD. pronounced in individuals ascertained from clinics than from the community, and this difference attenuates during adult- hood (2). The reasons for the difference in childhood preva- lence are unclear. Here, we present a series of analyses aimed at elucidating the basis for this difference. Sex-Speciﬁc Heterogeneity One possibility is that female ADHD is qualitatively different from male ADHD. Although the majority of twin studies have not detected any quantitative or qualitative sex differences in ADHD heritability (9–12), this does not necessarily imply that the same genetic variants are involved in ADHD etiology in both sexes. If ADHD in clinically diagnosed male individuals is In children, boys show a two to seven times higher ADHD diagnosis rate than girls (1,8). The male excess is more ABSTRACT ABSTRACT BACKGROUND: Attention-deﬁcit/hyperactivity disorder (ADHD) shows substantial heritability and is two to seven times more common in male individuals than in female individuals. We examined two putative genetic mechanisms underlying this sex bias: sex-speciﬁc heterogeneity and higher burden of risk in female cases. ABSTRACT BACKGROUND: Attention-deﬁcit/hyperactivity disorder (ADHD) shows substantial heritability and is two to seven times more common in male individuals than in female individuals. We examined two putative genetic mechanisms underlying this sex bias: sex-speciﬁc heterogeneity and higher burden of risk in female cases. METHODS: We analyzed genome-wide autosomal common variants from the Psychiatric Genomics Consortium and iPSYCH Project (n = 20,183 cases, n = 35,191 controls) and Swedish population register data (n = 77,905 cases, n = y g p g y g METHODS: We analyzed genome-wide autosomal common variants from the Psychiatric Genomics Consortium and iPSYCH Project (n = 20,183 cases, n = 35,191 controls) and Swedish population register data (n = 77,905 cases, n = 1,874,637 population controls). RESULTS: Genetic correlation analyses using two methods suggested near complete sharing of common variant effects across sexes, with rg estimates close to 1. Analyses of population data, however, indicated that female in- dividuals with ADHD may be at especially high risk for certain comorbid developmental conditions (i.e., autism spectrum disorder and congenital malformations), potentially indicating some clinical and etiological heterogeneity. Polygenic risk score analysis did not support a higher burden of ADHD common risk variants in female cases (odds ratio [conﬁdence interval] = 1.02 [0.98–1.06], p = .28). In contrast, epidemiological sibling analyses revealed that the siblings of female individuals with ADHD are at higher familial risk for ADHD than the siblings of affected male individuals (odds ratio [conﬁdence interval] = 1.14 [1.11–1.18], p = 1.5E-15). ( [ ] [ ] p ) CONCLUSIONS: Overall, this study supports a greater familial burden of risk in female individuals with ADHD and some clinical and etiological heterogeneity, based on epidemiological analyses. However, molecular genetic analyses suggest that autosomal common variants largely do not explain the sex bias in ADHD prevalence. Keywords: ADHD, Epidemiology, GWAS, Neurodevelopmental disorders, Polygenic risk score analysis, Sex bias https://doi.org/10.1016/j.biopsych.2017.11.026 Keywords: ADHD, Epidemiology, GWAS, Neurodevelopmental disorders, Polygenic risk score analysis, Sex bias https://doi.org/10.1016/j.biopsych.2017.11.026 https://doi.org/10.1016/j.biopsych.2017.11.026 Attention-deﬁcit/hyperactivity disorder (ADHD) is a common (w5% childhood prevalence), highly heritable (70%–80%) neurodevelopmental disorder (1,2). Recent genome-wide association studies (GWASs) implicate thousands of genetic risk variants across the allele frequency spectrum (3–7). Genetic Data Data for ADHD cases and controls were available from the Psychiatric Genomics Consortium (PGC) and the Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH). See GWAS publication for full details of quality control, imputation, and principal components analysis (7) and the Supplement for a more detailed description of all methods. The iPSYCH samples were genotyped, processed, and analyzed in 23 separate waves. See Supplemental Table S1 for sex-stratiﬁed sample sizes for each PGC study and iPSYCH wave. The total sample size after all quality control was n = 20,183 cases (25% female) and n = 35,191 pseudocontrols/ population controls (38% female). Analyses that were restricted to European-only samples consisted of 19,099 cases and 34,194 controls. ADHD GWAS summary statistics were also available from research participants of the personal genetics company 23andMe Inc. (n = 5857 self-reported ADHD cases, n = 70,393 controls) (Mountain View, CA). Data for ADHD cases and controls were available from the Psychiatric Genomics Consortium (PGC) and the Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH). See GWAS publication for full details of quality control, imputation, and principal components analysis (7) and the Supplement for a more detailed description of all methods. The iPSYCH samples were genotyped, processed, and analyzed in 23 separate waves. See Supplemental Table S1 for sex-stratiﬁed sample sizes for each PGC study and iPSYCH wave. The total sample size after all quality control was n = 20,183 cases (25% female) and n = 35,191 pseudocontrols/ population controls (38% female). Analyses that were restricted to European-only samples consisted of 19,099 cases and 34,194 controls. ADHD GWAS summary statistics were also available from research participants of the personal genetics company 23andMe Inc. (n = 5857 self-reported ADHD cases, n = 70,393 controls) (Mountain View, CA). Genetic Investigation of Sex Bias in ADHD distinct from ADHD in diagnosed female individuals, this could yield differences in prevalence. Sex-based genetic heteroge- neity in common variants has been shown for several complex human traits (e.g., blood pressure, waist-hip ratio) (13,14). Here, we assessed the genome-wide autosomal genetic cor- relation of ADHD in male and female individuals to determine whether genetic heterogeneity from common variation con- tributes to the observed biased prevalence. prevalence in ADHD. First, autosomal common variant data were used to estimate the genetic correlation of ADHD in male and female individuals. We then used population register data to examine whether female individuals with ADHD are at an increased risk for comorbid developmental conditions compared with affected male individuals. Next, genome-wide autosomal SNP data were used to test whether female in- dividuals diagnosed with ADHD carry a higher burden of common risk variants than affected male individuals. Finally, using register data, we tested whether relatives of female in- dividuals with ADHD are at an increased risk for ADHD compared with relatives of diagnosed male individuals. p The absence of extensive sequencing data currently pre- cludes analogous analyses of rare genetic variants. Instead, to evaluate whether such variants play differential roles in male and female individuals with ADHD, we used risk for comorbid brain-related developmental disorders (i.e., autism spectrum disorder [ASD], intellectual disability [ID], epilepsy, motor developmental delay) and rare syndromic phenotypes (i.e., congenital malformations, syndromes related to chromosomal abnormalities) as proxies for possible presence of de novo or rare segregating alleles. Rare, highly deleterious (including noninherited) genetic variation has been implicated in such phenotypes (15–25). Indeed, comorbid ID has been associated with an increased likelihood that an individual with ADHD is a carrier of a large, rare copy number variant (CNV) (26). It has also long been known that rare genetic syndromes (e.g., fragile X syndrome, velocardiofacial syndrome) are associated with ADHD (27,28). Evidence for an increase in comorbid conditions in female individuals with ADHD, when compared with affected male individuals, would imply a more severe syndromal phenotypic presentation of ADHD in a higher proportion of female individuals. Such more complex presentations are arguably more likely to be linked to deleterious rare mutations. A higher rate of these comorbidities in female individuals would also be consistent with clinical heterogeneity, which may pertain to the observed prevalence differences. Female Protective Effect Sex-speciﬁc logistic regression GWAS analyses of imputed autosomal data were performed in each PGC study and iPSYCH wave separately. Results were ﬁltered for each study/ wave based on minor allele frequency, imputation quality, call rate, and expected minor allele frequency in cases. Sex- speciﬁc GWAS results for samples of European ancestry were meta-analyzed and also ﬁltered based on sample size and presence of each SNP in both sets of results. This yielded results for n = 7,531,543 common variants (hereafter PGC 1 iPSYCH). GWAS summary statistics can be downloaded at https://www.med.unc.edu/pgc/results-and-downloads. Aside from heterogeneity, prevalence differences may be caused by a female protective effect, whereby female in- dividuals are resilient to developing ADHD and thus require a higher burden of genetic liability to develop it. Three family studies have observed indirect evidence for this hypothesis in the form of increased risk of ADHD in ﬁrst-degree relatives of affected female individuals compared with affected male in- dividuals, suggesting that families with an affected female in- dividual may have a higher burden of genetic risk (29–31). Not all studies report an increase in the recurrence rate of ADHD in relatives of female probands, however (32,33). Two molecular genetic studies tested this hypothesis more directly using ADHD GWAS discovery data to calculate the burden of com- mon risk alleles, as estimated by polygenic risk scores (PRSs), in independent samples. In both studies, female children with ADHD-related phenotypes had higher scores for ADHD than affected male individuals (3,34). Although these preliminary studies are consistent with the family studies mentioned above, they were based on small discovery studies. Additional tests using large GWAS datasets are needed to test whether there is an increased burden of common genetic risk variants in female individuals with ADHD. SEE COMMENTARY ON PAGE e55 1044 ª 2017 Society of Biological Psychiatry. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal 1044 ª 2017 Society of Biological Psychiatry. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal ISSN: 0006-3223 Testing the Female Protective Effect Hypothesis Testing the Female Protective Effect Hypothesis A leave-one-study/wave-out approach was used to maximize power and maintain independent target and discovery samples for PRS analyses using the standard approach (39,40). GWAS results from 23andMe were also included in discovery meta- analyses. A PRS was calculated for each individual in each target sample (European-only samples) by scoring the number of alleles weighted by log(odds ratio [OR]) across the set of clumped, meta-analyzed SNPs in PLINK version 1.9 (see Supplemental Table S2). PRSs were standardized using z-score transformations; ORs can be interpreted as increase in risk of the outcome per standard deviation in PRS. Logistic regression analyses including principal components were used to test for association of PRS with sex in cases (female in- dividuals were coded as 1). Results of these leave-one-study/ wave-out analyses were meta-analyzed. Figure 1. Genetic correlation estimates for attention-deﬁcit/hyperactivity disorder in male and female individuals obtained from genomic related- ness matrix restricted maximum likelihood (GREML) and linkage disequi- librium score regression (LDSC) for the Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH), Psychiatric Genomics Con- sortium (PGC), and combined PGC 1 iPSYCH datasets. Because of strict restrictions on raw individual genotype access and transfer, GREML ana- lyses could only be performed separately in the PGC and iPSYCH samples. Bars display standard errors. The horizontal dashed line indicates a genetic correlation of 1. The estimator was left unconstrained for these analyses to allow for an unbiased assessment of the standard errors of the estimates; as such, some of the estimates exceed 1. Epidemiological Analyses Analyses of Swedish register data were based on all in- dividuals of known parents born in Sweden between 1987 and 2006, living in Sweden at least until 12 years of age (n = 77,905 ADHD cases and n = 1,874,637 population controls). See Supplement for details of the sample and analyses. PGC dataset was lower, but large standard errors were seen in this dataset for both methods. Additional cross-dataset and cross-sex LDSC genetic cor- relation estimates were used to assess the extent of heteroge- neity across PGC and iPSYCH (see Supplemental Figure S1 and Table S5). The genetic correlation between these subsamples was not signiﬁcantly different from 1 [rg (SE) = 1.13 (0.22)]. Cross- datasetrg values forPGCmaleindividuals withiPSYCH maleand female individuals were also not signiﬁcantly different from 1. Estimates for PGC female individuals with iPSYCH female in- dividuals were lower (signiﬁcantly different from 0 and 1), although this is likely related to the small sample size of the PGC female individuals (n = 1067 cases and n = 5178 controls). Testing for Sex-Speciﬁc Heterogeneity Bivariate linkage disequilibrium (LD) score regression (LDSC) (35,36) analyses were run on the sex-speciﬁc meta-analyzed summary statistics. The primary analyses (with greatest power) are those for the full PGC 1 iPSYCH sample; we also exam- ined estimates in the PGC and iPSYCH samples separately using LDSC and a second method, genomic relatedness matrix restricted maximum likelihood (GREML), using genome- wide complex trait analysis (37). Because of strict restrictions on access to individual genotypes, bivariate GREML analyses (38) were performed separately in PGC and iPSYCH. Analyses were restricted to European-only samples. Sex-speciﬁc We present a series of analyses to test the qualitative and quantitative difference hypotheses for the biased sex 1045 Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal Biological Psychiatry Biological Psychiatry Genetic Investigation of Sex Bias in ADHD Figure 1. Genetic correlation estimates for attention-deﬁcit/hyperactivity disorder in male and female individuals obtained from genomic related- ness matrix restricted maximum likelihood (GREML) and linkage disequi- librium score regression (LDSC) for the Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH), Psychiatric Genomics Con- sortium (PGC), and combined PGC 1 iPSYCH datasets. Because of strict restrictions on raw individual genotype access and transfer, GREML ana- lyses could only be performed separately in the PGC and iPSYCH samples. Bars display standard errors. The horizontal dashed line indicates a genetic correlation of 1. The estimator was left unconstrained for these analyses to allow for an unbiased assessment of the standard errors of the estimates; as such, some of the estimates exceed 1. heritability was estimated using univariate models (see Supplement for detailed methods). Testing the Female Protective Effect Hypothesis We estimated whether risk of ADHD in siblings of female in- dividuals with ADHD was higher than that in siblings of affected male individuals after adjusting for sex of the com- parison sibling. Analyses stratiﬁed by sex of the comparison sibling are also presented. Analyses were restricted to pairs of full siblings where at least one child had an ADHD diagnosis (n = 71,691 observations, including n = 21,784 unique index individuals). Testing for Sex-Speciﬁc Heterogeneity We assessed sex-speciﬁc differences in association between ADHD and the following categories of developmental disor- ders/syndromes: ID, ASD, developmental coordination disor- der, epilepsy, congenital malformations, and chromosomal abnormalities (see Supplemental Table S3 for details, including International Classiﬁcation of Diseases codes). Generalized estimating equations were used to test for the effect of an ADHD-by-sex interaction term on each outcome, while accounting for related samples. SNP-h2 was estimated at 0.123 (SE = 0.025) in female in- dividuals and 0.247 (SE = 0.021) in male individuals (Supplemental Figure S2 and Table S4). Downsampling male cases and controls randomly to match the female sample size and case-control ratio showed more similar SNP-h2 estimates (see Supplemental Figure S2 and Table S6). Results varying the relative population prevalence assumed are shown in Supplemental Table S7. Figure 2 summarizes these results, illustrating the impact of the assumed male:female ratio (which affects assumed sex-speciﬁc population prevalence rates) and sample size on SNP-h2 estimates; SNP-h2 estimates increased in male individuals and decreased in female individuals as the ratio was increased, and downsampling male cases and con- trols gave similar SNP-h2 estimates in both sexes. Sex-Speciﬁc Heterogeneity Genome-Wide Association Studies. Sex-speciﬁc quantile–quantile and Manhattan plots are shown in Figure 3. Three loci were genome-wide signiﬁcant in the male-only GWAS (n = 14,154 cases, n = 17,948 controls). No SNPs surpassed the genome-wide signiﬁcance threshold in the female-only GWAS (n = 4945 cases, n = 16,246 controls). (See Supplemental Table S8 for top 10 LD-independent SNPs for each GWAS, annotated with the nearest gene.) Female Protective Effect Hypothesis Figure 2. Sex-speciﬁc single nucleotide polymorphism (SNP) herita- bility estimates for attention-deﬁcit/hyperactivity disorder, varying the assumed population prevalence based on different male:female ratios (ranging from 1:1 to 7:1). Estimates are presented for the total available sample of male individuals as well as for a downsampled set of male cases and controls to match the available sample size in female individuals. Figure 2. Sex-speciﬁc single nucleotide polymorphism (SNP) herita- bility estimates for attention-deﬁcit/hyperactivity disorder, varying the assumed population prevalence based on different male:female ratios (ranging from 1:1 to 7:1). Estimates are presented for the total available sample of male individuals as well as for a downsampled set of male cases and controls to match the available sample size in female individuals. Figure 2. Sex-speciﬁc single nucleotide polymorphism (SNP) herita- bility estimates for attention-deﬁcit/hyperactivity disorder, varying the assumed population prevalence based on different male:female ratios (ranging from 1:1 to 7:1). Estimates are presented for the total available sample of male individuals as well as for a downsampled set of male cases and controls to match the available sample size in female individuals. PRS Analysis. Results of meta-analyses of each leave-one- study/wave-out regression analysis for ADHD PRS are shown in Figure 4. There was no association of ADHD PRS with sex in cases (OR [conﬁdence interval (CI)] = 1.02 [0.98–1.06], p = .28, mean R2 [SE] = .0019 [.00039]). Sensi- tivity tests were run excluding the data from 23andMe and non-European ancestry individuals and then also not including sex as a covariate in the discovery GWAS analyses; results remained similar (Supplemental Figure S7). There was no association of ADHD PRS with sex in controls (OR [CI] = 0.99 [0.96–1.01], p = .23, mean R2 [SE] = .0011 [.00024]) (Supplemental Figure S8). Mothers and fathers (in parent- offspring trio design studies) also did not differ in ADHD PRS (Supplemental Table S11). Several secondary analyses support the high genetic cor- relation, suggesting that there is little or no difference in the ADHD results for male and female individuals. First, no genome-wide signiﬁcant heterogeneity is observed when meta-analyzing the sex-speciﬁc GWAS results (Supplemental Figure S3). Second, a GWAS of sex-by-genotype interactions for ADHD identiﬁes no variants with differential effects by sex, nor does it show any deviation from the null distribution of test statistics genome-wide (Supplemental Figure S4). Genetic Correlation. Genetic Correlation. Figure 1 displays genetic correlation (rg) results for male and female ADHD from bivariate analyses using GREML and LDSC (see Supplemental Table S4 for exact estimates). The LDSC rg estimate in the full dataset (PGC 1 iPSYCH) was near 1. Similar results were found for bivariate GREML analyses in both iPSYCH and PGC and for the LDSC analyses in the iPSYCH dataset. The LDSC estimate in the Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal 1046 Genetic Investigation of Sex Bias in ADHD Figure 2. Sex-speciﬁc single nucleotide polymorphism (SNP) herita- bility estimates for attention-deﬁcit/hyperactivity disorder, varying the assumed population prevalence based on different male:female ratios (ranging from 1:1 to 7:1). Estimates are presented for the total available sample of male individuals as well as for a downsampled set of male cases and controls to match the available sample size in female individuals. comorbidities. Supplemental Table S9 displays the frequency of the disorder categories examined and the proportion of individuals affected overall and split by ADHD case status and sex. The male:female ratio for ADHD was 2:1. ADHD cases were at higher risk for all diagnostic categories, as compared with sex-matched controls (Table 1). Signiﬁcant ADHD-by-sex interactions were observed for ASD and congenital malformations, suggesting that although in the context of ADHD both sexes are at increased risk for these comorbid problems, the increase in risk is even higher in female individuals compared with controls. A nominally sig- niﬁcant association was observed for the interaction term for ID, which did not survive correction for multiple testing (Bonferroni correction for six independent tests: p-value threshold = .0083). Secondary analyses of severity of ID (where information was available) indicated that this weak association signal came from mild ID (IQ = 50–70), not moderate (IQ = 35–49) or severe/profound (IQ , 35) ID (Supplemental Table S10). Interaction terms were nonsignif- icant for epilepsy, developmental coordination disorder, and chromosomal abnormalities. Female Protective Effect Hypothesis Similarly, GWAS results for ADHD in the combined sample with or without including sex as a covariate are nearly perfectly correlated, with a low standard error (rg = .97, SE = .007). Narrowing the focus to only ADHD cases also ﬁnds no genome-wide signiﬁcant differences between male and female cases (Supplemental Figure S5). Although some genome-wide inﬂation is observed for this ﬁnal analysis in the iPSYCH sample, it is not replicated in the PGC data and appears to be attributable to one locus driven by a single low-frequency genotyped SNP (minor allele frequency = 0.02). Investigation of this locus shows no support for differences between male and female cases in neighboring genotyped SNPs, suggesting that the signal is likely a technical artifact (Supplemental Figure S6). There was a clear association of ADHD PRS with ADHD case status, with similar differences in PRS between cases and controls observed for female individuals (OR [CI] = 1.57 [1.50–1.64], p = 4.1xE-91, mean R2 [SE] = .039 [.0034]) and male individuals (OR [CI] = 1.50 [1.46–1.53], p = 7.3xE-216, mean R2 [SE] = .032 [.0024]). In this analysis, several individual PGC studies did not show a signiﬁcant association with case- control status in female individuals; this is likely due to low power (e.g., n = 27 female case/pseudocontrol pairs in the Canadian study). Epidemiological Analysis. To test for evidence of increased risk for ADHD in siblings of female individuals with ADHD, we used Swedish population data to select sibling pairs where at least one child had ADHD. The results showed that siblings of female individuals diagnosed with ADHD are at higher risk for ADHD than siblings of affected male in- dividuals (OR [CI] = 1.14 [1.11–1.18], p = 1.5E-15); results stratiﬁed by comparison sibling sex were similar for male individuals (OR [CI] = 1.18 [1.13–1.23], p = 1.8E-13) and female individuals (OR [CI] = 1.09 [1.01–1.16], p = .017). Covarying for presence of ASD, congenital malformations, Epidemiological Analyses. We examined the association between having an ADHD diagnosis and risk of having a comorbid developmental phenotype using Swedish register data. We were interested in whether there is an interaction between sex and ADHD as it pertains to these Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal 1047 Biological Psychiatry Genetic Investigation of Sex Bias in ADHD Figure 3. Quantile–quantile and Manhattan plots for sex-speciﬁc genome-wide association meta-analyses. (A) Female case-control analysis quantile– quantile plot. Female Protective Effect Hypothesis (B) Female case-control analysis Manhattan plot. (C) Male case-control analysis quantile–quantile plot. (D) Male case-control analysis Manhattan plot. In (B) and (D), the horizontal red (upper) line indicates genome-wide signiﬁcance (p , 5E-8) and the horizontal green (lower) line indicates suggestive subthreshold signals (p , 5E-6). Figure 3. Quantile–quantile and Manhattan plots for sex-speciﬁc genome-wide association meta-analyses. (A) Female case-control analysis quantile– quantile plot. (B) Female case-control analysis Manhattan plot. (C) Male case-control analysis quantile–quantile plot. (D) Male case-control analysis Manhattan plot. In (B) and (D), the horizontal red (upper) line indicates genome-wide signiﬁcance (p , 5E-8) and the horizontal green (lower) line indicates suggestive subthreshold signals (p , 5E-6). and ID in the proband did not affect the overall results (OR [CI] = 1.15 [1.11–1.19], p = 7.3E-16). and ID in the proband did not affect the overall results (OR [CI] = 1.15 [1.11–1.19], p = 7.3E-16). and ID in the proband did not affect the overall results (OR [CI] = 1.15 [1.11–1.19], p = 7.3E-16). The observed high SNP-based genetic correlation (consis- tent across two methods) suggests that largely the same common autosomal risk variants are involved in ADHD for both sexes. While sex-speciﬁc heterogeneity from common auto- somal variants seems unlikely based on our results, we cannot rule out the possibility that heterogeneous effects exist for rare or nonautosomal variation or that with increased sample sizes, weaker effects of common variant heterogeneity could be detected. DISCUSSION We tested two speciﬁc hypotheses for the male bias in ADHD: ﬁrst, that sex-speciﬁc genetic heterogeneity may affect prev- alence rates via clinical heterogeneity in ADHD diagnosis by sex, and second, that female individuals affected with ADHD may carry an increased burden of genetic risk variants compared with affected male individuals. We analyzed auto- somal common variant data from the largest available ADHD case-control GWAS sample (n = 55,374 individuals) and Swedish population register data (n = 1,952,542 individuals). We demonstrated a high level of genome-wide autosomal genetic correlation for ADHD across sex and found no in- crease of polygenic burden in affected female individuals compared with affected male individuals. However, in register- based data, we observed that siblings of female individuals with ADHD are at increased risk for ADHD compared with siblings of affected male individuals. The results also sug- gested that female individuals diagnosed with ADHD may be at an especially high risk for certain comorbid developmental conditions. Indeed, the epidemiological analyses of Swedish population data suggest some degree of clinical and/or etiological het- erogeneity. ADHD was associated with comorbid diagnoses of developmental conditions in both sexes. The strength of as- sociation was greater in female individuals for ASD, congenital malformations, and (to a lesser extent) mild ID, but it was similar for both sexes for motor problems, epilepsy, and chromosomal abnormalities. There are several possible ex- planations for these ﬁndings. First, female individuals with ADHD may have a higher than expected risk of comorbid severe conditions and so may have a higher level of clinical heterogeneity as compared with male cases. This could indirectly point to a greater role of rare deleterious variants in female individuals with ADHD because such variants are strongly implicated in some of the comorbid Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal 1048 Genetic Investigation of Sex Bias in ADHD Table 1. Results of Logistic Regression Analyses of ADHD Case Status on Comorbid Developmental Conditions, Stratiﬁed by Sex, in the Swedish Population Sample (Total N = 1,952,542) Table 1. DISCUSSION Results of Logistic Regression Analyses of ADHD Case Status on Comorbid Developmental Conditions, Stratiﬁed by Sex, in the Swedish Population Sample (Total N = 1,952,542) Outcome Sex % of ADHD Cases With Outcome Sex-Speciﬁc Association ADHD by Sex Interaction OR LCI UCI p OR LCI UCI p ASD Male 14.82 18.91 18.33 19.50 , 2.2E-308a 1.52 1.44 1.61 2.9E-50a Female 12.04 28.68 27.38 30.04 , 2.2E-308a DCD Male 1.86 17.90 16.38 19.57 , 2.2E-308a 0.97 0.82 1.14 .71 Female 1.08 17.61 15.25 20.33 , 2.2E-308a ID Male 5.30 9.82 9.38 10.28 , 2.2E-308a 1.11 1.03 1.20 .0090 Female 4.89 10.89 10.23 11.60 , 2.2E-308a Epilepsy Male 2.57 3.27 3.09 3.47 , 2.2E-308a 1.08 0.99 1.19 .099 Female 2.85 3.55 3.29 3.82 6.6E-238a Congenital Malformations Male 8.20 1.40 1.36 1.45 1.8E-109a 1.11 1.05 1.17 2.5E-04a Female 6.46 1.54 1.47 1.62 8.9E-72a Chromosomal Abnormalities Male 0.60 2.96 2.63 3.34 2.7E-70a 0.84 0.68 1.03 .096 Female 0.52 2.48 2.07 2.96 9.5E-24a Sex is coded as 0 for male and 1 for female. Birth year is included as a covariate. ADHD, attention-deﬁcit/hyperactivity disorder; ASD, autism spectrum disorder; DCD, developmental coordination disorder; ID, intellectual disability; LCI, lower conﬁdence interval; OR, odds ratio; UCI, upper conﬁdence interval. aEstimated p value below a Bonferroni-adjusted threshold for multiple testing (p , .0083). Sex is coded as 0 for male and 1 for female. Birth year is included as a covariate. ADHD, attention-deﬁcit/hyperactivity disorder; ASD, autism spectrum disorder; DCD, developmental coordination disorder; ID, intellectual disability; LCI, lower conﬁdence interval; OR, odds ratio; UCI, upper conﬁdence interval. aEstimated p value below a Bonferroni-adjusted threshold for multiple testing (p , .0083). likely to be diagnosed with ADHD if they have a more severe phenotypic presentation, could be involved. Individuals who receive one diagnosis become the focus of clinical attention and are more likely to receive subsequent diagnoses, whereas individuals with less complex phenotypes might not come to clinical attention. If female individuals are routinely under- diagnosed with ADHD or other neurodevelopmental disorders, this issue may disproportionately affect ascertainment of likely to be diagnosed with ADHD if they have a more severe phenotypic presentation, could be involved. Individuals who receive one diagnosis become the focus of clinical attention and are more likely to receive subsequent diagnoses, whereas individuals with less complex phenotypes might not come to clinical attention. Genetic Investigation of Sex Bias in ADHD Despite these differences, we ﬁnd very high genetic correlation between PGC and iPSYCH, suggesting that overall these diagnostic differences have no perceptible impact on the involvement of common risk variants. PGC-only and iPSYCH-only sex-speciﬁc analysis results were similar, with the caveat that the PGC study was smaller and results had larger conﬁdence intervals. p Epidemiological analyses also showed that siblings of fe- male individuals diagnosed with ADHD were at higher risk for being diagnosed with ADHD than siblings of diagnosed male individuals. This conﬁrms results from previous family studies (29–31) indirectly supporting the hypothesis that female in- dividuals require a greater burden of genetic risk to manifest ADHD. Although these analyses do not distinguish between genetic and shared environmental effects, twin studies have consistently demonstrated high heritability of ADHD with typically nonsigniﬁcant contributions from shared environ- mental factors (2), suggesting that this effect is most likely to be genetic in origin. However, the effect sizes were not large (OR = 1.09–1.18), suggesting that any increased burden of inherited genetic variation might be only a small contribution to the sex bias in ADHD prevalence. These results could also occur if clinicians had a higher threshold for diagnosing ADHD in female individuals or were more likely to diagnose it if accompanied by a comorbid disorder. However, the results did not attenuate when comorbid conditions were accounted for. Alternatively, sex-speciﬁc ascertainment biases could inﬂate estimated risk of ADHD in siblings of female individuals. Although female cases were more likely to be ascertained if they had an already diagnosed brother (64.6%) than vice versa, the sex-stratiﬁed results indicate that such biases cannot fully explain our results. A limitation of the analyses was restricting the sample to full siblings, which may have led to a conservative estimate of the effect size and the possibility that the results might not generalize to families with one child or half-siblings. Another possible contribution to sex bias, beyond the scope of the current study, is the role of sex hormones and chro- mosomes. There is evidence for a speciﬁc role of sex hor- mones (e.g., estrogen) and sex chromosomes (e.g., X chromosome aneuploidy) in early brain development and neurodevelopmental disorders such as ADHD (28,50,51), suggesting that future efforts to examine the role of sex chromosomes and their downstream products in ADHD may be worthwhile. Genetic Investigation of Sex Bias in ADHD obscured. Although the variance in ADHD PRS did not appear to differ between male and female cases (p = .31; see Supplement), limited power of this analysis prevents us from conclusively ruling out this possibility. obscured. Although the variance in ADHD PRS did not appear to differ between male and female cases (p = .31; see Supplement), limited power of this analysis prevents us from conclusively ruling out this possibility. female cases, leading to the observed pattern of results. Other possible sources of bias include typical exclusion criteria for diagnosing ADHD (e.g., ID, ASD) and the possibility of an inﬂated false positive rate of diagnoses due to diagnostic un- certainty and change over time. We limited the impact of the latter by considering diagnoses only in individuals with at least two reported diagnoses. Although the results are consistent with increased etiological heterogeneity in female individuals with ADHD, studies of rare variation are required to rule out these alternative explanations. y g p y Although the focus of this paper is on possible genetic sources of inﬂuence on ADHD sex bias, other factors, such as ascertainment and diagnostic biases, may play an important role. Female individuals are more likely to be diagnosed with the predominantly inattentive subtype of ADHD, whereas male individuals are more likely to be diagnosed with the combined subtype of ADHD and present with hyperactive-impulsive and disruptive behavioral problems (46–49). Relative prevalence rates also vary by diagnostic instrument used and case ascertainment. For example, the ratio of male:female cases in the Swedish population was 2:1, somewhat lower than that in the iPSYCH Danish population (2.8:1) and PGC clinical data (3.5:1). Individuals with moderate to severe ID (IQ , 50) were excluded from iPSYCH. ADHD cases in the PGC studies were primarily ascertained from clinics; ADHD was conﬁrmed with structured interviews, and children with comorbid ASD, epi- lepsy, ID (IQ , 70), and other conditions were excluded. As such, the false positive rate for an ADHD diagnosis is likely higher in the iPSYCH and Swedish register-based datasets than in the PGC studies, while the latter is likely underrepre- sented for individuals with neurodevelopmental comorbidities. Another major difference is that many of the PGC studies used DSM criteria and thus included children with inattentive and hyperactive-impulsive subtypes of ADHD, whereas the Swedish and iPSYCH studies (and some European PGC studies) used the stricter International Classiﬁcation of Dis- eases deﬁnition. DISCUSSION If female individuals are routinely under- diagnosed with ADHD or other neurodevelopmental disorders, this issue may disproportionately affect ascertainment of conditions assessed (15–25) and this effect has been seen in other neurodevelopmental disorders (15–19). However, this needs to be tested directly because common variants also play an important role in such complex disorders (41,42), and little is currently known about the contribution of rare variants to ADHD. An alternative explanation for the results is that ascertain- ment and diagnostic biases, where female individuals are more Figure 4. Forest plots of meta-analysis results for logistic regression analyses of attention-deﬁcit/hyperactivity disorder polygenic risk scores with case sex as the outcome (A), case-control status in female individuals (B), and case-control status in male individuals (C). Box sizes reﬂect sample sizes. Refer to Table 1 for exact sample sizes. CIs, conﬁdence intervals; iPSYCH, Lundbeck Foundation Initiative for Integrative Psychiatric Research; PGC, Psychiatric Genomics Consortium. Figure 4. Forest plots of meta-analysis results for logistic regression analyses of attention-deﬁcit/hyperactivity disorder polygenic risk scores with case sex as the outcome (A), case-control status in female individuals (B), and case-control status in male individuals (C). Box sizes reﬂect sample sizes. Refer to Table 1 for exact sample sizes. CIs, conﬁdence intervals; iPSYCH, Lundbeck Foundation Initiative for Integrative Psychiatric Research; PGC, Psychiatric Genomics Consortium. Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal 1049 Biological Psychiatry Genetic Investigation of Sex Bias in ADHD Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal ACKNOWLEDGMENTS AND DISCLOSURES This work was supported by the National Human Genome Research Insti- tute of the National Institutes of Health (NIH) (Grant No. R44HG006981 to the 23andMe Team), the Wellcome Trust (Grant No. 106047 to JM). the Australian National Health and Medical Research Council (Grant Nos. 1078901 and 1087889 to NRW). The Broad Institute and Massachusetts General Hospital investigators acknowledge support from the Stanley Medical Research Institute and NIH grants: Grant No. 1R01MH094469 (principal investigator, BMN) and Grant No. 1R01MH107649-01 (principal investigtor, BMN). The iPSYCH team acknowledges funding from the Lundbeck Foundation (Grant Nos. R102-A9118 and R155-2014-1724), the Stanley Medical Research Institute, the European Research Council (Project No. 294838), the European Community’s Horizon 2020 Programme (H2020/ 2014-2020) under Grant No. 667302 (Comorbid Conditions of ADHD), the Novo Nordisk Foundation for supporting the Danish National Biobank resource, and grants from Aarhus and Copenhagen universities and uni- versity hospitals, including support to the Centre for Integrative Sequencing, the GenomeDK HPC facility, and the Centre for Integrated Register-Based Research at Aarhus University. Consortium members (listed alphabetically; for consortium authors’ af- ﬁliations, see Supplement): JM contributed to study conception, data analysis, interpretation of the results, and drafting of the manuscript. RKW, DD, and MM contributed to data analysis, interpretation of the results, and critical revision of the manuscript. AT, MCO, and BMN contributed to study conception, inter- pretation of the results, and critical revision of the manuscript. IB and LG contributed to data analysis and critical revision of the manuscript. SHL and NRW contributed to interpretation of the results and critical revision of the manuscript. ADB contributed to data generation, design of the overall project, and critical revision of the manuscript. TW, PBM, MGP, OM, MN, DMH, and JB-G contributed to data generation and design of the overall project. HL, PL, ER, NE, BF, and SVF contributed to critical revision of the manuscript. Psychiatric Genomics Consortium: ADHD Subgroup—Özgür Albayrak, Richard J.L. Anney, Alejandro Arias Vasquez, Maria Jesús Arranz, Philip Asherson, Tobias Banaschewski, Tobias J. Banaschewski, Claiton Bau, Joseph Biederman, Preben Bo Mortensen, Anders Børglum, Jan K. Buite- laar, Miguel Casas, Alice Charach, Bru Cormand, Jennifer Crosbie, Soeren Dalsgaard, Mark J. Daly, Ditte Demontis, Astrid Dempﬂe, Alysa E. Doyle, Richard P. Ebstein, Josephine Elia, Stephen V. Faraone, Stephen V. ARTICLE INFORMATION From the Department of Medical Epidemiology & Biostatistics (JM, IB, LG, HL, PL) and Centre for Psychiatry Research (MM), Department of Clinical Neuroscience, Karolinska Institutet, and Stockholm Health Care Services (MM), Stockholm County Council, Stockholm; and School of Medical Sci- ences (HL), Örebro University, Örebro, Sweden; Stanley Center for Psychi- atric Research (JM, RKW, ER, BMN), Broad Institute, Cambridge, and Analytic and Translational Genetics Unit (JM, RKW, ER, BMN), Massachu- setts General Hospital, Boston, Massachusetts; 23andMe Inc. (NE, 2RT), Mountain View, California; Departments of Psychiatry and of Neuroscience and Physiology (SVF), SUNY Upstate Medical University, Syracuse, New York; MRC Centre for Neuropsychiatric Genetics and Genomics (JM, MCO, AT), Cardiff University, Cardiff, United Kingdom; Lundbeck Foundation Initiative for Integrative Psychiatric Research [iPSYCH] (DD, MM, TW, PBM, MGP, OM, MN, DMH, JB-G, ADB); Centre for Integrative Sequencing [iSEQ] (DD, MM, PBM, ADB), Department of Biomedicine–Human Genetics (DD, MM, ADB), National Centre for Register-Based Research (PBM, MGP), and Centre for Integrated Register-Based Research (PBM, MGP), Aarhus Uni- versity, Aarhus; Institute of Biological Psychiatry (TW), MHC Sct. Hans, Mental Health Services Copenhagen, Roskilde; Department of Clinical Medicine (TW), Mental Health Services in the Capital Region of Denmark (MN), Mental Health Center Copenhagen, University of Copenhagen, and Center for Neonatal Screening (DMH, JB-G), Department for Congenital Disorders, Statens Serum Institut, Copenhagen; and Psychosis Research Unit (OM), Aarhus University Hospital, Risskov, Denmark; Queensland Brain Institute (SHL, NRW), University of Queensland, Brisbane, Queensland, School of Environmental and Rural Science (SHL), University of New En- gland, Armidale, New South Wales, and Centre for Population Health Research (SHL), School of Health Sciences and Sansom Institute of Health Research, University of South Australia, Adelaide, Australia; Departments of Human Genetics and Psychiatry (BF), Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands; and K.G. Jebsen Centre for Research on Neuropsychiatric Disorders (SVF), University of Bergen, Bergen, Norway. Genetic Investigation of Sex Bias in ADHD Genetic Investigation of Sex Bias in ADHD The high genetic correlation and lack of difference in poly- genic burden between male and female ADHD cases support combining GWAS data from both sexes in ADHD meta-analyses and further suggest that current clinical practices of diagnosing ADHD are capturing a clinical phenotype that is similar at the level of common genetic risk variants in both sexes. Thus, our results indicate that genome-wide autosomal common variants largely do not explain the observed sex bias in ADHD preva- lence. On the other hand, the epidemiological results also suggest some degree of clinical heterogeneity, with ADHD showing a stronger phenotypic association with comorbid ASD, congenital malformations, and mild ID in female individuals. We also found evidence for a modest increase in familial risk for ADHD in female individuals based on sibling analysis. Further work simultaneously examining variants across the spectrum of frequencies is needed to comprehensively determine the role of genetic risk in the sex bias in ADHD prevalence. Press: Straight Talk About Your Child’s Mental Health; Oxford University Press: Schizophrenia: The Facts; and Elsevier: ADHD: Non-Pharmacologic Interventions. He is principal investigator of www.adhdinadults.com. All other authors report no biomedical ﬁnancial interests or potential conﬂicts of interest. Genetic Investigation of Sex Bias in ADHD Future studies should also examine the degree of shared genetic risks across ADHD and other developmental conditions (e.g., ASD, ID, congenital abnormalities) in a sex- speciﬁc manner as well as the role of rare variants (e.g., CNVs, single nucleotide mutations) in ADHD sex bias. Contrary to smaller studies (3,34), we did not ﬁnd an enrichment of polygenic burden from common variants in fe- male individuals compared with male individuals with ADHD. Consistently, analyses in ASD have not found an increased burden from common variants in affected female individuals (43,44), in contrast to rare variant studies (15–19). One possibility is that a higher degree of genetic hetero- geneity within female individuals masked any differences in PRS burden by sex in the current study. Common and rare variants may contribute additively to ADHD risk, with non-CNV carrier cases having lower ADHD PRSs than cases with large rare CNVs (45). Thus, if female cases are more likely to have a complex syndromic phenotype (as suggested by the register- based analyses), and given that such phenotypes are more likely to be associated with rare variants (15–25), this subgroup of female individuals could have on average lower PRSs than male individuals with ADHD. On the other hand, affected fe- male individuals with a less severe phenotypic presentation, who are not carriers of such rare variants, could have higher PRSs than affected male individuals. If this were the case, any overall differences in PRS between the sexes could be The results of this study demonstrate a clear polygenic contribution from common autosomal genetic variants to ADHD in both sexes, as evidenced by moderate SNP-h2 esti- mates using two methods (similar in both sexes once nonrandom sample ascertainment is taken into account via sample size matching), clear deviation of test statistics on sex- speciﬁc GWAS quantile–quantile plots, and signiﬁcantly higher overall PRSs in cases compared with sex-matched controls. Top hits from sex-speciﬁc GWAS analyses corroborate results from a combined analysis of both sexes (7). 050 Biological Psychiatry June 15, 2018; 83:1044–1053 www.sobp.org/journal 1050 REFERENCES 1. 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ACKNOWLEDGMENTS AND DISCLOSURES Far- aone, Manuel Föcker, Barbara Franke, Christine Freitag, Joel Gelernter, Michael Gill, Eugenio Grevet, Jan Haavik, Hakon Hakonarson, Ziarih Hawi, Johannes Hebebrand, Beate Herpertz-Dahlmann, Amaia Hervas, Anke Hinney, Sarah Hohmann, Peter Holmans, Mara Hutz, Abel Ickowitz, Stefan Johansson, Lindsey Kent, Sarah Kittel-Schneider, Henry Kranzler, Jonna Kuntsi, Nanda Lambregts-Rommelse, Kate Langley, Gerd Lehmkuhl, Klaus- Peter Lesch, Sandra K. Loo, Joanna Martin, James J. McGough, Sarah E. Medland, Jobst Meyer, Eric Mick, Frank Middletion, Ana Miranda, Fernando Mulas, Aisling Mulligan, Benjamin M. Neale, Stan F. Nelson, T. Trang Nguyen, Michael C. O’Donovan, Robert D. Oades, Michael J. Owen, Haukur Palmason, Josep Antoni Ramos-Quiroga, Andreas Reif, Tobias J. Renner, Luis Rhode, Marta Ribasés, Marcella Rietschel, Stephan Ripke, Olga Rivero, Herbert Roeyers, Marcel Romanos, Jasmin Romanos, Nina Roth Mota, Aribert Rothenberger, Cristina Sánchez-Mora, Russell Schachar, Helmut Schäfer, André Scherag, Benno G. Schimmelmann, Joseph Sergeant, Judith Sinzig, Susan L. Smalley, Edmund J S Sonuga-Barke, Hans-Christoph Steinhausen, Patrick F. Sullivan, Anita Thapar, Margaret Thompsom, Alex- andre Todorov, Irwin Waldman, Susanne Walitza, Raymond Walters, Yufeng Wang, Andreas Warnke, Nigel Williams, Stephanie H. Witt, Li Yang, Tetyana Zayats, and Yanli Zhang-James. We thank the research participants and employees of 23andMe for making this work possible. An earlier version of this manuscript is available as a preprint on bioRxiv. BMN is a member of Deep Genomics Scientiﬁc Advisory Board and has received travel expenses from Illumina. He also serves as a consultant for Avanir and Trigeminal Solutions. BF has received educational speaking fees from Merz and Shire. HL has served as a speaker for Eli Lilly and Shire and has received research grants from Shire, all outside of the submitted work. PL has served as a speaker for Medice. During the past year, SVF received income, potential income, travel expenses, and continuing education sup- port and/or research support from Lundbeck, Rhodes, Arbor, KenPharm, Ironshore, Shire, Akili Interactive Labs, CogCubed, Alcobra, VAYA, Suno- vion, Genomind, and NeuroLifeSciences. With his institution, he has U.S. patent US20130217707 A1 for the use of sodium-hydrogen exchange in- hibitors in the treatment of ADHD. During previous years, he received sup- port from Shire, Neurovance, Alcobra, Otsuka, McNeil, Janssen, Novartis, Pﬁzer, and Eli Lilly. 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https://openalex.org/W2290708267	https://europepmc.org/articles/pmc4772538?pdf=render	English	null	Quercitrin-nanocoated titanium surfaces favour gingival cells against oral bacteria	Scientific reports	2,016	cc-by	7,251	Quercitrin-nanocoated titanium surfaces favour gingival cells against oral bacteria g Manuel Gomez-Florit1,2, Miguel A. Pacha-Olivenza3,4, Maria C. Fernández-Calderón3,5, Alba Córdoba1,2, Maria L. González-Martín3,4, Marta Monjo1,2 & Joana M. Ramis1,2 g Manuel Gomez-Florit1,2, Miguel A. Pacha-Olivenza3,4, Maria C. Fernández-Calderón3,5, Alba Córdoba1,2, Maria L. González-Martín3,4, Marta Monjo1,2 & Joana M. Ramis1,2 received: 11 December 2015 accepted: 15 February 2016 Published: 01 March 2016 received: 11 December 2015 accepted: 15 February 2016 Published: 01 March 2016 Many dental implants fail due to the infection and inflammation that walk hand in hand with poor healing and soft tissue integration. Titanium surfaces were nanocoated with quercitrin, a natural flavonoid, with the aim to improve soft tissue integration and increase dental implants success. Streptococcus mutans attachment and biofilm formation was analysed. Then, the anti-inflammatory properties and the potential of quercitrin-nanocoated surfaces to boost soft tissue regeneration were tested using human gingival fibroblasts. An inflammatory situation was mimicked using interleulin-1-beta. We found that quercitrin-nanocoated surfaces decreased initial bacterial adhesion while increasing human gingival fibroblasts attachment. Furthermore, quercitrin-nanocoated Ti increased collagen mRNA levels and decreased matrix metalloproteinase-1/tissue inhibitor of metalloproteinanse-1 mRNA ratio, which is related to a reduced metalloproteinase-mediated collagen degradation, while also decreasing the pro-inflammatory prostaglandin E2 release under basal and inflammatory conditions. These results suggest that quercitrin-nanocoated surfaces could enhance the soft tissue integration and increase dental implants success. Success of a dental implant depends on osseo- and soft tissue-integration. Many dental implants fail due to the infection and inflammation that walk hand in hand with poor healing and soft tissue integration1,2. Soft tissue integration to dental implants establishes a biological seal between the oral environment and the implant which prevents microbial invasion, gingival recession and inflammatory peri-implantitis2–4. Therefore, the development of antibacterial, anti-inflammatory and tissue-regenerative surface modifications constitutes a major challenge to guarantee implant success.f g Research efforts to develop a new generation of dental implants are focused in bioactive surface coatings intended to provoke a defined cellular reaction5. A number of modified surfaces with integrated antibiotics, growth factors, antiresorptive drugs, synthetic peptides or complex formulations of artificial extracellular matrix (ECM) components have been developed. However, biocompatibility of most anti-bacterial surfaces is still uncer- tain while the lack of stability of growth factors after implantation is the main reason for the absence of such a product from the current implant market6,7.i p p An interesting option for surface modification is the use of natural multi-target molecules. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 Results Bacterial adhesion and biofilm formation. QUER surfaces significantly decreased S. mutans adhesion after 30 min compared to Ti and A controls. After 60 and 90 min A and QUER surfaces decreased bacterial adhe- sion compared to Ti control. Unexpectedly, A Red and QUER Red surfaces increased bacterial adhesion at all time points compared to Ti control and to non-reduced surfaces (Fig. 2). Moreover, in all surfaces studied and times, the bacterial viability was not compromised.ifif y p We did not find statistical differences in biofilm formation between the different groups. However, A and QUER groups decreased biofilm formation by 12 and 6% respectively, compared to Ti control (Fig. 2). Higher biofilm formation was observed for A Red and QUER Red surfaces compared to the non-reduced surfaces. Furthermore, SEM images confirmed these trends (Fig. 2). gi ( g ) Due to the increased bacterial adhesion of A Red and QUER Red surfaces, we decided to select A and QU surfaces for further analysis. Cell adhesion. We found that QUER surfaces increased hGF adhesion compared to Ti and A controls after 30 min although we did not observe this clear effect at longer adhesion times, i.e. 60 and 120 min (Fig. 3). When comparing the total number of cells adhered to the surfaces, A surfaces decreased cell number by almost the half while QUER surfaces increased it almost 20% compared to Ti. Furthermore, QUER surfaces increased cell adhe- sion compared to A control surfaces.ti p Confocal images reveal that after 30 min, hGFs on QUER surfaces showed the typical spindle-shaped fibro- blastic morphology while on Ti and A surfaces were more rounded. Also, hGFs on QUER surfaces showed higher vinculin staining and filopodia than cells on Ti and A surfaces (Fig. 3). Response of hGF to the different surfaces. In basal conditions, QUER surfaces increased collagens mRNA levels compared to Ti and A controls and decreased MMP1/TIMP1 mRNA ratio and COX2 mRNA levels compared to Ti. When hGF growing on Ti surfaces were challenged with IL-1β , we observed decreased colla- gens and TIMP1 mRNA levels while increasing COX2 mRNA. Under this challenging condition, QUER surfaces increased the mRNA levels of collagens while decreasing MMP1/TIMP1 mRNA ratio and COX2 mRNA com- pared to Ti and A controls (Fig. 4).t p g After 14 days of cell culture, PGE2 released to culture media was increased on the groups cultured under inflammatory conditions (Fig. Results 5). Furthermore, A and QUER surfaces decreased PGE2 production, compared to Ti surfaces. Effect of disinfection on the bioactivity of the different surfaces. We hypothesized that the disin- fection process with dry heat at 100 °C would not affect the coatings bioactivity since this temperature is lower than the melting temperature of quercitrin (176–179 °C) and lower than the boiling temperatures of APTES and quercitrin (223 °C and 316 °C respectively). To confirm this hypothesis, we compared the response of hGF on dis- infected (heated) surfaces to non-heated surfaces (see Supplementary Fig. S1). Disinfection process did not affect coatings bioactivity since we only found that COX2 mRNA on Ti disinfected surfaces was decreased compared to Ti non-heated surfaces. Quercitrin-nanocoated titanium surfaces favour gingival cells against oral bacteria Natural mole- cules, which promise higher therapeutic efficacy and safety8, represent an alternative to pharmaceuticals and animal-derived compounds due to low immunogenicity and toxicity9. Flavonoids are natural phenolic com- pounds derived from plants with antioxidant10, anti-inflammatory11 and antimicrobial capacity12,13, among other functions9. In previous studies, we screened among different flavonoids and selected quercitrin due to its potential in soft and hard periodontal tissues regeneration and its anti-inflammatory activity14. Furthermore, we successfully grafted quercitrin to titanium (Ti) surfaces, producing a quercitrin-nanocoating, and observed that the resulting surfaces have promising biological activities in vitro15. In a posterior work, to increase the nanocoating stability and reproducibility, quercitrin grafting conditions were optimized (Fig. 1). The opti- mized quercitrin-nanocoated Ti surfaces enhance human mesenchymal stem cells initial adhesion and promote 1Group of Cell Therapy and Tissue Engineering, Research Institute on Health Sciences (IUNICS), University of Balearic Islands, Palma de Mallorca, Spain. 2Instituto de Investigación Sanitaria de Palma, 07010 Palma, Spain. 3Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Badajoz, Spain. 4Department of Applied Physics, Faculty of Science, University of Extremadura, Av. Elvas s/n, 06071 Badajoz, Spain. 5Department of Biomedical Sciences, University of Extremadura, Badajoz, Spain. Correspondence and requests for materials should be addressed to J.M.R. (email: joana.ramis@uib.es) Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 1 www.nature.com/scientificreports/ Figure 1. Schema of surface nanocoatings. Ti, titanium passivated surfaces; A, aminosilanised surfaces; QUER, quercitrin-nanocoated surfaces; QUER Red, reduced quercitrin-nanocoated surfaces. Figure 1. Schema of surface nanocoatings. Ti, titanium passivated surfaces; A, aminosilanised surfaces; QUER, quercitrin-nanocoated surfaces; QUER Red, reduced quercitrin-nanocoated surfaces. osteoblast differentiation16. One step closer for the exploitation of quercitrin-nanocoated Ti-surfaces for their use in dental implant abutments would be the evaluation of their antibacterial, anti-inflammatory and soft tissue-regenerative properties.f In the present study, we aimed at evaluating the effects of quercitrin-nanocoated Ti-surfaces in bacterial adhe- sion and biofilm formation using Streptococcus mutans. Furthermore, human gingival fibroblasts were used to evaluate the effects of quercitrin-nanocoated Ti-surfaces in cell adhesion, anti-inflammatory properties and soft tissue regeneration in normal and in inflammatory microenvironments. Discussion One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti control at each time point; (b) versus A control at each time point; (c) effect of reduction within each group at each time point. attachment. Furthermore, quercitrin-nanocoated surfaces increased collagen mRNA levels and decreased MMP1/ TIMP1 mRNA ratio while decreasing COX2 mRNA and PGE2 release under basal and inflammatory conditions. These results suggest that quercitrin-nanocoated surfaces could enhance the soft tissue integration and increase dental implants success.tt p Peri-implant soft tissue healing follows the four overlapping phases of wound healing3. After an implant placement a blood coagulum is formed which provides a provisional ECM. Inflammatory cells migrate, phago- cytise foreign particles and release inflammatory mediators to either finalise the inflammation or to amplify it. Fibroblasts then invade the fibrin network and produce collagen fibres to form a connective tissue in contact with the implant surface. The final phase involves collagen remodelling which can produce either a scarred (repaired) tissue or a structural and functional regenerated tissue, depending on the microenvironmental signals. During this process, bacterial accumulation (dental plaque formation), may trigger the inflammatory process. However, it depends on the soft tissue integration to resist the deletereous effects of plaque accumulation, thus, avoiding peri-implantitis to guarantee long-term efficacy of dental implants.ll p p g gfi y p Peri-implant tissue is colonized by the same flora as the periodontium17. Among oral micro flora, S. mutans is one of the initial colonizers during dental plaque formation, which prepare a favourable environment for the late colonizers, and it is a recognised biofilm forming specie18. We found that quercitrin-nanocoated surfaces inhibited initial S. mutans attachment, in line with the results obtained on glass and hydroxyapatite surfaces using S. mutants suspensions in tea extract solutions (containing flavonoids, tannins and indolic compounds)19. Furthermore, biofilm formation was slightly inhibited on quercitrin-nanocoated surfaces. In previous studies, quercitrin had a bacteriostatic effect on Staphylococcus epidermidis14 while Hasan et al.20 found that quercitrin decreased S. mutans biofilm formation. However, in both studies quercitrin concentrations were much higher than the amount of biomolecule grafted to our surfaces, which was 0.2 to 0.8 nmol of quercitrin per Ti coin on QUER surfaces16. It is interesting to highlight that aminosilanized surfaces (A group) also decreased bacterial adhesion, in agreement with previous reports21. Discussion In this study we present a surface nanocoated with quercitrin, a natural flavonoid with multi-target actions, designed to improve soft tissue integration and to increase dental implants long-term success. Quercitrin-nanocoated Ti surfaces decreased initial bacterial adhesion while increasing human gingival fibroblasts Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 2 www.nature.com/scientificreports/ Figure 2. Bacterial adhesion and biofilm formation. (A) Dinamic bacterial adhesion: quantitative analysis of the live bacteria adhered to the control and modified surfaces after 30, 60 and 90 min. (B) Biofilm formation: quantitative analysis of the percentage of biofilm formation on the different surfaces after 24 hours. (C) Scanning electron microscope images of the biofilm formed on the different surfaces (scale bar = 100 μm). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti control at each time point; (b) versus A control at each time point; (c) effect of reduction within each group at each time point. Figure 2. Bacterial adhesion and biofilm formation. (A) Dinamic bacterial adhesion: quantitative analysis of the live bacteria adhered to the control and modified surfaces after 30, 60 and 90 min. (B) Biofilm formation: quantitative analysis of the percentage of biofilm formation on the different surfaces after 24 hours. (C) Scanning electron microscope images of the biofilm formed on the different surfaces (scale bar = 100 μm). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti control at each time point; (b) versus A control at each time point; (c) effect of reduction within each group at each time point. Figure 2. Bacterial adhesion and biofilm formation. (A) Dinamic bacterial adhesion: quantitative analysis of the live bacteria adhered to the control and modified surfaces after 30, 60 and 90 min. (B) Biofilm formation: quantitative analysis of the percentage of biofilm formation on the different surfaces after 24 hours. (C) Scanning electron microscope images of the biofilm formed on the different surfaces (scale bar = 100 μm). Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 Discussion It has been shown that bacterial adhesion depends on the sur- face’s terminal functionality and that, in fact, decreases on amino (NH2)-terminated surfaces22. Further studies on anti-bacterial properties of surfaces should confirm the present results. p pi p Our surfaces reduced with sodium cyanoborohydride (Red groups) showed an increased bacterial adhe- sion and biofilm formation. In a previous work, the amount of quercitrin grafted on QUER Red surfaces was around 0.1 nmol per Ti coin, less than that grafted to the non-reduced QUER surfaces16. The hydrolytic stability Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 3 www.nature.com/scientificreports/ Figure 3. Adhesion of hGF to the different surfaces. (A) Quantitative analysis of the cells adhered to the different surfaces after 15, 30, 60 and 120 minutes. (B) Cells were allowed to adhere to the surfaces for 30 min. Then, cells were stained with phalloidin-FITC (a–c; green) or with anti-vinculin antibodies (d–f; red) and nuclei were stained with DAPI (blue). Ti (a,d); A (b,e); QUER (c,f) surfaces (scale bars: upper = 100 μm; bottom = 25 μm). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti for each time point; (b) versus A for each time point; (c) the total number of cells on the different surfaces was compared. Figure 3. Adhesion of hGF to the different surfaces. (A) Quantitative analysis of the cells adhered to the different surfaces after 15, 30, 60 and 120 minutes. (B) Cells were allowed to adhere to the surfaces for 30 min. Then, cells were stained with phalloidin-FITC (a–c; green) or with anti-vinculin antibodies (d–f; red) and nuclei were stained with DAPI (blue). Ti (a,d); A (b,e); QUER (c,f) surfaces (scale bars: upper = 100 μm; bottom = 25 μm). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti for each time point; (b) versus A for each time point; (c) the total number of cells on the different surfaces was compared. of siloxanes at pH 7.5 has been reported to be poor23. Discussion Therefore, the grafting conditions used to produce QUER Red and A Red samples, at pH 7.5, may favour the hydrolisis of APTES grafted to Ti; on one hand, decreasing the availability of amino-terminal groups on A Red surfaces, and, on the other hand, decreasing the amount of quer- citrin that can be grafted on QUER Red surfaces, all in all, increasing bacterial adhesion and biofilm formation. For these reasons, we decided to select QUER surfaces for further analysis.ti y After installation of a dental implant, gingival fibroblasts are the preferred cells to repopulate the wound and to form a collagen-rich connective tissue24. We found that quercitrin-nanocoated surfaces increased initial hGF attachment. This fact together with the decreased bacterial attachment on quercitrin-nanocoated surfaces, increase hGF possibilites to win the “race for the surface” against oral bacteria25 since it depends on both an increased biomaterial surface area cell coverage and a decreased number of bacteria present on the surface26,27. Compared with often-reported monofunctional surface coatings on which bacterial adhesion and biofilm forma- tion is discouraged or they promote host tissue integration7,28, quercitrin-nanocoated surfaces decrease bacterial adhesion while increasing cell attachment. Furthermore, quercitrin-nanocoated surfaces avoid toxicity concerns of some anti-bacterial coatings. Therefore, the surfaces presented herein may form the required connective tissue around the implant to establish the biological seal that could prevent further bacterial colonization and ultimately preventing the implant loss by peri-implantitis.lf Inflammation is necessary for the effective defence against pathogens and to set in motion tissue repair fol- lowing injury29. However, excessive inflammation has been shown to delay healing and to result in increased scarring, compromising tissue regeneration30. Thus, a controlled inflammatory process after an implant place- ment is critical for their success. Here, we found that quercitrin-nanocoated surfaces effectively inhibited COX2 expression and decreased its functional product PGE2 under basal and inflammatory conditions. Furthermore, the antioxidant properties of quercitrin14 could also contribute to the inflammation resolution since oxidative stress is considered one of the major causes of inflammation and inhibition of tissue regeneration31. l Collagen synthesis and remodelling is a requisite for complete wound healing. Here we found that quercitrin-nanocoated surfaces increased COL1A1 and COL3A1 mRNA levels in basal and inflammatory conditions. Moreover, in inflammatory conditions MMPs are upregulated while TIMPs, their inhibitors, are downregulated, all together boosting collagen degradation32. Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 Discussion Quercitrin-nanocoated surfaces decreased MMP1 and increased TIMP1 mRNA levels in inflammatory conditions, thus decreasing inflammation-driven Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 4 www.nature.com/scientificreports/ Figure 4. Gene expression of hGF on the different surfaces. Gene expression analysis was performed after 14 days of cell culture. Cells were cultured with IL-1β to mimic inflammation (inflammatory group) or without it (basal group). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti within each condition; (b) versus A within each condition; (c) effect of IL-1β addition for each surface. Figure 4. Gene expression of hGF on the different surfaces. Gene expression analysis was performed after 14 days of cell culture. Cells were cultured with IL-1β to mimic inflammation (inflammatory group) or without it (basal group). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P< 0.01 and P< 0.001, respectively: (a) versus Ti within each condition; (b) versus A within each condition; Figure 4. Gene expression of hGF on the different surfaces. Gene expression analysis was performed after 14 days of cell culture. Cells were cultured with IL-1β to mimic inflammation (inflammatory group) or without it (basal group). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti within each condition; (b) versus A within each condition; (c) effect of IL-1β addition for each surface. Figure 5. PGE2 release of hGF on the different surfaces. The release of PGE2 to the culture media was evaluated after 14 days of cell culture. Cells were cultured with IL-1β to mimic inflammation (inflammatory group) or without it (basal group). One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti within each condition; (b) versus A within each condition; (c) effect of IL-1β addition for each surface. Figure 5. PGE2 release of hGF on the different surfaces. The release of PGE2 to the culture media was evaluated after 14 days of cell culture. Cells were cultured with IL-1β to mimic inflammation (inflammatory group) or without it (basal group). Methods Materials Methods Materials. Machined Ti disks, c.p. grade IV, 6.2 mm diameter and 2 mm height were purchased from Implantmedia (Lloseta, Spain). (3-Aminopropyl)triethoxysilane (APTES), quercitrin and sodium cyanoborohy- dride (NaCNBH3) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Surface nanocoatings. Surfaces were prepared as previously described (Table 1)15,16. Briefly, Ti disks were passivated by immersion in 30% (v/v) HNO3 for 30 min followed by immersion in H2O for 24 h (Ti group). Immediately after Ti passivation, aminosilanization was performed with 2% (v/v) APTES solution in dry toluene for 24 h under a dry nitrogen atmosphere. Then, disks were gently rinsed with dry toluene, acetone, and ethanol, and finally dried under a nitrogen flow. Aminosilanised Ti disks (A group) were then immersed either in quer- citrin (1 mM) at pH 5.0 (QUER group), in NaCNBH3 (100 μM) at pH 7.5 (A Red group) or in quercitrin (1 mM) and NaCNBH3 (100 μM) at pH 7.5 (QUER Red group) aqueous solutions. Samples were stirred for 1 h (150 rpm), gently rinsed with water, and dried under a nitrogen flow. NaCNBH3 was used to reduce the imine bond, resulting from the reaction between the terminal amino group of APTES and the ketone group of quercitrin, to a single -C–N- bond to decrease bond reactivity and increase coating reproductibility16. This reduction is known to be selective for imine bonds at mildly basic pH. For bacterial experiments, surfaces were polished and cleaned35 prior to surface modification. Some samples were disinfected with dry heat (100 °C, 30 min) previous to bacteria/ cell seeding. Bacterial experiments. The bacterial strain used in this study was Streptococcus mutans ATCC 25175 (S. mutans). The strain was maintained in brain heart infusion (BHI; Sigma–Aldrich) agar plates and cultured in BHI broth for 24 h at 37 °C and 5% CO2. Bacterial adhesion. Bacterial suspension was homogenized through a 3 min ultrasonic bath (Ultrasons; JP Selecta, Barcelona, Spain) and harvested by centrifugation, washed two times with artificial saliva36 precondi- tioned at 37 °C, and resuspended at a concentration of 3 × 108 bacteria/ml in artificial saliva. The flow system was a modified Robbins device where disks were fixed to removable ports that allow contact between the surfaces under study and a flow of suspended bacteria in laminar conditions. Before each experiment, the whole system was filled with artificial saliva and preconditioned at 37 °C. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Group Treatment Ti Titanium passivated A Ti + APTES A Red Ti + APTES + NaCNBH3 QUER Ti + APTES + QUER QUER Red Ti + APTES + QUER + NaCNBH3 Table 1. Groups included in the experiment. Table 1. Groups included in the experiment. ECM-destruction. Noteworthy, quercitrin decreased the expression of pro-fibrotic markers in a wound healing in vitro model14 and higher proportions of type III collagen are related to more-regenerative than more-scarring responses33. Thus, data suggest that quercitrin-nanocoated surfaces may induce connective tissue forma- tion around a dental implant even in the inflammatory conditions found after a dental implant installation. Furthermore, in previous studies of our group using mesenchymal stem cells, quercitrin-nanocoated surfaces increased cell adhesion and osteoblastic differentiation16. Therefore, quercitrin-nanocoated surfaces could also increase the osseointegration process of dental implants. Further clinical studies should confirm the potential of quercitrin-nanocoated surfaces to enhance soft and hard tissue integration of dental implants. ECM-destruction. Noteworthy, quercitrin decreased the expression of pro-fibrotic markers in a wound healing in vitro model14 and higher proportions of type III collagen are related to more-regenerative than more-scarring responses33. Thus, data suggest that quercitrin-nanocoated surfaces may induce connective tissue forma- tion around a dental implant even in the inflammatory conditions found after a dental implant installation. Furthermore, in previous studies of our group using mesenchymal stem cells, quercitrin-nanocoated surfaces increased cell adhesion and osteoblastic differentiation16. Therefore, quercitrin-nanocoated surfaces could also increase the osseointegration process of dental implants. Further clinical studies should confirm the potential of quercitrin-nanocoated surfaces to enhance soft and hard tissue integration of dental implants. qt g p In concusion, nanocoating of titanium surfaces with quercitrin decreased initial bacterial adhesion and increased human gingival fibroblasts attachment. Furthermore, quercitrin-nanocoated Ti increased colla- gen mRNA levels and decreased MMP1/TIMP1 mRNA ratio while also decreasing COX2 mRNA and PGE2 release under basal and inflammatory conditions. These results are in agreement with previous studies where we found that quercitrin had positive effects on cells from soft and hard tissue when added to the culture media34. From a clinical perspective, quercitrin-nanocoated surfaces could increase dental implants efficacy by enhanc- ing peri-implant soft tissue regeneration and decreasing peri-implantitis. Furthermore, this study constitutes an example that quercitrin, and flavonoids in general, could be used in combination with other biomaterials, i.e. hydrogels or scaffolds required in severe cases of periodontal and peri-implant diseases. Discussion One, two and three symbols represent a significant difference between two groups with P ≤ 0.05, P < 0.01 and P < 0.001, respectively: (a) versus Ti within each condition; (b) versus A within each condition; (c) effect of IL-1β addition for each surface. Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 5 Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 Methods Materials Primary human gingival fibroblasts (hGF) were purchased from Provitro GmbH, Berlin, Germany). Three different donors were used (range 19–47 years; male:female ratio 2:1). Provitro assures that cells were obtained ethically and legally and that all donors provided written informed consent. Cells were routinely cultured at 37 °C and 5% CO2, and maintained in fibroblast growth medium (Provitro GmbH) supplemented with foetal calf serum (10%; Provitro GmbH), amphotericin (50 ng/ml) and gentamicin (50 μg/ml). Experiments were performed with hGF between passages 7 and 8 after isolation and media was supplemented with ascorbic acid (100 μM; Sigma-Aldrich).hfh μ g The different coins were placed in 96-well plates in sterile conditions. Three replicates for each donor were seeded at a density of 7.0 × 103 cells on each coin (n = 9). For cell adhesion experiments, four replicates from one randomly selected donor were used (n = 4).l In order to create inflammatory conditions, interleukin-1 beta (1 ng/ml) (IL-1β ; R&D systems, Abingd UK) was added 1 d after cell seeding and kept until day 3, according to previous studies34. Cell adhesion and vinculin immunostaining. Once seeded, hGFs were allowed to adhere for 15, 30, 60 and 120 minutes to the different surfaces. Then, unbounded cells were removed by washing twice with PBS. Cells were fixed with paraformaldehyde (4%, 15 min) and permeabilised with Triton X-100 (0.25%, 10 min). For nucleus counting, cells were stained with Phalloidin-FITC (5 μg/ml, 30 min). For vinculin immunostaining, cells were blocked with bovine serum albumin (5%, 1 h; Sigma-Aldrich) followed by incubation with anti-vinculin recombinant rabbit monoclonal antibody (4 μg/ml, 3 h, Invitrogen) and then labelled with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (5 μg/ml, 30 min, Thermo Scientific, Rockford, IL, USA). Samples were then mounted with DAPI-Fluoroshield (Sigma-Aldrich) and visualized under a fluorescence microscope and a confo- cal microscope. For nucleus counting, two pictures from the same positions of each coin were taken. Cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA). RNA isolation and real-time RT-PCR analysis. After, 14 days of cell culture, total RNA was isolated using Tripure (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s protocol. Total RNA was quantified at 260 nm using a nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Methods Materials Afterward, the bacterial suspension was allowed to flow through the system at a flow rate of 2 ml/min, corresponding to a shear rate of 0.97 s−1 at the wall of the flow chamber (unpublished results). This shear rate is such that the specific interactions that could occur between the cells and the different substrata dominate over the drag force of the fluid flow. The bacterial suspension was perfused through the system with recirculation for 30, 60 and 90 min. Samples were carefully removed from the flow chamber and bacteria were stained with the kit Live/Dead Baclight L-7012 (Invitrogen, Camarillo, CA, USA) according to the manufacturer. Then, 5–6 pictures at randomly chosen locations from each sample were taken under fluorescence and the number of bacteria per unit area was counted. The experiments were run in triplicate from separately grown bacterial suspensions. iofilm formation. Biofilm formation was quantified as previously described37. Briefly, bacteria were resus- ended at a concentration of 3 × 108 bacteria/ml in BHI. Ti-modified surfaces were disinfected and placed in Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 6 www.nature.com/scientificreports/ 96-well plates in sterile conditions. Then, bacterial suspension (200 μl) was cultured on the surfaces at 37 °C and gentle shaking (20 rpm). After 90 min, bacterial suspension was carefully removed and fresh BHI was added. Samples were cultured at 37 °C and gentle shaking (20 rpm) for 24 h. Biofilm formation was quantified using BacTiter-Glo (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. Culture media was removed and BacTiter-Glo (200 μl) was added to the wells and incubated (10 min). Then, the content of each well was transferred to wells 96-well white polystyrene flat-bottomed microtiter plates. The light emission (luciferin-luciferase reaction) was measured in a microplate reader. Each assay was performed in duplicate and repeated two times from separately grown bacterial suspension in order to confirm reproducibility. Scanning electron microscopy. To visualise biofilm formation on the surfaces, bacteria were fixed with glutaral- dehyde in PBS (4%, 2 h) and washed with distilled water. At 30 min intervals, the samples were dehydrated by the addition of 50%, 70%, 90% and 100% ethanol solutions. Samples were left at room temperature to evaporate the remaining ethanol before sputter gold coating. A scanning electron microscope (SEM; Hitachi S-3400 N, Krefeld, Germany) using secondary electrons, low vacuum conditions and 15 kV of voltage was used to acquire images. Cell culture. Methods Materials The same amount of RNA (0.2 μg) was reverse transcribed to cDNA (42 °C, 60 min) according to the pro- tocol of the supplier (High Capacity RNA-to-cDNA kit, Applied Biosystems, Foster City, CA, USA). Aliquots of each cDNA were frozen (− 20 °C) until the PCR reactions were carried out. Real-time PCR was performed for two reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTBL2), and target genes (Table 2). Real-time PCR was performed in a thermocy- cler (Lightcycler 480, Roche Diagnostics) using SYBR green detection. Each reaction contained 7 μl of master mix (Lightcycler 480 SYBR Green I Master, Roche Diagnostics), the sense and the antisense specific primers (0.5 μM) and cDNA sample (3 μl) in a final volume of 10 μl. The amplification program consisted of a pre-incubation step for denaturation of the template cDNA (5 min 95 °C), followed by 45 cycles consisting of a denaturation step (10 s 95 °C), an annealing step (10 s 60 °C) and an extension step (10 s 72 °C). After each cycle, fluorescence was meas- ured at 72 °C. A negative control without cDNA template was run in each assay. g p y All samples were normalized by the geometric mean of the expression levels of ACTBL2 and GAPDH and fold changes were related to the control groups using the following equation: ratio = Etarget ΔCp target (mean control – sample)/ Ereference ΔCp reference (mean control – sample) adapted from38, where Cp is the is the crossing point of the reaction amplifi- cation curve and E is the eficiency from the given slopes using serial dilutions, as determined by the software (Lightcycler 480 software, Roche Diagnostics). Stability of reference genes was calculated using a statistical tool (BestKeeper software, Technical University of Munich, Weihenstephan, Germany)39. References Phytomedicine 18, 990–993 (2011).l p y y 3. Cushnie, T. P. T. & Lamb, A. J. Recent advances in understanding the antibacterial properties of flavonoids. Int. J. Antimicrob. Agent 38, 99–107 (2011).i 4. Gómez-Florit, M., Monjo, M. & Ramis, J. M. Identification of quercitrin as potential therapeutic agent for periodontal applications J. Periodontol. 85, 966–974 (2014).il 15. Córdoba, A. et al. Flavonoid-modified surfaces: multifunctional bioactive biomaterials with osteopromotive, anti-inflammatory, and anti-fibrotic potential. Adv. Healthc. Mater. 4, 540–549 (2015). 16. Córdoba, A., Monjo, M., Hierro-Oliva, M., González-Martín, M. L. & Ramis, J. M. Bioinspired quercitrin nanocoatings: a fluorescence-based method for its surface quantification, and its effect on stem cell adhesion and differentiation to the osteoblastic lineage. ACS Appl. Mater. Interfaces (2015) doi: 10.1021/acsami.5b05044.i ineage. ACS Appl. Mater. Interfaces (2015) doi: 10.1021/acsami.5b0 g pp f 7. Hultin, M. et al. Microbiological findings and host response in patients with peri-implantitis. Clin. Oral Implants Res. 13, 349–58 (2002).i 8. Lévesque, C. M. et al. Involvement of sortase anchoring of cell wall proteins in biofilm formation by Streptococcus mutans. Infect Immun. 73, 3773–3777 (2005).fi 9. Wang, Y., Lee, S. M. & Dykes, G. A. Potential mechanisms for the effects of tea extracts on the attachment, biofilm formation and cel size of Streptococcus mutans. Biofouling 29, 307–18 (2013). p f g 0. Hasan, S., Singh, K., Danisuddin, M., Verma, P. K. & Khan, A. U. Inhibition of major virulence pathways of Streptococcus mutan by quercitrin and deoxynojirimycin: a synergistic approach of infection control. PLoS One 9, e91736 (2014). d l ll d l h l f f b d l l ll d 1. Rodríguez-Cano, A. et al. Controlled silanization-amination reactions on the Ti6Al4V surface for biomedical applications. Colloid Surf. B Biointerfaces 106, 248–257 (2013).il 2. Katsikogianni, M. G. & Missirlis, Y. F. Bacterial adhesion onto materials with specific surface chemistries under flow conditions. In J. Mat. Sci. Mater. Med. 21, 963–968 (2010). g pil J. Mat. Sci. Mater. Med. 21, 963–968 (2010). 23. Silverman, B. M., Wieghaus, K. a. & Schwartz, J. Comparative properties of siloxane vs phosphonate monolayers on a key titanium alloy Langmuir 21 225–228 (2005) 3. Silverman, B. M., Wieghaus, K. a. & Schwartz, J. Comparative properties of siloxane vs phosphonate monolayers on a key titanium alloy. Langmuir 21, 225–228 (2005).if y g 4. Palaiologou, A. A., Yukna, R. A., Moses, R. & Lallier, T. E. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Gene Primer sequence (5′–3′) Product size (bp) Beta-actin (ACTBL2) S: CTGGAACGGTGAAGGTGACA 136 A: AAGGGACTTCCTGTAACAATGCA Collagen I α 1 (COL1A1) S: AGAGCATGACCGATGGATTC 122 A: TTCTTGAGGTTGCCAGTC Collagen III α 1 (COL3A1) S: GGCCTACTGGGCCTGGTGGT 190 A: CCACGTTCACCAGGGGCACC Cyclooxygenase-2 (COX2) S: ATGGGGTGATGAGCAGTTGT 221 A: GAAAGGTGTCAGGCAGAAGG Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) S: TGCACCACCAACTGCTTAGC 87 A: GGCATGGACTGTGGTCATGAG Matrix metalloproteinase-1 (MMP1) S: TGTCAGGGGAGATCATCGGGACA 177 A: TGGCCGAGTTCATGAGCTGCA Metalloproteinase inhibitor-1 (TIMP1) S: TTCCGACCTCGTCATCAGGG 144 A: TAGACGAACCGGATGTCAGC Table 2. Sense (S) and antisense (A) primers used in the real-time PCR of reference and target genes. USA) and GraphPad Prism (version 6, La Jolla, CA, USA) were used. Results were considered statistically signifi- cant at P ≤ 0.05. 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Methods Materials g p y All samples were normalized by the geometric mean of the expression levels of ACTBL2 and GAPDH and fold changes were related to the control groups using the following equation: ratio = Etarget ΔCp target (mean control – sample)/ E f ΔCp reference (mean control – sample) adapted from38 where Cp is the is the crossing point of the reaction amplifi- g g g g g Ereference ΔCp reference (mean control – sample) adapted from38, where Cp is the is the crossing point of the reaction amplifi- cation curve and E is the eficiency from the given slopes using serial dilutions, as determined by the software (Lightcycler 480 software, Roche Diagnostics). Stability of reference genes was calculated using a statistical tool (BestKeeper software, Technical University of Munich, Weihenstephan, Germany)39. Prostaglandin E2 quantification. 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Cathepsin C, matrix metalloproteinases, and their tissu crevicular fluid from periodontitis-affected patients. J. Dent. Res. 81, 174–178 (2002).i lf 33. Satish, L. & Kathju, S. Cellular and molecular characteristics of scarless versus fibrotic wound healing. Dermatol. Res. Pract. 2010, 790234 (2010). ( ) 34. Gómez-Florit, M., Monjo, M. & Ramis, J. M. Quercitrin for periodontal regeneration: effects on human gingival fibroblasts and mesenchymal stem cells. Sci. Rep. Author Contributions M.G.F., M.P.O., M.F.C., A.C., M.G.M., M.M. and J.M.R. contributed to the conception and design. M.G.F. and A.C. produced the samples. M.G.F., M.P.O. and M.F.C. acquired and analysed data. M.G.F. wrote the main manuscript text. M.G.F., M.P.O., M.F.C., A.C., M.G.M., M.M. and J.M.R. contributed to interpretation of the results and reviewed the manuscript. Acknowledgementsh g This work was supported by the Conselleria d’Educació, Cultura i Universitats of the Balearic Islands Government and the European Social Fund (contract to JMR - PD/018/2014), by the Osteology Foundation (Grant Number: 13-069) and by the Instituto de Salud Carlos III (CIBER-BBN Mobility grant to MGF). The authors thank Dr. Ferran Hierro (University of the Balearic Islands) for his technical contribution with SEM. g This work was supported by the Conselleria d’Educació, Cultura i Universitats of the Balearic Islands Government and the European Social Fund (contract to JMR - PD/018/2014), by the Osteology Foundation (Grant Number: 13-069) and by the Instituto de Salud Carlos III (CIBER-BBN Mobility grant to MGF). The authors thank Dr. Ferran Hierro (University of the Balearic Islands) for his technical contribution with SEM. Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare the following conflict of interests: M.G.F., A.C., M.M. and J.M.R. are inventors of a pending patent application based on some aspects of this work (PCT/EP2013/058116). Competing financial interests: The authors declare the following conflict of interests: M.G.F., A.C., M.M. and J.M.R. are inventors of a pending patent application based on some aspects of this work (PCT/EP2013/058116). How to cite this article: Gomez-Florit, M. et al. Quercitrin-nanocoated titanium surfaces favour gingival cells against oral bacteria. Sci. Rep. 6, 22444; doi: 10.1038/srep22444 (2016). How to cite this article: Gomez-Florit, M. et al. Quercitrin-nanocoated titanium surfaces favour gingival cells against oral bacteria. Sci. Rep. 6, 22444; doi: 10.1038/srep22444 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:22444 \| DOI: 10.1038/srep22444 9
https://openalex.org/W4234435811	https://myukk.org/SM2017/sm_pdf/SM1889.pdf	English	null	Thermal Design and In Situ Temperature Measurement of Heterogeneous Material during Ultrafast Laser Scribing	Sensors and materials	2,019	cc-by	4,364	(Received March 26, 2018; accepted April 3, 2019) Keywords: in situ temperature, ultrafast laser, heterogeneous material, surface temperature distribution Keywords: in situ temperature, ultrafast laser, heterogeneous material, surface temperature distributions In this paper, an in situ technique is developed to measure the temperature distribution during the ultrafast laser scribing (machining) of heterogeneous materials using embedded sensors. The materials investigated in this study are an epoxy molding compound (EMC), solder bumps, and a specially designed ultrathin printed circuit board (PCB). The laser machining processes involved in this study are cutting/scribing through EMC and PCB, and trenching/scribing through EMC alone. Small thermocouples have been designed and inserted inside the heterogeneous materials to record the in situ temperature distribution during ultrafast laser irradiation and the thermal effect is carefully investigated. The highest temperature of 100.94 °C is recorded at the corner scribing position. For comparison, a forward-looking infrared (FLIR) thermal imaging camera was applied to capture the real-time surface temperature distributions of PCB and EMC. Owing to the ambient heat radiation, material emissivity, and reflective apparent temperature, the peak surface temperature detected by FLIR would be slightly lower than the in situ temperature measured by an embedded sensor. The thermal design in this jam-packed area is then applied to a standard laser manufacturing process. Corresponding author: e-mail: hchsu@isu.edu.tw https://doi.org/10.18494/SAM.2019.2328 1667 1667 Sensors and Materials, Vol. 31, No. 5 (2019) 1667–1677 MYU Tokyo Sensors and Materials, Vol. 31, No. 5 (2019) 1667–1677 MYU Tokyo S & M 1889 Sensors and Materials, Vol. 31, No. 5 (2019) 1667–1677 MYU Tokyo Thermal Design and In Situ Temperature Measurement of Heterogeneous Material during Ultrafast Laser Scribing Wen-Fei Lin,1 Hsiang‑Chen Hsu,2,3,4 Shih-Jeh Wu,4 and Boen Houng1 Wen-Fei Lin,1 Hsiang‑Chen Hsu,2,3,4* Shih-Jeh Wu,4 and Boen Houng1 1Department of Material Science and Engineering, I-Shou University, No. 1, Sec. 1, Syuecheng Rd., Dashu District, Kaohsiung City 84001, Taiwan, ROC 2Department of Mechanical and Computer-Aided Engineering, St. John’s University, No. 499, Sec. 4, Tam King Rd., Tamsui District, New Taipei City 25135, Taiwan, ROC 3Department of Industrial Management, I-Shou University, No. 1, Sec. 1, Syuecheng Rd., Dashu District, Kaohsiung City 84001, Taiwan, ROC 4Department of Mechanical and Automation Engineering, I-Shou University, No. 1, Sec. 1, Syuecheng Rd., Dashu District, Kaohsiung City 84001, Taiwan, ROC 1. Introduction The epoxy molding compound (EMC) has been widely applied to the IC packaging material in the semiconductor industry for decades.(1–3) EMC consists of 20 raw materials, such as epoxy resin (organic resin), fused silica (inorganic filler), catalysts, mold release material, pigment, flame retardants, adhesion promoters, ion traps, and stress relievers.(1) Usually, epoxy and filler dominate the mechanical property of EMC, that is, 12–15 wt% epoxy resin provides good machining quality and 70–80 wt% filler increases the strength and thermal conduction, Corresponding author: e-mail: hchsu@isu.edu.tw https://doi.org/10.18494/SAM.2019.2328 Corresponding author: e-mail: hchsu@isu.edu.tw https://doi.org/10.18494/SAM.2019.2328 ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ 1668 Sensors and Materials, Vol. 31, No. 5 (2019) and reduces the coefficient of thermal expansion. After the components on the printed circuit board (PCB) are encapsulated by EMC and singulated as one single unit, the whole package of heterogeneous materials undergoes a burn-in test process at the early stage of the mass product period. This test purposely induces certain failures under stringent conditions, such as that of the end product, and determines the reliability of the end product.(4,5) Traditionally, saw cutting is the main solution to singulate the integrated circuit unit. As new technology innovation emerges, there are new challenges; for example, in cutting irregular shapes and rigid and flexible materials, a mechanical saw can no longer meet the requirement, and a laser has become a new solution for singulation.(6) As a mechanical saw has to be operated in cutting fluid media, a laser works with air similarly. However, that is no comparison to the heat effect related to product reliability.(7) For instance, laser scribing creates a warmer environment than a normal burn-in temperature, i.e., 250 °C in most cases, and a regular burn-in test should be exercised under a more rigorous condition in the cases related to the laser process. There will be more laser applications to come during the assembly of system- in-package (SiP) products. More applications need more components in a regular PCB area, which would result in the undesired crosstalk of signals. 1. Introduction SiP is a design with high-density components on PCB, so high-giga components need to be isolated to prevent the noise effect on other components by shielding.(8) The specific structure of SiP shielding in a compound, which is hard to build by molding, can be created by laser scribing.(9) It is interesting to researchers and engineers to investigate how to maintain the performance of the components shielded in the package after the laser machining of the shielding structure. High temperature causes defects on the cured epoxy, such as material melting, deterioration, cracks, and loss of adhesion to copper.(10) Using a short or ultrashort pulse laser is a solution for semiconductor processes.(11) The main reason for this is that they cause a minimal thermal effect during the scribing processes. Those reasons explain why both researchers and engineers are interested in how high the temperature is during laser scribing. In this study, the authors present the design of the in situ temperature measurement of heterogeneous materials during ultrafast laser scribing. A noncontact forward-looking infrared (FLIR) thermal imaging camera is capable of scanning and visualizing the temperature distribution of an entire surface in many industrial applications.(12,13) However, there is still some difficulty encountered when FLIR is detecting the temperature of the surface during laser machining. Thus, the use of K-type thermocouples inserted inside the material near laser scribing has been demonstrated to be an accurate approach to measure the temperature in situ. 2. Materials and Methods A nanosecond UV laser (355 nm wavelength, 20 W power, <1.3 M2 beam quality, 200 μJ pulse energy, and 90–110 kHz base frequency) was used, and the optimal machining parameters for the cutting and trenching of the molding compound and PCB of SiP were explored. In laser scribing, the target material is a mixture of heterogeneous materials of EMC and PCB. The epoxy compound with a thickness of 0.8 mm in an ultrafine filler size range of 15–35 μm and PCB with a thickness of 0.36 mm were tested to investigate thermal behaviors during laser Sensors and Materials, Vol. 31, No. 5 (2019) 1669 scribing. There are four copper layers in PCB, where three copper layers are in the cutting street and the one copper layer is solder-printed for trenching. Each copper layer is 0.5 oz, and between EMC and PCB, there is a solder of about 45 μm height printed on the copper pad for the trenching dump, as shown in Fig. 1. Note that a solder microball has been deformed by laser trenching. 2.1 Optics system The original laser beam is insufficient for cutting through a SiP structure and needs optics to support the scribing process. A galvo-scanner head was used for high-feed-rate scribing, and a telecentric lens was used for a more vertical wall side, which was installed with a laser. Figure 2(a) schematically illustrates an innovated optical installation developed in this study.(14) A focal shifter helps in the installation, which easily makes the beam focus on the material bottom and is fixed during scribing. The theoretical spot size is designed to be 13.7 μm for this system. Fig. 1. (Color online) Cross section of SiP material. Fig. 1. (Color online) Cross section of SiP material. Fig. 2. (Color online) (a) Schematic illustration of optics design system. (b) Actual installation inside the laser machine. (a) (b) (b) (a) (b) (a) Fig. 2. (Color online) (a) Schematic illustration of optics design system. (b) Actual installation inside the las machine. 1670 Sensors and Materials, Vol. 31, No. 5 (2019) Figure 2(b) shows the actual optics system used in this study. First, a laser beam is generated by the laser resonator. The beam was manipulated by a focal shifter and a galvo to scribe on the position designed on the material, which will be described in Sect. 2.3. The telecentric lens makes the beam a small spot with a machine ability on the material. 2.2 Laser trench/cutting in scribing process For trenching, the parameters are as follows: a feed speed of 800 mm/s, a repetition rate of 100 kHz, and an output average power of 17.5 W measured under the lens. The scribing process used 146 passes to create the trench, which is shown in Fig. 3. For the cutting process, the parameters measured under the lens are as follows: a feed speed of 800 mm/s, a repetition rate of 100 kHz, and an output average power of 17.5 W. The scribing process was repeated 100 times to cut through the material. After trenching and removing the material, the top view of the test sample is shown in Fig. 4. The cutting shape is a rectangle and the trenching shape is a turned L shape from the top view of the SiP. The total cutting length is 108 mm with four corners, and the total trenching length is 14 mm with one corner. Note again that there are three copper layers in the cutting street and one more copper layer in the trenching position for solder-printed trenching. 2.3 In situ temperature measurement K-type thermocouples were used for in situ measurement in this study. The SiP was drilled with some 1-mm-diameter holes and then thermocouples were inserted into the holes. All the holes were drilled from PCB side with different depths as shown in Fig. 5, i.e., (a) a sensor inserted into the middle of EMC, (b) a sensor inserted into the middle of PCB, and (c) a sensor inserted into the middle of a solder bump. Note that the K-type thermocouple is the most common sensor calibration type providing the widest operating temperature range and generally works in most applications. A thermocouple Fig. 4. (Color online) Top view of test sample for scribing (trenching and cutting). Fig. 3. (Color online) Cross section of trenching structure designed in SiP. Fig. 3. (Color online) Cross section of trenching structure designed in SiP. Fig. 4. (Color online) Top view of test sample for scribing (trenching and cutting). 1671 Sensors and Materials, Vol. 31, No. 5 (2019) Fig. 5. (Color online) Cross sections of drilling holes with different depths for a thermocouple to measure (a) EMC, (b) PCB, and (c) solder bump. (a) (b) (c) (c) (b) (a) (b) (c) (a) Fig. 5. (Color online) Cross sections of drilling holes with different depths for a thermocouple to measure (a) EMC, ( PCB, and (c) solder bump. circuit contains two alloy junctions, such as wire sand connectors and a voltage-measuring device. When two different temperatures were detected, measurable current flows through the circuit. The current is related to the temperature differential, which presents the detected temperature. Normally, the output signals from the K-type thermocouple are very small and need to be carefully calibrated before use. Owing to its reliability and accuracy, the K-type thermocouple is used extensively at temperatures up to 1260 °C. However, the K-type thermocouple with a temperature range of 0–275 °C was used in this study. For EMC and PCB temperature measurements, the in situ temperature was assessed for both cutting and trench processes, respectively. For solder temperature measurement, the in situ temperature was assessed only for the trench process. The thermocouples’ positions were gauged, as shown in Fig. 6, and designed 200 μm away from the scribing area, which is a normal position where an electronic component is located. For the temperature measured in a printed solder bump, Fig. 2.3 In situ temperature measurement 6(c) illustrates the thermocouples measured for the trenching process only, which were only inserted at the trenching position. There would be four thermocouples inserted in four different holes, labeled T1 to T4 in Fig. 6(a), to collect temperature data at the same time in one cutting process, and three thermocouples in three different holes, labeled T1 to T3 in Fig. 6(b), to collect temperature data at the same time in one trenching process. The positions of three thermocouples used to collect the in situ temperature data of the solder in the trenching process are labeled T1 to T3, as shown in Fig. 6(c). 2.4 Design of experiment (DOE) Three materials (EMC, PCB, and solder), in situ temperature measurement (positions labeled T1 to T5), and two scribing processes (cutting and trenching) were used to create five DOEs, which are summarized in Table 1. Note that there is no temperature measured in the solder bump for the cutting process. Each DOE was carried out individually, namely, DOE1 to DOE3 show one trench process to obtain three temperatures from three thermocouples for one material, and DOE4 to DOE5 show one cutting process to obtain four temperatures from four thermocouples for one material. Sensors and Materials, Vol. 31, No. 5 (2019) 1672 (a) Fig. 6. (Color online) Schematic diagrams of thermocouples’ positions of (a) cutting, (b) trenching, and (c) solder bump in trenching. (a) (b) (c) (c) (b) (b) (c) (a) Fig. 6. (Color online) Schematic diagrams of thermocouples’ positions of (a) cutting, (b) trenching, and (c) solder bump in trenching. Table 1 DOEs used in this study. DOE No. Process Material Position label of in situ temperature measurement DOE1 Trench EMC T1 T2 T3 — DOE2 Trench PCB T1 T2 T3 — DOE3 Trench Solder T1 T2 T3 — DOE4 Cutting EMC T1 T2 T3 T4 DOE5 Cutting PCB T1 T2 T3 T4 Table 1 DOEs used in this study. 2.5 Surface temperature real-time detection An alternative real-time temperature detected using a FLIR thermal imaging camera was used for comparison. The FLIR thermal imaging camera can distinguish fixed forward-looking thermal images from sideways-tracking infrared systems, which can very easily generate a real-time high-resolution image. A noncontact FLIR thermal imaging camera can scan and visualize the temperature distribution of an entire surface. However, the system requires material emissivity and reflective apparent temperature to precisely detect the temperatures of the target materials. Figure 7 shows the material emissivity calibration scheme used in this study. The object under test (PCB) was inserted with a K-type thermocouple and placed on a heating plate. The FLIR thermal imaging camera was aimed toward the target PCB with the thermocouple. The real-time temperature was recorded as the heating plate was continuously heating. Once the temperature in the thermal image was exactly the same as that detected by the K-type thermocouple, the material emissivity was then determined using the FLIR thermal imaging camera system. The calibrated material emissivity for PCB is 0.96. Figure 8 shows the real- time temperature detection equipment used in this study. 1673 Sensors and Materials, Vol. 31, No. 5 (2019) Fig. 7. (Color online) Emissivity calibration for PCB material. Fig. 8. (Color online) Real-time surface temperature detection using FLIR 440 cameras. Fig. 7. (Color online) Emissivity calibration for PCB material. Fig. 8. (Color online) Real-time surface temperature detection using FLIR 440 cameras. 3. Results and Discussion Figure 2(b) shows the main devices for laser machining. The test samples (workpiece) were mounted on an XY table and exposed to the atmosphere (air). At the upper right corner, there is an air blower (nozzle) to remove ash, dust, and residues from the machined surface. The temperature of the laser’s front end is assumed to be high enough to melt the workpiece. However, the temperature of the machined surface will be easily transferred to the operating ambient. Hence, the real-time surface temperature measured using the FLIR thermal imaging camera would be relatively lower than the actual laser machining temperature. Therefore, the embedded K-type thermocouple would be appropriate for measuring the in situ temperature along the cut depth. The thermocouples were connected to a monitor and recorded the accumulated temperature in the sample during laser scribing. It was one cycle with four steps, i.e., start of thermocouple sampling, start of laser scribing, end of laser scribing, and finally, end of thermocouple sampling. The temperature-time chart for each DOE was then automatically graphed. When laser scribing started, the temperature also rose at the same time. The temperature immediately fell after laser scribing. The measured peak temperature for every positioned thermocouple was also reported for the five DOEs. The temperatures measured in situ are shown in Figs. 9–13. After the 5 DOEs were carried out, the maximum temperature from the thermocouple sampled in one cycle received for all 25 graphs. Results were collected and are listed in Table 2. The obtained temperature variances were as follows: DOE1, 23.62 ℃; DOE2, 19.65 ℃; DOE3, 32.45 ℃; DOE4, 12.60 ℃; DOE5, 19.38 ℃. This implies that the area near the solder bump would cause a large thermally induced warpage owing to the mismatch of the coefficient of thermal expansion among heterogeneous materials. For comparison in the cutting process, the FLIR thermal imaging camera system with a thermal imaging system was used to detect the surface real-time temperature distributions of PCB and EMC, which are depicted in Figs. 14(a) and 14(b), respectively. PCB is a typical multilayer material consisting of copper foil, resin matrix, reinforcement, prepreg, and fillings. The temperature captured on PCB may vary owing to laser cutting on a metal or resin. Also, Sensors and Materials, Vol. 31, No. 5 (2019) 1674 Fig. 11. (Color online) DOE3: temperatures of solder measured in situ during trenching (℃). Fig. 12. 3. Results and Discussion (Color online) DOE4: temperatures of EMC measured in situ during cutting (℃). Fig. 9. (Color online) DOE1: temperatures of EMC measured in situ during trenching (℃). Fig. 10. (Color online) DOE2: temperatures of PCB measured in situ during trenching (℃). Fig. 9. (Color online) DOE1: temperatures of EMC measured in situ during trenching (℃). Fig. 10. (Color online) DOE2: temperatures of PCB measured in situ during trenching (℃). Fig. 9. (Color online) DOE1: temperatures of EMC measured in situ during trenching (℃). Fig 10 (Color online) DOE2: temperatures of PCB measured in situ during trenching (℃) Fig. 9. (Color online) DOE1: temperatures of EMC measured in situ during trenching (℃). Fig. 9. (Color online) DOE1: temperatures of EMC measured in situ during trenching (℃). Fig. 10. (Color online) DOE2: temperatures of PCB measured in situ during trenching (℃). Fig. 10. (Color online) DOE2: temperatures of PCB measured in situ during trenching (℃). Fig. 11. (Color online) DOE3: temperatures of solder measured in situ during trenching (℃). Fig. 12. (Color online) DOE4: temperatures of EMC measured in situ during cutting (℃). Fig. 12. (Color online) DOE4: temperatures of EMC measured in situ during cutting (℃). 1675 Sensors and Materials, Vol. 31, No. 5 (2019) Fig. 13. (Color online) DOE5: temperatures of PBC measured in situ during cutting (℃). Fig. 13. (Color online) DOE5: temperatures of PBC measured in situ during cutting (℃). Fig. 14. (Color online) Cutting temperature distributions captured using FLIR camera system: (a) PCB and (b) EMC. Table 2 In situ temperature of laser scribing (unit: ℃). DOE No. Process Material Position label T1 T2 T3 T4 T5 DOE1 Trench EMC 62.56 72.37 63.98 53.21 64.89 DOE2 Trench PCB 67.63 78.77 87.28 77.66 69.37 DOE3 Trench Solder 68.49 100.94 91.99 79.30 75.32 DOE4 Cutting EMC 71.21 81.63 76.36 69.03 72.22 DOE5 Cutting PCB 72.8 91.18 86.82 82.28 75.45 (a) (b) Fig. 14. (Color online) Cutting temperature distributions captured using FLIR camera system: (a) PCB and (b) EMC. Table 2 In situ temperature of laser scribing (unit: ℃). DOE No. 3. Results and Discussion Process Material Position label T1 T2 T3 T4 T5 DOE1 Trench EMC 62.56 72.37 63.98 53.21 64.89 DOE2 Trench PCB 67.63 78.77 87.28 77.66 69.37 DOE3 Trench Solder 68.49 100.94 91.99 79.30 75.32 DOE4 Cutting EMC 71.21 81.63 76.36 69.03 72.22 DOE5 Cutting PCB 72.8 91.18 86.82 82.28 75.45 (a) (b) Table 2 In situ temperature of laser scribing (unit: ℃). DOE No. Process Material Position label T1 T2 T3 T4 T5 DOE1 Trench EMC 62.56 72.37 63.98 53.21 64.89 DOE2 Trench PCB 67.63 78.77 87.28 77.66 69.37 DOE3 Trench Solder 68.49 100.94 91.99 79.30 75.32 DOE4 Cutting EMC 71.21 81.63 76.36 69.03 72.22 DOE5 Cutting PCB 72.8 91.18 86.82 82.28 75.45 Table 2 In situ temperature of laser scribing (unit: ℃). Table 2 Fig. 14. (Color online) Cutting temperature distributions captured using FLIR camera system: (a) PCB and (b) EMC. (a) (b) (b) (a) (a) (b) Fig. 14. (Color online) Cutting temperature distributions captured using FLIR camera system: (a) PCB and (b) EMC. the in-depth temperature of PCB was difficult to reflect on the surface owing to the various conductivities of the materials. The temperature of EMC is slightly higher than the temperature measured by the thermocouple. This may be caused by the ambient radiation effects on direct laser cutting. the in-depth temperature of PCB was difficult to reflect on the surface owing to the various conductivities of the materials. The temperature of EMC is slightly higher than the temperature measured by the thermocouple. This may be caused by the ambient radiation effects on direct laser cutting. 1676 Sensors and Materials, Vol. 31, No. 5 (2019) Acknowledgments This work was supported by ASE Kaohsiung, Taiwan and ENR Corporation. The authors thank Bear Jiang and Peter Ku for providing the test materials. We also thank the ENR RD team for providing an integral laser scribing machine. 4. Conclusions In this paper, the authors describe the temperature response of a material and how it was detected by sensors. Engineers would very much like to have more space to mount more components and ICs in a very limited space in PCB. During cutting, the highest temperature T2 was observed by T2 on a trench pad, i.e., 81.63 ℃ for EMC and 91.18 ℃ for PCB. During the trenching, the highest temperature was obseved at T2 in the corner, i.e., 72.37 ℃ for EMC, 78.77 ℃ for PCB, and 100.94 ℃ for a solder bump. During laser cutting, the solder on the trench pad would be the key medium to dissipate more heat to the T2 position shown in Fig. 6(a). During trenchire, heat was spread from two sides to the T2 position shown in Fig. 6(b). All the temperatures measured in situ during the laser process are lower than the burn-in test temperature and even lower than the curing temperature of EMC. Thermal effects from laser irradiation will not cause any damage to the electronic components under the parameter provided in Sect. 2, i.e., 0.2 mm. The FLIR thermal imaging camera system is capable of measuring the real-time temperature distributions near the laser cutting surface. However, the system highly depends on the ambient radiation, material emissivity, and reflective apparent temperature. The in situ temperature measurement with embedded thermocouples inside the material developed in this research is much more feasible and reliable. References 1 H. C. Hsu, S. J. Wu, W. F. Lin, and B. Houng: Polym. Polym. Compos. 26 (2018) 1. 1 H. C. Hsu, S. J. Wu, W. F. Lin, and B. Houng: Polym. Polym. Compos. 26 (2018) 1. 2 D. E. Lee, H. W. Kim, B. S. Kong, and H. O. Choi: J. Appl. Polym. Sci. 134 (2017) 45252. 3 Y. Huang, D. Bigio, and M. G. Pecht: IEEE Trans. Comp. Packag. Technol. 29 (2006) 364. 4 S. H. Park, J. Y. Park, and T. H. Kim: Electron. Mater. Lett. 9 (2013) 459. 5 J. A. Herbsommer: IEEE Trans. Comp. Packag. Technol. 2 (2012) 1293. 6 A. Alwaidh, M. Sharp, and P. French: Opt. Lasers Eng. 58 (2014) 109. 7 X. C. Wang, Z. L. Li, T. Chen, B. K. Lok, and D. K. Y. Low: Opt. Lasers Eng. 48 (2008) 5. . C. Wang, Z. L. Li, T. Chen, B. K. Lok, and D. K. Y. Low 8 J. M. Yannou, C. Zinck, E. Cheng, V. K. Liao, A. Chan, and N. Tien: Proc. Semicon West (2016). 10 K. Ahn, S. H. Park, and Y. H. Kim: Microelectron. Reliab. 78 (2017) 1.f 11 J. Meijer, K. Du, A. Gillner, D. Hoffmann, V. S. Kovalenko, T. Masuzawa, A. Ostendorf, R. Poprawe, and W Schulzc: CIRP Ann. 51 (2002) 2. ( ) 12 M. Popescu, A. Paino, K. Stone, and J. K. Keller: IEEE Symp. Computational Intelligence for Security and Defence Applications (IEEE, 2012) 1–5.l 12 M. Popescu, A. Paino, K. Stone, and J. K. Keller: IEEE Symp. Computational Intelligence for Security an Defence Applications (IEEE 2012) 1 5 13 FLIR System: https://www.flir.com/ (accessed July 2018). 14 W. F. Lin and C. H. Chang: ROC Patent No. M510208 (2015). 1677 Sensors and Materials, Vol. 31, No. 5 (2019) About the Authors Wen-Fei Lin received his B.S. degree from Chang Gung University, Taiwan, in 1997 and his M.S. degree from National Sun Yat-sen University, Taiwan, in 2000. Currently, he is a Ph.D. candidate of the Department of Material Science and Engineering, I-Shou University. Since 2015, he has been a director of Engineering, Process & Field Application at E&R Engineering Corporation, Taiwan. His research interests are in laser machining, material behavior, and assembly. Hsiang-Chen Hsu received his B.S. degree from National Cheng Kung University, Taiwan, in 1981 and his M.S. and Ph.D. degrees from North Carolina State University, USA in 1988 and 1993, respectively. After graduation, he was an associate professor and became a full professor at I-Shou University, Taiwan. Since 2017, he has been temporarily transferred to St. John’s University as vice president. His research interests are in IC packaging, reliability design, and automation. Shih-Jeh Wu received his B.S. degree from National Chen-Kung University, Taiwan, in 1982 and his M.S. and Ph.D. degrees from Pennsylvania State University, USA, in 1995 and 1997, respectively. Since 2000, he has been a faculty member and now an associate professor at I-Shou University, Taiwan. His research interests are in nondestructive evaluation, ultrasonics, laser engineering, bioengineering, and sensory systems. Boen Houng received his B.S. degree from Taipei Institute of Technology, Taiwan, in 1986 and his M.S. and Ph.D. degrees from Drexel University and Colorado School of Mines, USA, in 1991 and 1996, respectively. From 2003 to 2007, he was an assistant professor at I-Shou University, Taiwan. Since 2013, he has been a professor at I-Shou University. His research interests are in thin film technology, oxide ceramics, and electronic materials.

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